U.S. patent application number 12/130691 was filed with the patent office on 2008-12-04 for device for electrophoresis, device for transfer, device for electrophoresis and transfer, chip for electrophoresis and transfer, and method for electrophoresis, method for transfer, and method for electrophoresis and transfer.
This patent application is currently assigned to SHARP KABUSHIKI KAISHA. Invention is credited to Satonari Akutsu, Atsunori Hiratsuka, Hideki Kinoshita, Yuji Maruo, Koji Sakairi, Yutaka Unuma, Kenji Yokoyama.
Application Number | 20080296158 12/130691 |
Document ID | / |
Family ID | 40086890 |
Filed Date | 2008-12-04 |
United States Patent
Application |
20080296158 |
Kind Code |
A1 |
Maruo; Yuji ; et
al. |
December 4, 2008 |
DEVICE FOR ELECTROPHORESIS, DEVICE FOR TRANSFER, DEVICE FOR
ELECTROPHORESIS AND TRANSFER, CHIP FOR ELECTROPHORESIS AND
TRANSFER, AND METHOD FOR ELECTROPHORESIS, METHOD FOR TRANSFER, AND
METHOD FOR ELECTROPHORESIS AND TRANSFER
Abstract
A device for electrophoresis applies a voltage to a medium in
contact with a plurality of electric conductors so that a potential
of adjacent conductors is within a certain range. This allows
preventing decline in electrophoresis speed. A device for
electrophoresis and transfer includes an electrode having a
plurality of electrode regions being insulated one another and
arranged in a specific direction. This allows providing a practical
and easy-to-use device for electrophoresis and transfer. A device
for transfer alters an applied voltage or applied voltage duration
to a certain position to another position. This allows improving
transfer efficiency.
Inventors: |
Maruo; Yuji;
(Nagareyama-shi, JP) ; Unuma; Yutaka;
(Matsudo-shi, JP) ; Hiratsuka; Atsunori; (Tokyo,
JP) ; Kinoshita; Hideki; (Tokyo, JP) ;
Yokoyama; Kenji; (Tsukuba-shi, JP) ; Sakairi;
Koji; (Tokyo, JP) ; Akutsu; Satonari; (Tokyo,
JP) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
SHARP KABUSHIKI KAISHA
Osaka-shi
JP
National Institute of Advanced Industrial Science and
Technology
Tokyo
JP
TOPPAN PRINTING CO., LTD.
Tokyo
JP
|
Family ID: |
40086890 |
Appl. No.: |
12/130691 |
Filed: |
May 30, 2008 |
Current U.S.
Class: |
204/600 ;
204/450 |
Current CPC
Class: |
G01N 27/44704 20130101;
B01L 3/50273 20130101 |
Class at
Publication: |
204/600 ;
204/450 |
International
Class: |
C25B 7/00 20060101
C25B007/00; C25B 9/00 20060101 C25B009/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 31, 2007 |
JP |
2007-146215 |
May 31, 2007 |
JP |
2007-146235 |
May 31, 2007 |
JP |
2007-146276 |
Claims
1. A device for electrophoresis configured to separate separation
target components in a medium in which a plurality of electric
conductors are provided in contact with the medium, comprising:
voltage applying means configured to apply a voltage to the medium,
so that a potential difference between adjacent electric conductors
is more than 0V but not more than 0.3V.
2. The device for electrophoresis as set forth in claim 1, wherein:
the voltage applying means applies the voltage to each of the
electric conductors.
3. The device for electrophoresis as set forth in claim 1, wherein:
the electric conductor is line-shaped.
4. The device for electrophoresis as set forth in claim 3, wherein:
the line-shaped electric conductors are arranged in parallel one
another and extend in an orthogonal direction to a direction in
which the voltage applying means applies the voltage to the
medium.
5. A device for electrophoresis and transfer comprising: first
voltage applying means configured to apply a voltage with respect
to a specific direction in a first medium including separation
target components; and second voltage applying means configured to
apply a voltage to the first medium toward a direction of a second
medium being contact with the first medium, the second voltage
applying means including a first electrode having a plurality of
electrode regions being insulated one another and arranged in the
specific direction.
6. The device for electrophoresis and transfer as set forth in
claim 5, wherein: the electrode region is line-shaped.
7. The device for electrophoresis and transfer as set forth in
claim 6, wherein: the plurality of electrode regions are arranged
in parallel one another and extend in an orthogonal direction to
the specific direction.
8. The device for electrophoresis and transfer as set forth in
claim 5 further comprising: wire connection means configured to
switch the first electrode between connection and
disconnection.
9. The device for electrophoresis and transfer as set forth in
claim 5, wherein: the second voltage applying means further
includes a detachable second electrode.
10. The device for electrophoresis and transfer as set forth in
claim 9 further comprising: a detachable holder configured to hold
the second electrode and the second medium.
11. The device for electrophoresis and transfer as set forth in
claim 5, wherein: the second voltage applying means includes a
second electrode having a plurality of electrode regions being
insulated one another and arranged in the specific direction.
12. A chip for electrophoresis and transfer comprising: a
separation section configured to place a first medium including
separation target components, and a second medium in contact with
the first medium; a first buffer solution chamber and a second
buffer solution chamber sandwiching the separation section; and a
first electrode being provided on the separation section, and
including a plurality of electrode regions being insulated one
another and arranged in a specific direction specified by the first
buffer solution chamber and the second buffer solution chamber.
13. A method for electrophoresis and transfer comprising: (a)
applying a voltage to a first medium including separation target
components in a specific direction; and (b) applying a voltage to
the first medium toward a direction of the second medium being in
contact with the first medium, following the step (a), wherein in
the step (b), the voltage is applied with use of a first electrode
including a plurality of electrode regions being insulated one
another and arranged in parallel one another in the specific
direction.
14. A device for transfer configured to transfer transfer target
components in a first medium to a second medium comprising: voltage
applying means configured to apply a voltage to the first medium;
the voltage applying means applying the voltage to the first medium
in such a manner that a certain position and another position in
the first medium are provided different voltages or different
voltage durations.
15. The device for transfer as set forth in claim 14, wherein: the
voltage applying means increases the voltage stepwise or gradually
toward a specific direction specified by the first medium.
16. The device for transfer as set forth in claim 14, wherein: the
voltage applying means increases an applied voltage duration
stepwise or gradually toward a specific direction specified by the
first medium.
17. The device for transfer as set forth in claim 14, wherein: the
voltage applying means includes a first electrode and a second
electrode, each of which is divided into a plurality of electrode
regions being insulated one another.
18. The device for transfer as set forth in claim 17, wherein: the
electrode region is line-shaped.
19. The device for transfer as set forth in claim 18, wherein: the
electrode regions are arranged in parallel one another and extend
in an orthogonal direction to the specific direction.
20. The device for transfer as set forth in claim 17, wherein: the
voltage applying means applies the voltage to each of the electric
regions.
21. The device for transfer as set forth in claim 20, wherein: the
voltage applying means comprises at least one power supply, and a
plurality of conductive paths connecting the power supply and each
of the electrode regions conductively, and the conductive paths
have at least two kinds of resistance values.
22. The device for transfer as set forth in claim 20, wherein: the
voltage applying means alters a potential duration applied to each
of the electrode regions.
23. The device for transfer as set forth in claim 22, wherein: the
voltage applying means comprises a power supply and a mobile
electric conductive section conductively connecting the power
supply and the plurality of conductive regions, and the mobile
electric conductive section alters duration of potential
application to each of the electrode regions by its movement.
24. The device for transfer as set forth in claim 14, wherein: the
voltage applying means applies the voltage to the first medium via
an electric resistance layer, and a certain position and another
position on the electric resistance layer have different resistance
values.
25. A method for transfer for transferring transfer target
components in a first medium to a second medium, comprising:
applying a voltage to the first medium, in such a manner that a
certain position and another position in the first medium are
provided different voltages or different voltage durations.
Description
[0001] This Nonprovisional application claims priority under 35
U.S.C. .sctn. 119(a) on Patent Applications No. 146215/2007, No.
146235/2007, and No. 146276/2007 each filed in Japan on May 31,
2007, the entire contents of which are hereby incorporated by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to devices for
electrophoresis, transfer, and electrophoresis and transfer, and
methods for electrophoresis, transfer, and electrophoresis and
transfer.
BACKGROUND OF THE INVENTION
[0003] In the life science filed, electrophoresis is one of the
significantly useful separating and analyzing techniques. It is
used for separating biopolymers, such as protein, DNA, RNA, and the
like. It is also possible to separate cells. In addition to a
typical electrophoresis method with use of gel, a Capillary
electrophoresis method with use of buffer solutions containing a
polymer and a Free Flow Electrophoresis method carried out in free
solution have been performed.
[0004] As a technique to improve electrophoresis, Japanese Utility
Model Publication, Jitsukaihei, No. 5-4004 (date of publication:
Jan. 22, 1999) discloses that disruption in electrophoresis pattern
due to uneven electrophoresis field is prevented by providing a
wire-shaped electric conductor in an electrophoresis chamber.
[0005] Also, Japanese Utility Model Publication, Jitsukaihei, No.
5-4003 (date of publication: Jan. 22, 1993) discloses that
provision of an intermediate electrode in an electrophoresis
chamber actively controls distributions of electric fields and
electric potentials in the electrophoresis chamber.
[0006] As a result of study, however, the present inventors
identified that the electrophoresis devices having an electric
conductor in an electrophoresis chamber, such as the
electrophoresis devices in the above documents have a problem of
decline in electrophoresis speed.
[0007] Also, in Proteome analysis, it is necessary to simplify a
series of procedures including first electrophoresis, second
electrophoresis, and transferring a sample to a membrane, which are
carried out continuously.
[0008] Japanese Publication for Unexamined Patent Application,
Tokukai, No. 2007-64848 (date of publication: Mar. 15, 2007)
discloses an electrophoresis device for automating the first
electrophoresis and the second electrophoresis.
[0009] Japanese Publication for Unexamined Patent Application,
Tokukai, No. 2000-28578 (date of publication: Jan. 28, 2000)
discloses a device for electrophoresis and transfer.
[0010] However, a device disclosed in Japanese Publication for
Unexamined Patent Application, Tokukai, No. 2000-28578 (date of
publication: Jan. 28, 2000) still requires a complicated and
skillful procedure such as a removal of gel after an
electrophoresis.
[0011] Japanese Publication for Unexamined Patent Application,
Tokukai, No. 2006-71494 (date of publication: Mar. 16, 2006)
proposes an easy-operation device for electrophoresis and transfer
which does not require the removal of gel.
[0012] However, a practical and easy-operation device for
electrophoresis and transfer is not yet in existence.
[0013] According to the study of the present inventors,
electrophoresis devices merely having an electrode for transfer in
the middle of the electrophoresis path, such as a device disclosed
in Japanese Publication for Unexamined Patent Application, Tokukai,
No. 2006-71494 (date of publication: Mar. 16, 2006), have a
difficulty in performing an electrophoresis. Specifically, the
electrophoresis device with the above structure failed to apply a
voltage and migrate a sample in the process of electrophoresis.
[0014] Further, in analyzing a result of an electrophoresis, it is
widely performed that a sample in gel are transferred to a membrane
and carrying out an antigen antibody reaction on the membrane.
[0015] As a method for transferring a sample in gel to a membrane,
there are several methods such as a way of using capillary action
and a way of using a voltage difference. (Referring to a book
"DENKIEIDOU NARUHODO Q&A" ("Electrophoresis Q & A") P. 161
to 163, written by Michihiro Ofuji, published by Yodosha in
2005)
[0016] However, the present inventors identified that transfer
efficiency was declined in some samples in transferring them in gel
to a membrane under a conventional transfer method.
SUMMARY OF THE INVENTION
[0017] The first object of the present invention is to prevent
decline in electrophoresis speed in an electrophoresis device
including an electric conductor in an electrophoresis chamber.
[0018] As a result of diligent studies, the present inventors
identified that a cause of the decline in electrophoresis speed was
in a conventionally unidentified phenomenon. The phenomenon is that
air bubbles generated at an electric conductor in an
electrophoresis chamber detach gel from the electric conductor, and
a buffer solution goes into the gap between the gel and the
electric conductor, thereby attaining inhibition of a buffer
action.
[0019] Then, the present inventors studied further and found that
the air bubbles are not generated when an electric potential
difference between adjacent electric conductors is under a certain
voltage, and accomplished the invention based on the finding.
[0020] Namely, a device for electrophoresis of the present
invention is configured to separate separation target components in
a medium in which a plurality of electric conductors are provided
in contact with the medium, including voltage applying means
configured to apply a voltage to the medium, so that a potential
difference between adjacent electric conductors is more than 0V but
not more than 0.3V.
[0021] With this structure, the voltage applying means applies a
voltage so that a potential difference between adjacent electric
conductors is more than 0V but not more than 0.3V. If the potential
difference between adjacent electric conductors is not more than
0.3, no air bubbles generate as shown in embodiment examples
described later. Therefore, this structure is capable of
suppressing decline in electrophoresis speed due to the emergence
of air bubbles.
[0022] In the electrophoresis device with this structure, the
voltage applying means may apply the voltage to each of the
electric conductors.
[0023] According to this structure, the voltage applying means may
apply a potential to each of the electric conductors directly so
that it is easy to set a potential difference between adjacent
electric conductors more than 0V but not more than 0.3V.
[0024] It is preferable that the device for electrophoresis has 250
or more electric conductors.
[0025] According to this structure, it is easy to control a
potential difference between adjacent electric conductors not more
than 0.3V by only applying a voltage which is in normal range
voltage for electrophoresis (from 50V to 500V) to gel.
[0026] It is preferable that the electric conductor is line-shaped
in the device for electrophoresis.
[0027] With this structure, it is easy to control a potential
difference between adjacent electric conductors not more than 0.3V
with use of narrow line-shaped electric conductors which allow
arranging a number of electric conductors.
[0028] It is preferable that the line-shaped electric conductors
are arranged in parallel one another and extend in an orthogonal
direction to the direction in which the voltage applying means
applies a voltage to the medium.
[0029] According to this structure, the line-shaped electric
conductors are arranged in parallel one another and extend in an
orthogonal direction to the direction in which the voltage applying
means applies a voltage to the medium. Therefore, the electric
conductors do not affect a potential gradient toward the first
direction, thereby making it possible to separate the separation
target components in the medium favorably.
[0030] It is preferable that the line-shaped electric conductor is
not more than 10 .mu.m in thickness in the device for
electrophoresis.
[0031] With this structure, it is easy to control a potential
difference between adjacent electric conductors not more than 0.3V
with use of the narrow line-shaped electric conductors which allow
arranging a number of electric conductors even in a small sized
device.
[0032] It is preferable that the distance between adjacent electric
conductors is not more than 100 .mu.m in the device for
electrophoresis.
[0033] With this structure, it is easy to control a potential
difference between adjacent electric conductors not more than 0.3V
because of the narrow space between the electric conductors, which
allows arranging a number of electric conductors even in a small
sized device.
[0034] It is preferable in the device for electrophoresis that the
electric conductor is made of metal selected from the group
consisting of platinum, zinc, and copper.
[0035] This structure allows performing a method for
electrophoresis of the present invention repeatedly because the
electric conductor is less likely to be deteriorated.
[0036] The device for electrophoresis may be an isoelectric
electrophoresis device.
[0037] This structure allows forming a uniform pH gradient in a
medium in an isoelectric electrophoresis device.
[0038] A method for the electrophoresis of the present invention is
a method for separating separation target components in a medium in
which a plurality of electric conductors are provided in contact
with the medium, including applying a voltage to the medium so that
a potential difference between adjacent electric conductors is more
than 0V but not more than 0.3V.
[0039] With this arrangement, in the process of applying voltage, a
voltage is applied so that a potential difference between adjacent
electric conductors is more than 0V but not more than 0.3V. When
the voltage between the adjacent electric conductors is not more
than 0.3V, air bubbles do not generate. Therefore, this arrangement
allows preventing the decline in electrophoresis speed due to the
generation of air bubbles.
[0040] The second object of the present invention is to provide a
practical and easy-to-use device for electrophoresis and transfer,
a chip for electrophoresis and transfer, and a method for
electrophoresis and transfer.
[0041] As a result of diligent studies, the present inventors found
that it is possible to perform an electrophoresis with ease, even
though an electrode for transfer is provided in the middle of the
electrophoresis path, by using the electrode having a plurality of
electrode regions being insulated one another and arranged in a
direction of electrophoresis path and completed the invention.
[0042] Namely, a device for electrophoresis and transfer of the
present invention includes first voltage applying means configured
to apply a voltage with respect to a specific direction in the
first medium having separation target components, and second
voltage applying means configured to apply a voltage to the first
medium toward a direction of a second medium being in contact with
the first medium, the second voltage applying means including a
first electrode having a plurality of electrode regions being
insulted one another and arranged in the specific direction.
[0043] According to this structure, the device for electrophoresis
and transfer of the present invention can separate separation
target components in the first medium, and then transfer the
separated separation target components to the second medium. For
example, it is very useful to use the device for electrophoresis
and transfer which can perform both an electrophoresis and a
blotting easily in an analysis of biopolymer.
[0044] In a conventional device for electrophoresis and transfer,
however, the electrode, which is provided in the second voltage
applying means for transferring separation target components in a
first medium to a second medium, hampers the separation of the
separation target components in the first medium. Therefore, the
conventional device was not useful.
[0045] However, with this structure, the second voltage applying
means includes the first electrode having a plurality of electrode
regions being insulated one another and arranged in the specific
direction. Since the electrode regions in the first electrode are
insulated one another and arranged in the specific direction, the
separation of the separation target components in the first medium
is not hampered. Namely, with this structure, it is possible to
perform an electrophoresis and a transfer practically and
easily.
[0046] It is preferable that the electrode region is line-shaped in
the device for electrophoresis and transfer.
[0047] According to this structure, each of the electrode regions
is line-shaped which enables to fill up the surface easily when
they are arranged in the specific direction.
[0048] This allows transferring the separation target components in
the first medium to the second medium efficiently.
[0049] It is preferable that the plurality of electrode regions are
arranged in parallel one another and extend in an orthogonal
direction to the specific direction in the device for
electrophoresis and transfer.
[0050] The line-shaped electrode regions arranged in parallel one
another and extending in an orthogonal direction to the specific
directiondo not affect the voltage applied to the specific
direction. This allows separating separation target components in
the first medium favorably.
[0051] It is more preferable that the device for electrophoresis
and transfer further includes wire connection means configured to
switch the first electrode between connection and
disconnection.
[0052] With this structure, it is possible to control a potential
of the first electrode easily because the wire connection means is
capable of switching the first electrode between connection and
disconnection.
[0053] The second voltage applying means may further include a
detachable second electrode in the device for electrophoresis and
transfer.
[0054] According to this structure, the second voltage applying
means has a detachable second electrode as well as the first
electrode. This allows transferring the separation target
components in the first medium to the second medium with ease
without affecting the separation of separation target components in
the first medium.
[0055] The device for electrophoresis and transfer may include a
detachable holder configured to hold the second electrode and the
second medium.
[0056] According to this structure, the second medium is easy to be
used for further analysis since the second medium is
detachable.
[0057] The second voltage applying means may further include a
second electrode having a plurality of electrode regions being
insulated one another and arranged in the specific direction in the
device for electrophoresis and transfer.
[0058] According to this structure, the electrode regions in the
second electrode do not hamper the separation of the separation
target components in the first medium because the electrode regions
are insulated one another and are arranged in the specific
direction in which the separation target components in the first
medium are separated. Therefore, it is possible to transfer the
separation target components in the first medium to the second
medium with ease without affecting the separation of separation
target components in the first medium.
[0059] A chip for electrophoresis and transfer of the present
invention includes a separation section configured to place a first
medium having separation target components, and a second medium in
contact with the first medium, a first buffer solution chamber and
a second buffer solution chamber sandwiching the separation
section, and a first electrode being provided on the separation
section, and having a plurality of electrode regions being
insulated one another and arranged in the specific direction
specified by the first and the second buffer solution chambers.
[0060] According to this structure, it is possible to separate the
separation target components in the first medium toward the
specific direction by applying a voltage to the first medium
provided on the separation section via the electrodes arranged in
the first and second buffer solution chambers. At this time, the
first electrode, which is provided on the separation section, has
the electric regions being insulated one another and arranged in
the above direction. This allows not affecting to the separation of
the separation target components in the first medium.
[0061] The device realizes both electrophoresis and transfer
because the first electrode allows applying a voltage to the
separated separation target components easily, in a direction from
the first medium toward the second medium.
[0062] A method for electrophoresis and transfer of the present
invention includes (a) applying a voltage to the first medium
having separation target components in the specific direction, and
(b) applying a voltage to the first medium toward a direction of
the second medium being in contact with the first medium, following
the step (a), wherein in the step (b), the voltage is applied with
use of the first electrode including a plurality of electrode
regions being insulated one another and arranged in the specific
direction.
[0063] With this arrangement, it is possible to perform
electrophoresis and transfer favorably because the first electrode
which is used in the second voltage applying process do not
adversely affect the separation of the separation target components
in the first medium in the first voltage applying process.
[0064] The third object of the present invention is made in view of
the foregoing problem and is for providing a device for transfer
preventing decline in transfer efficiency.
[0065] As a result of diligent sturdy, the inventors of the present
invention found that there is a suitable transfer voltage depending
on a molecular weight of a sample to be transferred and also found
that it is possible to prevent decline in transfer efficiency by
adjusting an applied voltage to the medium including the sample
depending on positions. The present invention is accomplished based
on the finding.
[0066] Namely, a device for transfer of the present invention is
configured to transfer target components in the first medium to the
second medium, including voltage applying means configured to apply
a voltage to the first medium, the voltage applying means applying
the voltage to the first medium in such a manner that a certain
position and another position in the first medium are provided
different voltages or different voltage durations.
[0067] With this structure, the transfer target components in the
first medium such as agarose gel are transferred to the second
medium such as a membrane by applying a voltage to the first
medium. The voltage applied to the first medium varies depending on
positions in the first medium. This allows applying a suitable
voltage depending on a molecular weight of each of the transfer
target components in the first medium, for example, SDS-PAGE
separated polyacrylamide gel including transfer target components
whose molecular weights differ in position in the gel.
[0068] According to the findings of the present inventors, there is
a suitable voltage for transfer depending on a molecular weight of
a transfer target component. In other words, the transfer
efficiency declines when a voltage far from the suitable voltage is
applied to the transfer target component. This structure allows
applying a suitable voltage depending on the molecular weight of
each of the transfer target components and preventing decline in
transfer efficiency.
[0069] As described later, it is possible to obtain the same effect
by adjusting a voltage duration applied to the transfer target
components instead of adjusting an applied voltage to the transfer
target components.
[0070] It is preferable that the voltage applying means increases
the voltage stepwise or gradually toward the specific direction
specified by the first medium in the device for transfer.
[0071] According to this structure, the voltage applied to the
first medium increases toward the specific direction specified by
the first medium. Accordingly, it is possible to apply a suitable
voltage depending on the molecular weight of each of the transfer
target components in the first medium such as polyacrylamide gel
separated by SDS-PAGE including transfer target components in which
the distribution of the molecular weight increases in one way.
[0072] The voltage applying means may increase an applied voltage
duration stepwise or gradually toward the specific direction
specified by the first medium in the device for transfer.
[0073] As described above, it is possible to obtain the same effect
by adjusting a voltage duration applied to transfer target
components instead of adjusting an applied voltage to transfer
target components.
[0074] It is preferable that the voltage applying means includes
the first electrode and the second electrode, each of which is
divided into a plurality of electrode regions being insultated one
another in the device for transfer.
[0075] According to this structure, each of the first and the
second electrodes includes a plurality of electrode regions being
insulated one another. Therefore, it is possible to provide a
different potential to each of the electrode regions or a different
potential duration to each of the electrode regions with ease.
[0076] It is preferable that the voltage applying means includes
the first electrode and the second electrode, each of which is
divided into a plurality of electrode regions arranged in the
specific direction in the device for transfer.
[0077] According to this structure, each of the first and the
second electrodes is divided into a plurality of electrode regions
being insulated one another and arranged in the specific direction.
Therefore, it is possible to increase an applied voltage or a
voltage duration applied to the first medium toward the specific
direction with ease.
[0078] It is preferable that each divisional shape of the electrode
regions of the first electrode and each divisional shape of the
electrode regions of the second electrode are substantially equal
in the device for transfer.
[0079] According to this structure, it is possible to apply a
voltage to the first medium accurately because the divisional shape
of the electrode regions of the first electrode and the second
electrode are substantially equal. Namely, an electrode region of
the first electrode and a corresponding electrode region of the
second electrode have a substantially equal shape. Therefore, an
electric field applied by a potential difference between the first
and the second electrode regions is easily predictable because the
electric field is based on the shape of both electrode regions.
[0080] It is preferable that the electrode region is line-shaped in
the device for transfer.
[0081] According to this structure, it is possible to generate a
fine potential difference because each of the electrode regions is
line-shaped.
[0082] It is preferable that the line-shaped electrode regions are
arranged in parallel one another and extend in an orthogonal
direction to the specific direction in the device for transfer.
[0083] This structure allows dividing a potential finely toward the
specific direction.
[0084] The voltage applying means may apply the voltage to each of
the electrode regions in the device for transfer.
[0085] This structure allows the voltage applying means to apply a
potential to the electrode regions directly.
[0086] Therefore, it is possible to adjust an applied voltage in
accordance with the molecular weight of the transfer target
components with ease.
[0087] In the device for transfer, the voltage applying means
includes at least one power supply, and a plurality of conductive
paths connecting the power supply and each of the electrode regions
conductively, and the conductive paths may have at least two kinds
of resistance values.
[0088] According to this structure, the conductive paths
conductively connecting the power supply and each of the electrode
regions have two or more kinds of resistance values. This allows
applying two or more kinds of potentials to each of the electrode
regions depending on the molecular weight of the transfer target
components.
[0089] In the device for transfer, the voltage applying means may
alter a potential duration applied to each of the electrode
regions.
[0090] According to this structure, it is possible to apply a
voltage for a suitable time period to the transfer target
components depending on a molecular weight of each component
because the voltage applying means controls a potential duration
applied to the electrode regions.
[0091] In the device for transfer, the voltage applying means
includes a power supply and a mobile electric conductive section
conductively connecting the power supply and the plurality of
conductive regions, and the mobile electric conductive section may
alter duration of potential application to each of the electrode
regions by its movement.
[0092] This structure allows the voltage applying means to control
a potential duration applied to each of the electrode regions with
ease.
[0093] The mobile electric conductive section may be bar-shaped in
the device for transfer.
[0094] This structure allows the potential control means to control
a potential duration applied to each of the electrode regions with
ease.
[0095] In the device for transfer of the present invention, the
voltage applying means applies the voltage to the first medium via
an electric resistance layer, and a certain position and another
position on the electric resistance layer may have different
resistance values.
[0096] According to this structure, the voltage applying means
applies a voltage to the first medium via the electric resistance
layer and the electric resistance layer have various resistance
values depending on positions. This allows the voltage applying
means to apply different voltages to the first medium by positions
with ease.
[0097] It is preferable that the voltage applying means applies a
voltage to the first medium via the electric resistance layer in
which a resistance value decreases stepwise or gradually to the
specific direction specified by the first medium.
[0098] According to this structure, since the electric resistance
layer has a stepwise or gradually decreasing resistance value to
the specific direction specified by the first medium, the voltage
applying means is able to increase an applied voltage stepwise or
gradually to the first medium to the specific direction with
ease.
[0099] It is preferable that the device for transfer further
includes cooling means for cooling the voltage applying means.
[0100] According to this structure, the cooling means radiates heat
in the voltage applying means and maintains optimum temperatures
for the first and the second mediums, thereby making it possible to
prevent affecting both mediums and the transfer target components
qualitatively.
[0101] A method for transfer of the present invention is a method
for transferring transfer target components in a first medium to a
second medium including applying a voltage to the first medium, in
such a manner that a certain position and another position in the
first medium are provided different voltages or different potential
durations.
[0102] According to this structure, it is possible to prevent
decline in transfer efficiency by applying a suitable voltage
depending on a molecular weight of each of the transfer target
components same as the device for transfer of the present
invention.
[0103] Additional objects, features, and strengths of the present
invention will be made clear by the description below. Further, the
advantages of the present invention will be evident from the
following explanation with reference to the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0104] FIG. 1 is a schematic view illustrating a structure of a
device for electrophoresis according to an embodiment of the
present invention.
[0105] FIG. 2 is a perspective view illustrating a structure of an
isoelectric electrophoresis device according to another embodiment
of the present invention.
[0106] FIG. 3 is a plane view illustrating the structure of the
isoelectric electrophoresis device according to the embodiment of
the present invention.
[0107] FIG. 4 is a side view illustrating the structure of the
isoelectric electrophoresis device according to the embodiment of
the present invention.
[0108] FIG. 5 is an explanatory view of an electrophoresis method
according to further another embodiment of the present
invention.
[0109] FIG. 6 is a photograph of a plurality of electric conductors
according to an embodiment of the present invention.
[0110] FIG. 7 is a photograph of a result of the electrophoresis
method according to an embodiment of the present invention.
[0111] FIG. 8 is a graph of a result of the electrophoresis method
according to an embodiment of the present invention.
[0112] FIG. 9 is a schematic view illustrating a structure of a
device for electrophoresis and transfer according to an embodiment
of the present invention in electrophoresis operation.
[0113] FIG. 10 is a schematic view illustrating a structure of the
device for electrophoresis and transfer according to the embodiment
of the present invention in transfer operation.
[0114] FIG. 11 is an explanatory drawing illustrating an
electrophoresis operation of the device for electrophoresis and
transfer according to the embodiment of the present invention.
[0115] FIG. 12 is an explanatory drawing illustrating a transfer
operation of the device for electrophoresis and transfer according
to the embodiment of the present invention.
[0116] FIG. 13 is a schematic view illustrating another structure
of a device for electrophoresis and transfer according to an
embodiment of the present invention.
[0117] FIG. 14 is an explanatory view illustrating an operation of
a wire connector in the device for electrophoresis and transfer
according to the embodiment of the present invention.
[0118] FIG. 15 is an explanatory view illustrating an
electrophoresis operation of the device for electrophoresis and
transfer according to the embodiment of the present invention.
[0119] FIG. 16 is an explanatory view illustrating a transfer
operation of the device for electrophoresis and transfer according
to the embodiment of the present invention.
[0120] FIG. 17 is a schematic view illustrating a structure of an
automated two-dimensional Western blotting device according to an
embodiment of the present invention.
[0121] FIG. 18 is a photograph showing a result of electrophoresis
in the case where one side of the gel plates is removed.
[0122] FIG. 19 is a photograph showing another result of
electrophoresis in the case where one side of the gel plates is
removed.
[0123] FIG. 20 is a photograph of the device for electrophoresis
and transfer according to the embodiment of the present invention
which was taken right after an electrophoresis operation.
[0124] FIG. 21 is a photograph of the device for electrophoresis
and transfer according to the embodiment of the present invention
which was taken after a transfer operation.
[0125] FIG. 22 is a photograph of a transferred membrane of the
device for electrophoresis and transfer according to the embodiment
of the present invention which was taken right after a transfer
operation.
[0126] FIG. 23 is a photograph of a series of procedures of a
method for electrophoresis and transfer according to the embodiment
of the present invention.
[0127] FIG. 24 is a photograph showing a result of a transfer
performed by the device for electrophoresis and transfer according
to the embodiment of the present invention.
[0128] FIG. 25 is a perspective view illustrating an overall of a
device for transfer according to an embodiment of the present
invention.
[0129] FIG. 26 is a schematic view illustrating an operation of the
device for transfer according to the embodiment of the present
invention.
[0130] FIG. 27 is a schematic view illustrating an operation of a
device for transfer according to another embodiment of the present
invention.
[0131] FIG. 28 is a schematic view illustrating an operation of a
device for transfer according to further another embodiment of the
present invention.
[0132] FIG. 29 is a schematic view illustrating another operation
of the device for transfer according to the embodiment of the
present invention.
[0133] FIG. 30 is a perspective view illustrating an overall of a
device for transfer according to still further another embodiment
of the present invention.
[0134] FIG. 31 is a perspective view illustrating an overall of an
electric resistance sheet according to an embodiment of the present
invention.
[0135] FIG. 32 is a schematic view illustrating a structure of the
device for transfer according to the embodiment of the present
invention.
[0136] FIG. 33 is a perspective view illustrating an overall of a
device for transfer according to yet another embodiment of the
present invention.
[0137] FIG. 34 is a photograph showing a result of a transfer
performed by a conventional device for transfer.
[0138] FIG. 35 is a table showing optimum transfer voltages
depending molecular weights.
[0139] FIG. 36 is a graph showing optimum transfer voltages
depending on molecular weights.
[0140] FIG. 37 is a schematic view of a device for transfer
according to an embodiment of the present invention.
[0141] FIG. 38 is a schematic view of a device for transfer
according to an embodiment of the present invention.
[0142] FIG. 39 is a photograph of a device for transfer according
to an embodiment of the present invention.
[0143] FIG. 40 is a photograph showing a result of a transfer
performed by a device for transfer according to an embodiment of
the present invention.
[0144] FIG. 41 is a photograph of a result of a transfer performed
by a conventional device for transfer.
DESCRIPTION OF THE EMBODIMENTS
[1: Device for Electrophoresis and Method for Electrophoresis]
[0145] The following explanation deals with an embodiment of a
device for electrophoresis according to the present invention with
refer to the FIG. 1.
[0146] The device for electrophoresis 100 of the present embodiment
includes buffer solution chambers 101 and 102, a separation section
103, electrodes 104 and 105 (voltage applying means), a stripe
electrode 106 (electric conductors), and a lid member 107. The
buffer solution chambers 101 and 102 are filled with a buffer
solution and provided with the electrodes 104 and 105 therein,
respectively. The separation section 103 is sandwiched between the
buffer solution chambers 101 and 102. The stripe electrode 106 is
provided on the separation section 103, and a medium 120 is
provided on the stripe electrode 106 in the separation section
103.
[0147] As the buffer solution, a buffer solution which has a
composition generally used for electrophoresis is applicable. Also,
the medium 120 is made from a gel generally used for
electrophoresis such as an agarose gel, a polyacrylamide, and the
like and contains separation target components. The separation
target components are the components to be separated and analyzed
by electrophoresis and transfer. The separation target components
may be prepared preferably from biological materials such as bions,
biological fluid, cell strains, cultured tissues, and fragment
tissues.
[0148] The electrodes 104 and 105 apply a voltage to the medium 120
so that a potential difference between stripe electric conductors
106 is not more than 0.3 V. This makes it possible to prevent the
generation of air bubbles. The voltage applied by the electrodes
104 and 105 is calculated by the number of electric conductors in
the stripe electrode 106 and the following formula (I) is
satisfied. Note that the applied voltage should exceed 0V.
Applied voltage (V).ltoreq.(the number of electric
conductors-1).times.0.3 (V) (1)
[0149] As one aspect, the device for electrophoresis 100 of the
present embodiment may have potential control means (voltage
applying means), which is not illustrated in the drawings, for
controlling a potential of each electric conductor in a stripe
electric conductor 113. The potential control means, for example,
includes a power supply, and conductive paths. The power supply is
conductively connected to each of the electric conductors via a sub
power supply or conductive paths, each of which has a different
resistance value. This allows the potential control means to apply
a different voltage to each of the electric conductors. Note that
it is not necessary to control potentials of all the electric
conductors. Potentials of some electric conductors (called
intermediate electrodes hereinafter) may be controlled. In this
case, the other electric conductors sandwiched between the
intermediate electrodes have a potential based on a potential
gradient created by each of the intermediate electrodes. In any
cases, the potential should be applied so that the potential
difference of the adjacent electric conductors is not more than 0.3
V. This allows preventing the generation of air babbles.
[0150] In addition to the above effect, the potential control means
contributes to favorably carrying out the electrophoresis. For
example, the electrophoresis may be carried out with a greater
potential gradient in a low potential range and a smaller potential
gradient in a high potential range. The greater potential gradient
in the low potential range allows greater separation of high
polymer weight components which are difficult to separate, among
the separation target components in the medium 120, while the
smaller potential gradient in the high potential range retains in
the medium 120 such fast-migrating low polymer weight components
among the separation target components in the medium 120. This
allows favorable separation of the separation target components,
which include wide range of molecular weight components. Namely, in
electrophoresis, components migrate from a low potential side to a
high potential side. High polymer weight components remain on the
low potential side and low polymer weight components tend to
migrate to the high potential side. As described above, it is
possible to increase the degree of separation of high polymer
weight components by increasing the potential gradient of a low
potential range and hamper the migration of low polymer weight
components by decreasing the potential gradient of a high potential
range.
[0151] In another embodiment, a device for electrophoresis of the
present invention is an isoelectric electrophoresis device. The
isoelectric electrophoresis device 200 of the present embodiment is
illustrated in FIG. 2 (a perspective view), in FIG. 3 (a top view),
and in FIG. 4 (a side view). The isoelectric electrophoresis device
200 includes a separation chamber 201, electrodes 202 and 203
(voltage applying mean), stripe electrodes 204 and 205, and a lid
member 206. Medium 220 is sandwiched between the stripe electrodes
204 and 205 in the separation chamber 201.
[0152] It is preferable that the medium 220 includes ampholyte for
generating a pH gradient. When the stripe electrodes 204 and 205
apply a voltage to the medium 220, each of the electric conductors
of the stripe electrodes 204 and 205 has an individual potential
and an electric conductor of the stripe electrode 204 and an
electric conductor of the stripe electrode 205, both of which have
a same distance from respective electrodes, have a same potential.
Therefore, there is a uniform potential in the vicinity of each
electric conductor of the stripe electrodes 204 and 205.
Accordingly, ampholyte migrates in the medium 220 and generates a
uniform pH gradient.
[0153] In this case, the electrodes 202 and 203 apply a voltage so
that a potential difference between each pair of electric
conductors of the stripe electric conductors 213 and 214 is not
more than 0.3V, thereby preventing air babbles.
[0154] Further, in further another embodiment, a device for
electrophoresis 300 of the present invention applies a voltage to a
medium 320 with use of stripe electrodes 301 and 302. FIG. 5 is an
explanatory drawing of an operation of the device for
electrophoresis 300 of the present embodiment. As illustrated in
FIG. 5, the device for electrophoresis 300 performs electrophoresis
by applying a voltage only to adjacent corresponding electric
conductors of the stripe electrodes 301 and 302 and shifting the
voltage applied electric conductors sequentially.
[0155] In addition to a method for applying voltage to adjacent
corresponding electric conductors, it is possible to apply a few V
to 10V to a plurality of electric conductors leaving a certain
space and shift the voltage applied electric conductors
sequentially when a gap between electric conductors is narrow. For
example, in the case of 10V being applied, it is required to leave
space more than 34 electric conductors. This allows applying a
voltage so that the potential difference of the adjacent electric
conductors is not more than 0.3V.
[0156] It is possible to prevent the generation of air babbles by
adjusting the potential difference between adjacent electric
conductors 0.3V. Compared to the other embodiments, the present
embodiment less likely to have a problem caused by air babbles
because a potential is applied to limited electrodes only. Needless
to say, it is preferable that the potential difference between
adjacent electric conductors is not more than 0.3V.
Example 1
Making a Stripe Electrode
[0157] Cr binder was deposited on a 6 cm.times.5 cm glass plate 3
mm in thickness by a sputtering device and about 2000 .ANG. Pt was
also deposited. Then, a thickness of Pt electrode and a gap between
Pt electrodes were created to be approximately 100 .mu.m by a
Dicing saw (Disco) with a blade 100 .mu.m in thickness. In
addition, two kinds of stripe electrode substrates, (i) a thickness
of Pt wire and a gap between Pt wires were 100 .mu.m and (ii) a
thickness of Pt wire and a gap between Pt wires were 50 .mu.m, were
created by a CO.sub.2 laser carving machine under a following
cutting condition, laser power 7%, process speed 6%, and 3000 Hz
(FIG. 6). Note that the surface of the glass was little processed
by a laser under the above condition.
Example 2
Comparison of Substrates
[0158] A pair of 6 cm.times.5 cm gel plates (glass plate)
sandwiching 1 mm spacer therebetween was placed in a gel-making
container. A glass substrate was used as one side of the gel
plates. As for the other side of the gel plates, a stripe electrode
substrate plate in which a thickness of Pt wire and a gap between
Pt wires were 100 .mu.m, a substrate whose entire surface was
covered by Pt, or a glass substrate (positive control) was
used.
[0159] Next, after adding a processed resolving gel solution (13%
acrylamide mixture (acrylamide: bisacrylamide=29.2:0.8), 378 mM
Tris-HCl (pH 8.8), 0.05% APS, and 0.1% TEMED) up to 7 mm from the
tip, water was added to form a water layer thereon. After the
polymerization of the resolving gel solution, the water was
removed, and then the processed and concentrated resolving gel
solution (4% acrylamide mixture (acrylamide: bisacrylamide=29.2:
0.8), 125 mM Tris-HCl (pH 8.8), 0.05% APS, and 0.2% TEMED) was
added. Then, a sample comb was placed. As for a negative
electrophoresis buffer composition, 25 mM Tris, 192 mM glycine, and
0.1% SDS were used. As for a positive electrophoresis buffer
composition, 150 mM Tris-HCl (pH 8.8) was used. Samples were mixed
with the same amount of 0.5% agarose gel, and were injected inside
a sample well. After the coagulation, SDS-PAGE was performed.
[0160] The samples to be separated by SDS-PAGE were stained and
visualized by SeeBlue plus2 M. W. Marker (Invitrogen Corportation)
and fluorescent-labeled DyLight fluorescent protein molecular
weight marker (PIERCE).
[0161] As a result of applying 20 mA constant current for 45
minutes, clear protein bands were detected even in the case where
one side of the gel plates was a stripe electrode substrate (FIG.
7). On the other hand, proteins did not migrate in the case where
one side of the gel plates was a substrate entirely covered with
Pt. This confirmed that the voltage application was not possible in
this case (FIG. 8). Also, mobility was slower in the case of using
the stripe electrode substrate.
[0162] Further, in the stripe electrode, air babbles were generated
on the stripe electrode during SDS-PAGE. It was deduced that the
air bubbles detached gel from the stripe electrode and buffer
solution came into the gap between the gel and the stripe
electrode, so that buffer action was inhibited and mobility of
proteins became slow. Note that the air bubbles do not largely
affect separation degree, however, the air bubbles prevent applying
a uniform voltage to the stripe electrodes in the following
transfer process. Thus, a method for preventing generation of air
bubbles was studied.
Example 3
Studying a Method for Preventing the Generation of Air Bubbles
[0163] It was deduced that water electrolysis might occur between a
pair of electrodes due to a potential difference between the
electrodes (wires) of the stripe electrodes, thereby causing the
air bubbles. Then, it was predicted that the generation of air
bubbles could be prevented by controlling the potential difference
between each pair of electrodes below 1V, decomposition voltage of
water.
[0164] Then, with use of a stripe electrode substrate in which the
thickness of Pt wire and the gap between Pt wires were 100 .mu.m,
the generation of air bubbles was monitored when altering applied
voltage during SDS-PAGE. Table 1 shows the applied voltages, and
calculated voltages between each pair of electrodes when the
applied voltages were applied. Note that a voltage between a pair
of electrodes was calculated by an applied voltage divided by the
number of stripes.
TABLE-US-00001 TABLE 1 Voltage between Initial Device for each pair
The Electric making of electrodes Air Number of Currency stripe (V)
Bubbles electrodes Voltage L/S (.mu.m) (mA) electrode 0.2 none 251
50 100/100 15 Dicing saw 0.3 none 251 75 100/100 25 Dicing saw 0.4
generated 251 100 100/100 Dicing saw a little 0.6 generated 251 150
100/100 49 Dicing saw 0.8 generated 251 200 100/100 191 Dicing saw
a lot 0.3 none 501 150 50/50 56 CO.sub.2 laser carving machine
[0165] The result shows that the air bubbles were generated when
the voltage between each pair of electrodes was 0.4V or higher; and
the air bubbles was not generated when the voltage was 0.3V or
lower (applied voltage to gel was 75V or lower). Further, in the
case where a stripe electrode substrate in which the thickness of
Pt wire and the gap between Pt wires were 50 .mu.m, the air bubbles
was not generated when the applied voltage to gel was 150V (voltage
between each pair of electrodes was 0.3V). Therefore, it was clear
that the generation of air babbles is preventable on the condition
that the voltage between each pair of electrodes is 0.3 V or
lower.
[0166] Further, decomposition voltage of water is affected by a
material of electrodes and components in a buffer. It is deduced
that it is possible to increase an applied voltage between each
pair of electrodes by using materials such as zinc or copper which
have lower excessive voltage than Pt.
Example 4
Present Invention Worked with a Commercial Apparatus
[0167] A stripe electrode was created by using a mini-size gel
plate (10 cm.times.8.2 cm3 mm in thickness Bio-Rad) in the same
procedure as explained in Example 1. Five hundred stripes were
created to satisfy L/S=80 .mu.m/120 .mu.m within 10 cm substrate.
In this case, a potential difference between stripe electrodes was
0.2V. Gel was polymerized and SDS-PAGE was performed in the
following three cases, (i) both sides of the gel plates were stripe
electrode substrates; (ii) one side of the gel plates was a stripe
electrode substrate; and (iii) both sides of the gel plates were
glass substrates. SDS-PAGE was performed in a standard method by
using a Mini-Protian 3 Cell (Japan Bio Lad Laboratories, Inc.) at
100V for 1 hour. Under this condition, no air bubbles were
generated on the stripe electrode plate. Regardless of whether one
side of the gel plates was a stripe electrode substrate, or both
sides of the gel plates were stripe electrode substrates, clear-cut
bands could be obtained and electrophoresis speed was not declined
either.
[0168] The present invention makes it possible to prevent the
generation of air bubbles at electric conductors inside an
electrophoresis chamber and the decline in electrophoresis
speed.
[0169] The present invention provides an improved electrophoresis
technique which can be used in a filed of separating and analyzing
biomaterials.
[2: Device for Electrophoresis and Transfer and Method for
Electrophoresis and Transfer]
[0170] The following deals with an explanation of an embodiment of
the present invention with refer to drawings. FIG. 9 and FIG. 10
are schematic view of a device for electrophoresis and transfer 400
of an embodiment of the present invention. FIG. 9 illustrates a
structure of the device for electrophoresis and transfer 400 in
electrophoresis operation. FIG. 10 illustrates the device for
electrophoresis and transfer 400 in transfer operation.
[0171] As illustrated in FIG. 9 and FIG. 10, the device for
electrophoresis and transfer 400 includes buffer solution chambers
401 and 402, a separation section 403, a wire connecting section
404, electrodes 405 and 406 (first voltage applying means), a
supporter 407, and a stripe electrode 408 (second voltage applying
means, first electrode); in transfer operation, further includes
electrode 409 (second voltage applying means, second electrode), a
supporter 410, and a wire connector 411. The details of the stripe
electrode 408 is explained later.
[0172] The buffer solution chambers 401 and 402 are used for
filling a buffer solution therein. As for the buffer solution, a
buffer solution which has a composition generally used for
electrophoresis is available. The electrode 405 provided in the
buffer solution chamber 401 has a negative potential in
electrophoresis operation. The electrode 406 provided in the buffer
solution chamber 402 has a positive potential in electrophoresis
operation.
[0173] The stripe electrode 408 is provided on a region between the
buffer solution chambers 401 and 402. Also, the region is divided
into the separation section 403 and the wire connection section 404
by the holder 407. As illustrated in FIG. 9, the wire connection
section 404 is isolated from buffer solution chambers 401 and 402
by the supporter 407 for preventing entering the buffer solution.
Also, the upper part of the holder 407 is open so that the stripe
electrode 408 located on the wire connection section 404 is able to
contact with other members.
[0174] The first medium 420 including separation target components
is provided on the separation section 406 and is sustained by the
holder 405. The upper part of the holder 407 is open so that the
first medium 420 is able to contact with other members.
[0175] The separation target components may be any components to be
separated and analyzed by electrophoresis and transfer. The
separation target components may be preferably prepared from a
biological material such as bions, biological fluid, cell strains,
cultured tissues, or fragment tissues. Polypeptide and
polynucleotide are more preferable as the separation target
components. As for the first medium 420, agarose gel,
polyacrylamide gel, and any gel generally used for electrophoresis
are available. Note that when gel is used as the first medium, a
gelated first medium 420 may be placed on the separation section
406, or liquid material may be filled in the separation section 406
to be gelated there as the first medium 420 on the separation
section 406. Further, when the present invention is adopted to an
electrophoresis driven by a capillary electrophoresis method, a
buffer solution containing polymer, which is generally used in a
capillary electrophoresis method, is applicable.
[0176] In transfer operation, the stripe electrode 408 on the wire
connection section 404 is conductively connected by the wire
connector 411 and a negative potential is applied. Also, the holder
410 is provided on the first medium 420. The holder 410 includes
the electrode 409 and the second medium 421. The first medium 420
is in contact with the second medium 421 and the electrode 409 is
provided on top of them. The electrode 409 is applied a positive
potential.
[0177] As for the second medium 421, materials which are able to
fix the separation target components are available, and the shape
is not limited but a thin membrane is preferable. Specifically, a
nitrocellulose membrane, a PVDF membrane, a nylon membrane, and the
like, which are used in biological macromolecule analysis technique
such as a known western blotting method, are preferably used.
[0178] The stripe electrode 408 has such a structure that
line-shaped electric conductors are arranged in parallel. In the
device for electrophoresis and transfer 400, the stripe electrode
408 is provided in an orthogonal direction to the direction of
electrophoresis. The stripe electrode 408 should be made of a
conducting material which is not deteriorated even in contact with
the buffer solution is preferable. Specifically, it is not limited
to the following materials but the stripe electrode 408 can be made
of platinum, copper, zinc, or the like. The stripe electrode 408
includes a plurality of electric conductors, and the thickness and
length of each electric conductor, and the gap between conductors
are not limited.
[0179] The following explanation deals with the operation of the
device for electrophoresis and transfer 400 with refer to drawings.
FIG. 11 and FIG. 12 are schematic views of the device for
electrophoresis and transfer 400. FIG. 11 illustrates the device
for electrophoresis and transfer 400 in electrophoresis operation.
FIG. 12 illustrates the device for electrophoresis and transfer 400
in transfer operation.
[0180] As illustrated in FIG. 11, since the electrode 405 has a
negative potential and the electrode 406 has a positive potential
during the electrophoresis operation, the separation target
components in the first medium 420 migrate to the direction from
the electrode 405 to the electrode 406 (a direction indicated by an
arrow in FIG. 11). At this time, the stripe electrode 408 does not
prevent the migration of the separation target components.
[0181] For example, as shown in another embodiment described later,
if the flat-plate shaped electrode is used instead of the stripe
electrode 408, a potential in the first medium 420 becomes uniform
and the separation target components do not migrate.
[0182] On the other hand, the stripe electrode 408 includes a
plurality of electrode regions being insulated one another and
arranged in an orthogonal direction to the voltage applied
direction. Therefore, a potential to the voltage applied direction
never become uniform and the separation target components in the
first medium 420 can migrate as explained above.
[0183] As illustrated in FIG. 12, the electrodes 405 and 406 do not
apply a voltage to gel 420 in the transfer operation. The second
medium 421 and the electrode 409 are provided on the first medium
420 in the transfer operation. As a negative potential is applied
to the stripe electrode 408 and a positive potential is applied to
the electrode 409, the separation target components in the first
medium 420 migrate from the stripe electrode 408 to the electrode
409 (direction toward the second medium 421). This allows the
separation target components to be transferred to the second medium
421.
[0184] A known and commonly used method can be used for switching
potentials of the electrodes 405, 406, and 409 between the
electrophoresis operation and transfer operation. As described
before, the switching the potential of the stripe electrode 408 is
carried out by connecting or disconnecting the wire connector 411
to the stripe electrode 408 on the wire connection section 404.
However, the switching is not limited to this way. Any methods to
apply a negative potential to the stripe electrode 408 are
applicable, for example, a known circuit technique such as a switch
may be adopted.
[0185] The applied voltage in the electrophoresis and transfer
operation may be a voltage which is used for general
electrophoresis devices and transfer devices. A known and commonly
used technique can be used as a voltage applying means other than
the above explained part.
[0186] In the device for electrophoresis and transfer 400 of an
embodiment in accordance with the present invention, the second
medium 421 can be detached easily as explained before. Therefore,
the analysis (such as antibody-antigen reaction) with use of the
second medium 421 including transferred target components is
favorably performed.
[0187] The following explanation deals with another embodiment of
the device for electrophoresis and transfer in accordance with the
present invention with refer to drawings. FIG. 13 is a schematic
view of a device for electrophoresis and transfer 500 of the
present embodiment.
[0188] As illustrated in FIG. 13, a device for electrophoresis and
transfer 500 includes a buffer solution chambers 501 and 502, a
separation section 503, a wire connection section 504, electrodes
505 and 506 (first voltage applying means), stripe electrodes 507
and 508 (second voltage applying means, first and the second
electrodes), and a wire connector 509.
[0189] The separation section 503 is provided between the buffer
solution chambers 501 and 502, and holds the first medium 520 and
the second medium 521 by sandwiching them. The wire connection
section 504 is provided at an outer region between the buffer
solution chambers 501 and 502, and adjacent to the separation
section 503. In the area from the separation section 503 to the
wire connection section 504, the stripe electrodes 507 and 508 are
provided one above the other and hold the first medium 520 and the
second medium 521 therebetween at the separation section 503.
[0190] The explanation of the buffer solution chambers 501 and 502,
the electrodes 505 and 506, the first medium 520, the second medium
521, and the stripe electrodes 507 and 508 are the same as the
explanation of the device for electrophoresis and transfer 400
above mentioned.
[0191] The wire connector 509 is a part for applying a voltage to
the stripe electrodes 507 and 508 in transfer operation. FIG. 14 is
an explanatory drawing illustrating a mechanism how the wire
connector 509 applies a potential to the stripe electrodes 507 and
508. As illustrated in enlarged views in FIG. 14, the wire
connector 509 includes two separated regions (shaded areas in the
drawings) which are connected to not illustrated voltage supply
means (such as a power supply) having positive and negative
potentials, respectively. The wire connector 509 rotates in the
wire connection section 504 (rotation types 1 to 3 in the figure);
or the wire connector 509 is inserted between the stripe electrodes
507 and 508 (a tapered type in the figure). This allows the above
regions to connect the stripe electrodes 507 and 508 for applying a
positive potential and a negative potential, respectively. Note
that the present invention is not limited to the method above for
applying the positive potential and the negative potential to the
stripe electrodes 507 and 508, respectively. For example, a known
circuit technology such as a switch may be adopted.
[0192] The following explanation deals with an operation of the
device for electrophoresis and transfer 500 with reference to
drawings. FIG. 15 and FIG. 16 are schematic views of the device for
electrophoresis and transfer 500. FIG. 15 illustrates the device
for electrophoresis and transfer 500 in the electrophoresis
operation. FIG. 16 illustrates the device for electrophoresis and
transfer 500 in the transfer operation.
[0193] As illustrated in FIG. 15, the electrode 505 has a negative
potential and the electrode 506 has a positive potential in
electrophoresis operation. Therefore, separation target components
in the first medium 502 migrate in a direction from the electrode
505 to the electrode 506 (direction indicated by an arrow in FIG.
15). At this time, the stripe electrodes 507 and 508 do not prevent
the migration of the separation target components as described
before.
[0194] As illustrated in FIG. 16, in the transfer operation, while
the electrodes 505 and 506 do not apply a voltage to the first
medium 520, the negative potential is applied to the stripe
electrode 507 and the positive potential is applied to the stripe
electrode 508. Accordingly, the separation target components in the
first medium 520 migrate in a direction from the stripe electrode
507 to the stripe electrode 508 (direction indicated by an arrow in
FIG. 16). This allows the separation target components to be
transferred to the second medium 521.
[0195] The explanation above deals with the case where the first
medium is provided on the negative stripe electrode 507 side and
the second medium is provided on the positive electrode 508 side.
The present invention, however, may be oppositely configured such
that the first medium is provided on the positive electrode 508
side and the second medium are provided is provided on the negative
stripe electrode 507 side.
[0196] The present invention also provides an automated
two-dimensional electrophoresis-western blotting device (2 DGE-WB)
including the device for electrophoresis and transfer of the
present invention. FIG. 17 is a schematic view of the 2 DGE-WB
device as an embodiment in accordance with the present
invention.
[0197] As illustrated in FIG. 17, the 2 DGE-WB device 600 includes
a base material 614, a sample injection and swelling chamber 601,
an isoelectric electrophoresis chamber 602, an SDS equilibrium
chamber 603, gel storage for isoelectric electrophoresis 604, an
electrophoresis and transfer section 605 (device for
electrophoresis and transfer), a stripe electrode for transfer 606
(second voltage applying means, first electrode), a wire connection
member 607, a buffer liquid chambers 608 and 609, a reaction
chamber 610, an electrode for transfer 611 (second voltage applying
means, second electrode), and supporting members 612 and 613. A gel
for an isoelectric electrophoresis 620 is placed in the gel storage
for an isoelectric electrophoresis 604, and a gel for
two-dimensional electrophoresis 621 is placed in the
electrophoresis and transfer section 605. The supporting member 613
has a transfer base material 622 at a bottom part of the electrode
for transfer 611.
[0198] The supporting member 612 performs as follows, (i) obtaining
gel for an isoelectric electrophoresis 620 from the gel storage for
isoelectric electrophoresis 604; (ii) carrying the obtained gel for
an isoelectric electrophoresis 620 to the sample injection and
swelling chamber 601 and performing sample injection and swelling;
(iii) performing isoelectric electrophoresis to separate the
samples injected in the gel for an isoelectric electrophoresis 620
in the isoelectric electrophoresis chamber 602; (iv) SDS
equilibrating the gel for the isoelectric electrophoresis 620
including separated samples in the SDS equilibrium chamber 603 and
carrying the SDS equilibrated gel to the electrophoresis and
transfer section 605 for contacting the gel for two-dimensional
electrophoresis 621; (v) performing the two-dimensional
electrophoresis in the electrophoresis and transfer section 605;
(vi) transferring the samples from the gel for two-dimensional
electrophoresis 621 to the a transfer base material 622 in such a
manner that the transfer electrode 611 and the transfer base
material 622 were placed on the gel for two-dimensional
electrophoresis 621 by the supporting member 613, and the stripe
electrode for transfer 606 and connection member 607 are connected
with a wire; (vii) the supporting member 613 carrying the transfer
material 622 including transferred samples to the reaction chamber
610 and carrying out an antigen antibody reaction.
[0199] As explained above, since the 2 DGE-WB device 600 of the
present embodiment including the device for the electrophoresis and
transfer of the present invention, the same members can be used in
the processes of two-dimensional electrophoresis and transfer.
Therefore, it is not necessary to carry the gel for two-dimensional
electrophoresis 621. This allows the two-dimensional
electrophoresis and western blotting to be performed continuously
and favorably.
[0200] The present invention further provides a chip for
electrophoresis and transfer which is implemented in the device for
electrophoresis and transfer of the present invention. The chip for
electrophoresis and transfer of the present embodiment includes a
separation section for placing the first medium including
separation target components and a second medium in contact with
the first medium, a first buffer solution chamber and a second
buffer solution chamber sandwiching the separation section, and a
first electrode having a plurality of electrode regions being
insulated one another and arranged in a specific direction
specified by the first buffer solution chamber and the second
buffer liquid chamber. In this specification, a structure as
described above, which includes a separation section, a first and
second buffer solution chambers, and a first electrode, is called a
chip for electrophoresis and transfer.
[0201] The chip for electrophoresis and transfer of the present
embodiment is capable of separating separation target components in
the first medium in the above mentioned direction by placing
electrodes in the first and the second buffer solution chambers and
applying a voltage to the first medium located on the separation
section. Each of the electric regions in the first electrode
located on the separation section are arranged in the above
mentioned direction and are insulated one another. Therefore, the
electric regions do not affect the separation of the target
components.
[0202] It is easy to understand for a skilled person in the art
that the above-mentioned chip for electrophoresis and transfer is
easily implemented to the device for electrophoresis and transfer
of the present invention. Since the chip of the present embodiment
includes the separation section, the first and the second buffer
solution chambers, and the first electrode, it is possible to
create, for example, a device similar to the device for
electrophoresis and transfer 400 of an embodiment in accordance
with the present invention by implementing the chip to a device
including an electrode for electrophoresis which may be placed in
the first and the second buffer solution chambers and an electrode
for transfer which may be placed in the upper part of the
separation section.
[0203] Note that the chip for electrophoresis and transfer of the
present embodiment may be implemented in a device for
electrophoresis including first voltage applying means in
electrophoresis operation, or may be implemented in a device for
transfer including second voltage applying means in transfer
operation. The chip for electrophoresis and transfer is capable of
having the first medium and is easy to be carried between a device
for electrophoresis and a device for transfer.
Reference Example 1
Electrophoresis without One Side of the Gel Plates
[0204] Electrophoresis was performed by using the device for
electrophoresis and transfer 400 of an embodiment in accordance
with the present invention on the condition that one side of the
gel plates was removed. Before studying the device for
electrophoresis and transfer of the present invention, a
conventional electrophoresis device was investigated whether it was
able to perform electrophoresis or not on the condition that one
side of the gel plates was removed.
[0205] As illustrated in FIG. 18 and FIG. 19, as is the case with
having both gel plates, it was possible to obtain a protein
separation result even though one side of the gal plates was
removed.
[0206] The reason why the electrophoresis device without one side
of the gel plates obtained bands of separated proteins which were
not clear enough is considered that the separated proteins are
trapped in the polyacrylamide gel (PAG) due to the dryness of the
surface of the gel during SDS-PAGE. Therefore, it is preferable to
have a moisture control.
Example 5
Creation of a Stripe Electrode
[0207] According to Reference Example 1, it was clear that
electrophoresis was able to be performed without one side of the
gel plates. Therefore, further study was preceded.
[0208] After Cr binder was deposited on a 6 cm.times.5 cm glass
plate 3 mm in thickness by a sputtering device and about 2000 .ANG.
Pt was also deposited. Then, a thickness of Pt and a gap between Pt
electrodes were created to be approximately 100 .mu.m by a Dicing
saw (Disco) with a blade 100 .mu.m in thickness. In addition, two
kinds of stripe electrode substrates, (i) a thickness of Pt wire
and a gap between Pt wires were 100 .mu.m and (ii) a thickness of
Pt wire and a gap between Pt wires were 50 .mu.m, were created by a
CO.sub.2 laser carving machine under a following cutting condition,
laser power 1.75 W, process speed 10.8 cm/sec, and pulse cycle 3000
Hz (FIG. 6). Note that the surface of the glass was little
processed by a laser under the above condition.
Example 6
Comparison of the Substrates
[0209] A pair of 6 cm.times.5 cm gel plates (glass plate)
sandwiching 1 mm spacer therebetween was placed in a gel-making
container. A glass substrate was used as one side of the gel
plates. As for the other side of the gel plates, a stripe electrode
substrate plate in which a thickness of Pt wire and a gap between
Pt wires were 100 .mu.m, a substrate whose entire surface was
covered with Pt, or a glass substrate (positive control) was
used.
[0210] Next, after adding a processed resolving gel solution (13%
acrylamide mixture (acrylamide:bisacrylamide=29.2:0.8), 378 mM
Tris-HCl (pH 8.8), 0.05% APS, and 0.1% TEMED) up to 7 mm from the
tip, a layer of water was formed thereon. After the polymerization
of resolving gel solution, the water was removed, and then the
processed and concentrated resolving gel solution (4% acrylamide
mixture (acrylamide: bisacrylamide=29.2: 0.8), 125 mM Tris-HCl (pH
8.8), 0.05% APS, and 0.2% TEMED) was added, followed by a sample
comb placement. As for a negative electrophoresis buffer
composition, 25 mM Tris, 192 mM glycine, and 0.1% SDS were used. As
for a positive electrophoresis buffer composition, 150 mM Trus-HCl
(pH 8.8) was used. Samples were mixed with the same amount of 0.5%
agarose gel, and were introduced in sample wells. After the
coagulation, SDS-PAGE was performed.
[0211] The samples to be separated by SDS-PAGE were stained and
visualized by SeeBlue plus2 M. W. Marker (Invitrogen Corportation)
and fluorescent-labeled DyLight fluorescent protein molecular
weight marker (PIERCE).
[0212] As a result of applying 20 mA currency for 45 minutes, clear
protein bands were detected in the case where one side of the gel
plates was a stripe electrode substrate (FIG. 7). On the other
hand, proteins did not migrate in the case where one side of the
gel plates was the substrate entirely covered with Pt. This
confirmed that the voltage application was not possible in this
case (FIG. 8).
Example 7
Study of Protein Transfer with Use of a Chip
[0213] The present inventors studied whether a series of procedures
from SDS-PAGE to transfer is able to be performed in the same chip
by using an own device in which an electrophoresis chamber and a
chip-like gel plate whose one side of gal surface was empty were
separated each other.
[0214] After the proteins were separated by SDS-PAGE, the separated
proteins were transferred with use of a stripe electrode substrate
in which a thickness of Pt wire and a gap between Pt wires were 100
.mu.m as a negative electrode and a stainless plate as a positive
electrode. FIG. 20 is a photograph of a chip right after performing
SDS-PAGE, and FIG. 21 is a photograph of the chip after a transfer.
FIG. 22 is a photograph of a transfer membrane (Immobilon-FL, PVDF
membrane). The separation and transfer of proteins was performed,
as shown in those photographs. As above explained, it was confirmed
that a series of procedures from SDS-PAGE to transfer was able to
be performed in one chip without removing gel from gel plate.
[0215] FIG. 23 is a plurality of photographs showing each of the
processes. Step 1 is a photograph of filling gel in a chip with a
stripe electrode. Step 2 is a photograph of performing SDS-PAGE.
Step 3 is a photograph showing that the chip was placed in a
transfer cassette (a device for applying a voltage for transfer to
the chip) and a PVDF membrane was placed. Step 4 is a photograph of
setting a positive electrode. Step 5 is a photograph of removing
the positive electrode after a transfer. Step 6 is a photograph
after removing (obtaining) the PVDF membrane.
Example 8
Study of a Structure of Stripe Electrodes Sandwiching Gel
[0216] Polyacrylamide gel electrophoresis (PAGE) was performed by
using a chip in which stripe electrodes were provided on both sides
of the chip and a PVDF (plyvinylidene fluoride) membrane and gel
were provided. The following is an assessment of the effects of the
stripe electrodes and PVDF membrane on an electrophoresis
pattern.
[0217] The stripe electrode was an electrode prepared by depositing
Platinum on a 50 mm.times.60 mm crystal glass by photolithography
to create 50 .mu.m-thick Pt wires repeated 500 times with 50 .mu.m
intervals. As for a PVDF membrane, Immobilon FL made by Millipore
Corporation was used. As for a gel forming condition, the
concentrated gel was formed in 4% acrylamide density and resolving
gel was formed in 13% acrylamide density, both of which were gel 1
mm in thickness. As for electrophoresis test samples, a molecular
weight marker SeeBlue Pre-stained made by Invitrogen Corporation
was used.
[0218] On the base plate of the chip made of polymethyl
methacrylate, a stripe electrode with the electrode plane facing
upward, a PVDF membrane, a spacer glass in 1 mm thickness, and a
stripe electrode with the electrode plane facing downward were
layered in this order; and a chip lid covered on top. Acrylamide
monomer liquid filled the space in 1 mm thickness to form a
concentrated gel and a resolving gel. Then, an electrophoresis was
performed at 150V after injecting samples.
[0219] FIG. 24 shows the result thereof. As illustrated in FIG. 24,
in the polyacrylamide gel electrophoresis performed by the chip in
which the stripe electrodes were located on both sides of the chip
respectively and the PVDF membrane was placed, it was able to
obtain a separation pattern as in the case with a separation
pattern without a PVDF membrane.
[0220] The present invention allows performing both electrophoresis
and transfer without removing gel. Therefore, it is possible to
provide a practical and easy-to-use device for electrophoresis and
transfer.
[0221] The present invention allows providing a practical and
easy-to-use device for electrophoresis and transfer. For example, a
series of procedures from SDS-PAGE to Western blotting can be
preformed by one chip. Further, it is possible to provide an
automated two-dimensional electrophoresis--western blotting device
in combination with two-axis carrier type automation device.
[3: Device for Transfer and Method for Transfer]
[0222] Transfer of proteins is carried out in a vertical direction
to the direction of migration in electrophoresis. It is difficult
to uniformly transfer the separated protein after electrophoresis
because its molecular weight varies from several hundred kD to
several kD.
[0223] In SDS-PAGE or agarose gel electrophoresis, smaller
molecular weight proteins migrate further due to an effect of
molecular screening. Therefore, when a cytolytic liquid is
separated by these electrophoresis, bands are emerged on a surface
of gel in decreasing order of molecules weight, each band being
formed with proteins having the same molecular weight. For example,
when performing SDS-PAGE with use of 12% acrylamide polyacrylamide
gel, proteins are able to be separated and classified into about
200 kD to several kD molecules weight as a plurality of bands.
[0224] In transferring proteins separated into each molecular
weight, when the target transfer proteins are small or medium sized
molecular weights, it is possible to be transferred in short period
of time from 30 to 60 minutes. However, it takes more than 60
minutes in transferring high molecular weight proteins. Also,
longer period transfer requires more SDS in buffer, so SDS is
required to be added during a transfer buffer. SDS increases
transfer efficiency, however, causes the following problems. The
obtained protein bands after transfer are not clear enough. Low
molecular weight protein in transfer base material becomes unstable
and passes through the transfer base material.
[0225] The device for transfer of the present invention enables to
adjust voltage condition, transfer duration, and electric currency
condition depending on a molecular weight of each protein for
transferring proteins of various molecular weight from gel to a
transfer substrate simultaneously and efficiently.
[0226] Namely, the device for transfer of the present invention
applies a voltage to the first medium in such a manner that a
certain position and another position in the first medium are
provided different voltages or different voltage durations.
[0227] Further, in an embodiment of the device for transfer in
accordance with the present invention, a voltage applied to a
certain position in the first medium is the same as a voltage
applied to another position which is posteriorly-located from the
certain position in a specific direction specified by the first
medium, or higher. Namely, the above device for transfer increases
a voltage applied to the first medium stepwise or gradually in the
specific direction specified by the first medium.
[0228] Also, in another embodiment of the device for transfer in
accordance with the present invention, a voltage duration applied
to a certain position in the first medium is the same or longer to
that of another position located in upstream of the certain
position with regard to the specific direction specified by the
first medium. Namely, the above device for transfer increase a
voltage duration applied to the first medium stepwise or gradually
with respect to the specific direction specified by the first
medium.
[0229] The following explanation deals with embodiments of the
present invention in details with reference to drawings.
[0230] FIG. 25 is a perspective view illustrating an overall of a
device for transfer 10 according to an embodiment of the present
invention. The device for transfer 10 of the present embodiment
includes stripe electrodes 11 and 12 (voltage applying means), a
conductive path 13, and is capable of having a first medium 14 and
a second medium 15.
[0231] The first medium 14 is not limited and any mediums including
target transfer components are preferably used, such as agarose
gel, polyacrylamide gel, and the like which are generally used for
electrophoresis. The target transfer components are not limited and
biological materials such as bion, biological fluid, cell strains,
cultured tissues, and fragment tissues are preferably used. More
preferably, polypeptide or polynucleotide is used as the target
transfer components. The second medium 15 should be a material
which can hold the transferred transfer target components, and the
form is not limited but a form like a thin membrane is preferable.
Specifically, a nitrocellulose membrane, a PVDF membrane, and a
nylon membrane, which are generally used for biopolymer analysis
such as a known western blotting method, are preferably used as the
second medium 15. Also, the second medium is provided in contact
with the first medium. The above explanation about the first and
the second mediums are applicable to the following embodiments.
[0232] The stripe electrode 11 is an electrode divided into a
plurality of electrode regions in one direction which are insulated
one another. Each of the electrode regions is line-shaped. The
stripe electrode 12 is equivalent to the stripe electrode 11. A
size of the stripe electrode is adjustable to the size of a device
for transfer. For example, it is possible to adjust wires 0 .mu.m
to 200 .mu.m in thickness, 50 .mu.m to 200 .mu.m gap between wires,
and 30 to 300 mm in length. As for the material of the wire,
electric conductors such as platinum, copper, and zinc are
available. The stripe electrode may be made in such a manner that a
metal is deposited on a surface of insulator such as a glass plate
and a plurality of gloves are created by a Dicing saw or a CO.sub.2
laser carving machine. A voltage is applied to the stripe
electrodes 11 and 12 via a conductive path 13.
[0233] FIG. 26 is a schematic view explaining an operation of a
device for transfer 10. In the device for transfer 10, in the
region that high molecular weight transfer target components exist
(left side in the figure), the stripe electrodes 11 and 12 are
conductively connected to a high voltage power supply via the
conductive path 13; in the region that low molecular weight
transfer target components exist (right side in the figure), the
stripe electrodes 11 and 12 are connected to a low voltage power
supply via the conductive path 13. This makes it possible to apply
a suitable voltage to each of the transfer target components in the
first medium 14. This allows that the transfer target components
are transferred from the first medium 14 to the second medium 15
efficiently. It is possible to adjust a voltage depending on the
molecular weight of the transfer target components, e.g. from 1V to
500V. It is more preferable to increase the voltage from 5 to 40V
stepwise or gradually from the region having low molecular weight
transfer target components to the region having high molecular
weight transfer target components. Also, a skillful person in the
art easy to understand the most suitable applied voltage with
reference to the following examples.
[0234] The following explanation deals with further another
embodiment. First of all, a device for transfer 20 of the present
embodiment is similar to the device for transfer 10 in FIG. 25. The
device for transfer 20 includes stripe electrodes 21 and 22
(voltage applying means), and a conductive path 23 and is capable
of having a first medium 24 and a second medium 25. A shape and a
function of the conductive path 23, however, are different from a
shape and a function of the conductive path 13. Also, the
conductive path 12 is made of a resistance element.
[0235] FIG. 27 is a schematic view explaining an operation of the
device for transfer 20 of the present embodiment.
[0236] As illustrated in FIG. 27, the stripe electrodes 21 and 22
are conductively connected to a power supply via the conductive
path 23 which is a continuous resistance element. The conductive
path 23 which is conductively connected to the power supply is
connected to an edge part on the side of the high molecular weight
target components existing in the first medium 24. Therefore, a
potential gradient is created in such a manner that the potential
gradient decreases from the edge toward an opposite edge on the
side of the low molecular weight target components existing in the
first medium 24. This allows transferring the transfer target
components efficiently from the first medium 24 to the second
medium 25 by applying a suitable voltage to each of the target
components. A voltage range to be used is same as the voltage range
used in the device for transfer 10. A resistance value of the
conductive path 23 is adjustable depending on the applied voltage,
for example a resistive element whose resistance value 10.OMEGA. to
10 k.OMEGA./cm per unit length may be used.
[0237] The following explanation deals with still further another
embodiment of the present invention. An outline of a device for
transfer 30 of the present embodiment is similar to the device for
transfer 10 in FIG. 25. The device for transfer 30 includes stripe
electrodes 31 and 32 (voltage applying means) and a conductive path
33, and is capable of having a first medium 34 and a second medium
35. A shape and a function of the conductive path 34, however, are
different from those of the conductive path 13. Also, the
conductive path 33 is a mobile and stick-shaped member.
[0238] FIG. 28 is a schematic view explaining an operation of the
device for transfer 30 of the present embodiment. In the device for
transfer 30, the stripe electrodes 31 and 32 are applied a voltage
in contact with the mobile conductive pass 33. By sliding the
conductive path 33, the stripe electrodes 31 and 32 located
opposite side of the sliding direction are disconnected to the
conductive path 33 from the edge sequentially and a voltage is not
applied to the region sandwiched between the electrodes 31 and 32
in the first medium 34 because no potential is applied between the
electrodes 31 and 32. This allows applying a suitable voltage to
each of the transfer target components for a suitable duration,
thereby achieving efficient transfer of the transfer target
components from the first medium 24 to the second medium 25. FIG.
29 is a schematic view illustrating an intermediate step of the
slide. As illustrated in FIG. 29, when the conductive path 33
slides to somewhere between the ends of the electrodes 31 and 32, a
potential is not applied on the stripe electrodes 11 and 12 located
in the region, which has low molecular weight transfer target
components (right side of the figure), so that no voltage is
applied on the region any more. Namely, it is possible to apply a
voltage for a suitable duration in accordance with the molecular
weight of the transfer target components. As explained above, each
of the transfer target components has a suitable voltage for
transfer and the mobility of the transfer target component is in
proportion with the product of an applied voltage and a voltage
duration. Therefore, it is possible to optimize the mobility of the
target component by adjusting a time period for the voltage
application. The time period for the voltage application is set in
consideration of an applied voltage, a transfer target component,
and a composition and thickness of gel. The time period for the
voltage application should be increased from 1 minute to 3 hours,
or more preferably from 5 minutes to 60 minutes, from a region
having low molecular weight transfer target components to a region
having high molecular weight transfer target components.
[0239] FIG. 30 is a perspective view illustrating an overall of a
device for transfer 40. As illustrated in FIG. 30, the device for
transfer 40 includes electrodes 41 and 43 (voltage applying means)
and an electric resistance layer 42, and is capable of having a
first medium 44 and a second medium 45.
[0240] The electric resistance layer 42 is a layer including a
resistivity distribution. The resistance value of the electric
resistance layer 42 varies depending on regions which are defined
by an upper and a lower layers, the electrode 43 and the first
medium 44, respectively. Specifically, in the electric resistance
layer 42, the resistance value is set to be low over the region
which has relatively high molecular weight transfer target
components in the first medium 44, and is set to be high over the
region which has relatively low molecular weight transfer target
components in the first medium 44. Accordingly, in the medium 44, a
high voltage is applied to the region which has relatively high
molecular weight target components to be transferred, and a low
voltage is applied to the region which has relatively low molecular
weight components to be transferred. Namely, a suitable voltage to
each of the transfer target components can be applied. A resistance
value in the electric resistance layer 42 is not limited, but it
should be ranging from several thousands .OMEGA. to ten thousands
.OMEGA..
[0241] There are various methods to form a resistivity distribution
in the electric resistance layer 42. It is possible to adopt the
following methods, such as distributing a plurality of materials,
each of which has a different resistance value in the electric
resistance layer 42, or distributing a plurality of resistive
materials, each of which has a different thickness in the electric
resistance layer 42. As specific examples, resin materials
containing carbon materials and a known composition which is used
as a resistor in general electronic components.
[0242] FIG. 31 is a perspective view illustrating an overall of the
electric resistance layer 42. As illustrated in FIG. 31, a
plurality of cylindrical resistive elements 46 coupled by an
insulating or high-resistance binding agent 47 are arranged in the
electric resistance layer 42 in an embodiment of the present
invention. Namely, the resistance value has an aeolotropy in a
thickness direction and in a surface direction in the present
embodiment. Specifically, the electric resistance layer 42 in a
thickness direction has a high resistance value defined by the
cylindrical resistive element 46, and has a low resistance value
defined by the binding agent 47 in a surface direction.
[0243] The cylindrical resistive element 46 has a cylindrical shape
10 .mu.m to a few mm in diameter, or may have another shape which
has a substantially equal cross sectional area, and the height is
calculated in considering the resistivity value of the material of
the cylindrical resistive element 46 and a resistivity distribution
that the electric resistance layer 42 should have. As explained
above, depending on a placement region in the electric resistance
layer 42, a variety of the cylindrical resistive element 46 may be
used, which has a different resistance material, a different cross
sectional area, or a different density of distribution.
[0244] FIG. 32 is a schematic view explaining a structure of a
device for transfer 40. In FIG. 32, the electric resistance layer
42 and the first medium 44 are illustrated separately into a
plurality of miniregions as a concept. The following explanation is
based on an assumption that currency only flows inside each of the
miniregions and not to flow across a plurality of miniregions.
[0245] When applying voltage for transfer, an approximate voltage
applied to each of the regions in the first medium 44 is calculated
by subtracting an amount of voltage depression in the electric
resistance layer 42 from the applied voltage to electrodes 41 and
43. The amount of voltage depression is calculated by a currency
passing through each of the regions in the electric resistance
layer 42 multiplied by a resistance value of each of the regions
(Rr). If the resistance value is large, the amount of voltage
depression in the region increases, thereby resulting in that the
applied voltage to the first medium 44 decreases effectively.
Similarly, if the resistance value is small, the amount of voltage
depression in the region decreases, thereby resulting in that the
applied voltage to the first medium 44 increases effectively.
[0246] Namely, it is possible to achieve an optimal transfer
condition in each of the regions in the first medium 44 in such a
manner that (i) a low electrical field is applied to the small
molecular weight region by increasing the resistance value of the
electric resistance layer 42; and (ii) a high electrical filed is
applied to the large molecular weight region by decreasing the
resistance value of the electric resistance layer 42.
[0247] Nonuniformity might exist in transfer target components
distribution, ion distribution, and pH distribution in the first
medium 44; in material distribution of the first medium 44; and in
different contact state between layers such as the second medium 45
and electrodes. Because of these nonuniformities, regional
resistivity variation exists in each of the miniregions in the
first medium 44. In a conventional method, when a same voltage is
applied to the first medium 44 entirely for transfer, an
overcurrent surges to a region which has an extremely small
resistance value (Rg) on the surface of the first medium 44.
Therefore, there is a possibility that a fine transfer in the
entire first medium 44 may not be performed.
[0248] On the other hand, the device for transfer 40 of the present
embodiment, overcurrent surging to a local region is prevented by
the electric resistance layer 42. This allows a fine transfer in
the entire surface of gel. Namely, when an amount of current
sharply increases in a local region, voltage depression in a
corresponding region in electric resistance layer 42 increases.
Accordingly, the voltage applied to the first medium 44 will be
small. This prevents overcurrent.
[0249] Further, when polyacrylamide gel or the like is used as the
first medium, a sum total of Rg of the entire first medium 44 is 50
to 500.OMEGA.. In this case, when a sum total of Rr is several
thousands .OMEGA. to tens of thousands .OMEGA., a currency passing
though each region mostly depends on Rr. Therefore, the
overcurrency due to nonuniformity in resistance distribution in the
first medium 44, is suppressed.
[0250] As described above, the transfer device 40 including the
electric resistance layer 42 of the present embodiment is capable
of setting an optimal transfer condition depending on each of the
regions defined by a different molecular weight of the transfer
target components expanding distributed in the first medium 44, and
is also capable of controlling deviation from the optimal transfer
condition by preventing overcurrent surging in a local region. This
allows improving transfer efficiency.
[0251] Also, the device for transfer 40 may further include cooling
means for cooling the electrodes 41 and 43. This allows the first
medium 44 and the second medium 45 to release heat and to maintain
an optimum temperature, thereby avoiding affecting the medium or
the transfer target components qualitatively.
[0252] As the cooling means, cooling components such as a radiating
fin, a heat pipe, a Peltier element, a water-cooled block, and a
one using a water pollination; radiation methods such as an
air-cooling fan, and those combination may be used.
[0253] FIG. 30 illustrates a device for transfer 40 including a
radiating fin as the cooling means. FIG. 6 illustrates a structure
including a radiating fin, an anode electrode, an electric
resistance layer 42, a second medium 45, a first medium 44, and a
cathode electrode from the top. In addition, another structure, for
example, may include a radiating fin, (radiation side) a Peltier
element (cooling side), an anode electrode, an electric resistance
layer 42, a second medium 45, a first medium 44, a cathode
electrode; a radiating fin, (radiation side) a Peltier element
(cooling side), a cathode electrode, an electric resistance layer
42, a first medium 44, a second medium 45, an anode electrode; or a
radiating fin, an cathode electrode, an electric resistance layer
42, a first medium 44, a second medium 45, an anode electrode. The
radiation method or the cooling means may be provided in either
upper or lower side of the first medium 44, or in both sides.
[0254] In FIG. 30, the first medium 44 is placed in a horizontal
position, however, it may be placed in a vertical position for the
purpose of making a cooling efficiency uniform on both back and
front sides. Also, the electric resistance layer 42 may be provided
on one side in the first medium 44 or on both sides in the first
medium 44. The cooling means such as a radiating fin and an
electrode, both of which are provided adjacent to each other may be
made of the same metal body as a joint part.
[0255] The radiating means and the cooling means are provided for
maintaining an optimal temperature in the first medium 44 and the
first medium 45 in such a manner that the radiating means and the
cooling means radiate heat produced by the electricity flowing
through the electric resistance layer 42, the first medium 44, and
the first medium 45.
[0256] The radiating means and cooling means may not be used when a
temperature increase in the first medium 45 is within an allowance
range because a currency amount in transfer operation is small or
other reasons.
[0257] Note that the electric resistance layer 42 may have a single
piece construction with the electrodes 41 and 43, or may be
provided as an independent member as an electric resistance
sheet.
[0258] The cooling means is easy to be implemented in a device for
transfer of an embodiment.
[0259] FIG. 33 is a perspective view illustrating an overall of the
device for transfer 50 according to yet another embodiment of the
present invention. As illustrated in FIG. 33, the device for
transfer 50 of the present embodiment includes a plate electrodes
51 and 52 (voltage applying means) and is capable of having a first
medium 53 and a second medium 54.
[0260] As illustrated in FIG. 33, the plate electrode 51 includes a
plurality of electrode regions. Each of the electric region is
applied a potential and applies a voltage to the first medium 53
and the second medium 54 by sandwiching between upper and lower
electrode regions. A potential difference is controlled to become
large when the electric regions sandwich the region which has
relatively high molecular weight transfer target components in the
first medium 53, and to become small when the electric regions
sandwich the region which has relatively low molecular weight
transfer target components in the first medium 53. It is possible
to use a known technique to apply a certain potential to a certain
electrode region in the plate electrodes 51 and 52. This allows an
optimal voltage to be applied to each of the transfer target
components.
Reference Example 2
Study of Transfer Efficiency of Protein
[0261] A protein including a variety of molecular weights was
transferred by applying three different voltages 10V, 20V, and 40V
and each of the transfer amounts was compared. After an SDS-PAGE
with use of a commercial SDS-PAGE gel (ATTO Co.) in which SeeBlue
M.W. and Dylight M. W. were applied 5 .mu.each, the protein
including a variety of molecular weights was transferred by
applying three different voltages 10V, 20V, and 40V with use of a
commercial device for transfer under a standard method.
[0262] A transfer buffer: 0.1% SDS, 25 mM Tris, 192 mM Glysine, and
20% Methanol was used. As a result, intensity of 200 k-64 k protein
bands increased gradually as the applied voltage increased up to at
40V. On the other hand, in 50 k or less protein bands, the less
molecular weight proteins reduce their intensity of protein bands
and transfer amount after the applied voltage reached 20V. Note
that a few k proteins were not able to be detected when it was
transferred at 40V (FIG. 34).
[0263] The transfer buffer including 0.1% SDS has high transfer
efficiency of high molecular weight proteins. However, it is
deduced that low molecular weight proteins were not adsorbed onto
the transfer membrane and allowed to pass through the transfer
membrane, or diffuse on the transfer membrane. Then a similar
transfer efficiency test was conducted with use of a non-SDS
additive transfer buffer with the anticipation that the low
molecular weight protein would not pass through the transfer
membrane.
Reference Example 3
Study of a Suitable Voltage Range Depending on a Molecular
Weight
[0264] SeeBlue M. W. and Dylight M. W. were separated by a SDS-PAGE
device (ATTO), and then were transferred with use of a non-SDS
additive transfer buffer (25 mM Tris, 192 mM Glysine, and 20%
Methanol) under the following voltage conditions at 10V, 20V, and
30V or 40V. Also, Dylight M.W. was transferred 4 times of each and
CV (Coefficient of Variation) was calculated. As a result, the
fluorescence of 200 k, 116 k, 97 k, and 66 k proteins increased as
the voltage increasing from 10V to 30V. On the other hand, the
fluorescence of 200 k, 116 k, 97 k, and 66 k proteins decreased as
voltage increasing from 30V to 40V. Also, the fluorescence of 42 k,
36 k, and 28 k proteins peaked at 10V and decreased as the voltage
increasing (FIG. 35 and FIG. 36).
[0265] According to these results, in transfer of protein, the less
molecular weight protein tends to pass through a transfer membrane
or diffuse on the transfer membrane. Also, this phenomenon was
detected even though a PVDF membrane with high adsorbed amount of
protein, 20% methanol, and non-SDS additive transfer buffer were
used.
Example 9
[0266] On the basis of findings from the Reference Examples 2 and
3, the present inventors designed and produced a device for
transfer which is capable of transferring transfer target
components composed of different molecular weights favorably.
[0267] On a blue glass plate (100 mm.times.82 mm), platinum lines
were provided in 930 .mu.m in width and 70 .mu.m in space. This
stripe electrode is connected to a power supply controlled by
LabView via copper meshes to create an electrode which is capable
of generating a potential gradient (FIG. 37).
[0268] Filter papers and a PVDF membrane (Immobilon-FL, Millipore
Corporation) were placed on top of the stripe electrode as
illustrated in FIG. 38. FIG. 39 is the photograph.
[0269] 40V, 30V, 20V, and 10V were applied in the order of high
molecular weight protein. The result was illustrated in FIG. 40. In
addition, 20V was applied uniformly as a control. This result is
shown in FIG. 41.
[0270] In comparison between FIG. 40 and FIG. 41, the device for
transfer of the present invention had less proteins penetrating
through a transfer membrane and more proteins were fixed in the
PVDF membrane compared to a device for transfer which was applied
20V uniformly.
[0271] It is possible to provide a device for transfer preventing
decline in transfer efficiency because the present invention can
apply a suitable voltage depending on a molecular weight of a
sample.
[0272] The present invention allows providing a device for transfer
having improved transfer efficiency, which is useful for the field
of analysis apparatus and the like.
[0273] The embodiments and concrete examples of implementation
discussed in the foregoing detailed explanation serve solely to
illustrate the technical details of the present invention, which
should not be narrowly interpreted within the limits of such
embodiments and concrete examples, but rather may be applied in
many variations within the spirit of the present invention,
provided such variations do not exceed the scope of the patent
claims set forth below.
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