U.S. patent application number 10/892285 was filed with the patent office on 2004-12-16 for methods and kits for detecting protein kinases.
This patent application is currently assigned to Lumitech (UK) Limited. Invention is credited to Crouch, Sharon Patricia Mary, Slater, Kevin John.
Application Number | 20040253658 10/892285 |
Document ID | / |
Family ID | 9905234 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040253658 |
Kind Code |
A1 |
Crouch, Sharon Patricia Mary ;
et al. |
December 16, 2004 |
Methods and kits for detecting protein kinases
Abstract
Methods and kits for detecting kinase activity A method for
measuring protein kinase activity comprising: (a) providing a first
solution comprising ATP and a protein kinase to be tested, and a
second solution comprising ATP in the absence of said kinase to be
tested; (b) adding a substrate capable of being phosphorylated by
the protein kinase to be tested to the first and second solutions
of step (a); (c) measuring the concentration of ATP and/or ADP, or
the rate of change thereof with respect to time, in each of the
reaction mixtures formed in step (b) using a bioluminescence
reaction; and (d) using the information about the concentration of
ATP and/or ADP to determine the activity of the protein kinase to
be tested.
Inventors: |
Crouch, Sharon Patricia Mary;
(Nottingham, GB) ; Slater, Kevin John;
(Nottingham, GB) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Assignee: |
Lumitech (UK) Limited
Nottingham
GB
|
Family ID: |
9905234 |
Appl. No.: |
10/892285 |
Filed: |
July 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10892285 |
Jul 16, 2004 |
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10426973 |
May 1, 2003 |
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10426973 |
May 1, 2003 |
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10014816 |
Dec 14, 2001 |
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6599711 |
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Current U.S.
Class: |
435/15 |
Current CPC
Class: |
G01N 2500/04 20130101;
C12Q 1/485 20130101; A61P 35/00 20180101 |
Class at
Publication: |
435/015 |
International
Class: |
C12Q 001/68; C12Q
001/48 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 15, 2000 |
GB |
0030727.2 |
Claims
1-42. (Canceled)
43. A method for detecting protein kinase activity comprising (a)
establishing a reaction mixture comprising ATP, a protein kinase to
be tested and a substrate capable of being phosphorylated by the
protein kinase; and (b) using a bioluminescence reaction to detect
whether any change in ATP concentration occurs.
44. A method for determining protein kinase activity comprising (a)
establishing a reaction mixture comprising ATP, a protein kinase to
be tested and a substrate capable of being phosphorylated by the
protein kinase; (b) using a bioluminescence reaction to detect
whether any change in ATP concentration occurs, and (c) using the
detection of step (b) to obtain information for determining protein
kinase activity.
45. A method for identifying a compound which modulates the
activity of a protein kinase, said method comprising: (a)
establishing a reaction mixture comprising ATP, a protein kinase to
be tested, a substrate capable of being phosphorylated by the
protein kinase and a compound to be tested for an ability to
modulate the activity of the protein kinase; (b) using a
bioluminescence reaction to detect whether any change in ATP
concentration occurs; (c) using the detection of step (b) to obtain
information for identifying whether the compound modulates the
activity of the protein kinase.
46. The method of claim 43, 44 or 45 wherein the reaction mixture
is substantially cell-free.
47. The method of claim 45 wherein the compound to be tested is
identified as a protein kinase inhibitor if the activity of the
kinase is lower in the presence of the compound.
48. The method of claim 45 wherein the compound to be tested is
identified as a protein kinase activator if the activity of the
kinase is higher in the presence of the compound.
49. The method of claim 43, 44, or 45 wherein the kinase is
activated prior to step (a).
50. The method of claim 43, 44 or 45 wherein the reaction mixture
comprises a buffer.
51. The method of claim 50 wherein the buffer is Hepes buffer.
52. The method of claim 43, 44, or 45 wherein phosphorylation is
allowed to proceed at room temperature prior to step (b).
53. The method of claim 52 comprising a further step (a'), carried
out after step (a) and before step (b), of adding a reagent to the
reaction mixture for stopping phosphorylation of the substrate.
54. The method of claim 53 wherein the reagent for stopping
phosphorylation is selected from the group consisting of an acid,
EGTA and EDTA.
55. The method of claim 54 comprising a further step (a"), carried
out after step (a') and before step (b), of adjusting the pH of the
reaction mixture formed in step (a.varies.) to pH 7.0.
56. The method of claim 55 wherein step (a") comprises adding Hepes
buffer.
57. The method of claim 43, 44 or 45 wherein step (b) comprises:
(i) adding a bioluminescent reagent comprising luciferin or a
derivative thereof and a luciferase to said reaction mixture, said
luciferin or a derivative thereof emitting light in a
bioluminescent reaction with the luciferase in the presence of ATP;
and (ii) detecting a light intensity, or a change of light
intensity with time, emitted by the bioluminescent reaction.
58. The method of claim 57 wherein step (b) further comprises the
following steps carried out after the light intensity detected in
step (ii) has reached a substantially constant level: (iii) adding
a reagent that converts ADP to ATP; (iv) adding a bioluminescent
reagent comprising luciferin or a derivative thereof and luciferase
to said reaction mixture of step (iii), said luciferin or a
derivative thereof emitting light in a bioluminescent reaction with
the luciferase in the presence of ATP; and (v) detecting light
intensity emitted by the bioluminescent reaction wherein the
difference in the intensity of light in step (v) and the steady
state intensity of light in step (ii) is a measure of ADP
concentration in the reaction mixture of step (ii).
59. The method of claim 45 wherein the kinase is JNK-1 and the
substrate is GST-c-jun.
60. The method of claim 45 wherein the kinase is MAP Kinase-1
(ERK-1) and the substrate is myelin basic protein.
61. The method of claim 45 wherein the kinase is MAP Kinase-2
(ERK-2) and the substrate is myelin basic protein.
62. The method of claim 45 wherein the kinase is PKA and the
substrate is Kemptide.
63. The method of claim 45 wherein the kinase is JNK-2 and the
substrate is GST-c-jun.
64. The method of claim 45 wherein the kinase is MEK-1 and the
substrate is inactive MAP Kinase-2 (ERK-2).
65. The method of claim 45 wherein the kinase is JNK.alpha. and the
substrate is ATF-2.
66. The method of claim 45 wherein the kinase is JNK.alpha. and the
substrate is c-jun.
67. The method of claim 45 wherein the kinase is SAPK-3 and the
substrate is myelin.
68. A kit for detecting protein kinase activity comprising: (a) a
bioluminescent reagent comprising luciferin or a derivative thereof
and a luciferase, said luciferin or a derivative thereof emitting
light in a bioluminescent reaction with the luciferase in the
presence of ATP; (b) a kinase; (c) a substrate capable of being
phosphorylated by said kinase; and (d) ATP.
69. The kit of claim 68 further comprising one or more buffers for
reconstituting, diluting or dissolving the bioluminescent reagent,
kinase, substrate and/or ATP.
70. The kit of claim 68 further comprising a reagent capable of
stopping the reaction of said kinase with said substrate.
71. The kit of claim 68 further comprising one or more reagent(s)
which converts ADP to ATP.
72. The kit of claim 71, wherein the reagent which converts ADP to
ATP comprises pyruvate kinase and phosphoenol pyruvate.
73. The kit of claim 68 further comprising two or more different
kinases and corresponding substrates.
74. The kit of claim 68 wherein the reagent or reagents is or are
provided in lyophilised form.
75. The kit of claim 68 further comprising a multiwell microtitre
plate.
76. The kit of claim 75 wherein the multiwell microtitre plate
contains 96 wells or more.
77. A compound identified using a method according to claim 45.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to methods for detecting
protein kinase activity and kits for performing such methods.
[0003] 2. Description of Related Art
[0004] Protein kinases play crucial roles in the modulation of a
wide variety of cellular events. These enzymes act by transferring
phosphate residues to certain amino acids in intracellular
polypeptides, to bring about the activation of these protein
substrates, and set in motion a cascade of activation controlling
events including the growth, differentiation and division of cells.
Protein kinases have been extensively studied in the field of
tumour biology. A lack of controlled activity of kinases in cells
is believed to lead to the formation of tumours. The pharmaceutical
industry is constantly in search of drugs that target these
kinases, to help with the treatment of a wide variety of tumours.
There are at least 1200 protein kinases that are involved in the
regulation of cell functions. They occur as both transmembrane and
cytosolic enzymes and they phosphorylate serine, threonine and
tyrosine amino acid residues. Based on these substrate
specificities the kinases are divided into two groups, the
serine/threonine kinases and tyrosine kinases. This has led to the
development of a number of techniques that focus on the ability of
these proteins to take a phosphate group and attach it to a
protein/peptide.
[0005] One of the most widely used techniques is a radio-isotope
method, that utilises either .sup.32P or .sup.33P gamma phosphates.
In the presence of an active kinase, the labelled phosphate is
transferred from the ATP to the protein or peptide substrate. These
assays need to be performed in the presence of ATP labelled to a
high specific activity. This results from keeping the concentration
of unlabelled ATP in the micromolar concentration range. Also in
order to achieve the required sensitivity the peptide substrate has
to be used at high concentrations (5-20 .mu.M). The increased
radioactivity on the resulting phosphoproteins can then be detected
using scintillation counters after capture on phosphocellulose
paper.
[0006] Other methods include immunoprecipitation procedures. During
these assays the kinase, ATP and substrate reaction is allowed to
proceed and is then stopped using a buffer, such as Laemmli buffer.
The protein is then run out using SDS/PAGE electrophoresis. The gel
is then blotted onto a nitrocellulose membrane and probed for
phosphorylated substrate, using an antibody to the phosphorylated
amino acid of choice. The presence of the phosphorylated band can
be visualised using a secondary antibody conjugated to horseradish
peroxidase, followed by the use of a chemiluminescence detection
system, and exposure onto photographic film. As in the case of many
of the methods that have been proposed as an alternative to the
radioisotope assays, however, the above western blotting technique
lacks sensitivity and is quite laborious.
[0007] The use of luminescent detection, either by bioluminescence
or chemiluminescence allows for a highly sensitive detection
system. Lehel et al. (1997) Anal. Biochem. 244, 340-346 reported
the use of a chemiluminescent microtitre plate assay for detection
of protein kinase activity. This assay is based on the use of
biotinylated substrate peptides captured on a streptavidin-coated
microplate, together with monoclonal antibodies. The authors chose
protein kinase A (PKA) to develop the assay, but also found
reliable results with chose protein kinase C (PKC),
calcium/calmodulin-dependent protein kinase II, receptor
interacting protein and src activities. These assays were performed
in the presence of 20 .mu.M ATP and the kinase of interest
+/-inhibitor, the kinase reaction was allowed to proceed to
completion. The plates were then washed prior to antibody binding
and chemiluminescence detection with a secondary antibody
conjugated to horseradish peroxidase with chemiluminescence
determined using a Tropix (RTM) (USA) chemiluminescent substrate
kit. This assay still relies upon the availability of specific
substrates, and also antibodies to the phosphoproteins
generated.
[0008] Another approach that has been taken, is to adopt
microchip-based technology. Cohen et al. (1999) Anal. Biochem. 273,
89-97, reported an assay for PKA based on photolithographic
techniques. Performing an on-chip electrophoretic separation of the
fluorescently labelled peptide substrate and product allowed for
determination of the movement of the phosphate group from ATP to
the serine residue of the heptapeptide, Kemptide. This technology
was developed for the detection of PKA activity.
[0009] Eu et al (1999) Anal. Biochem. 271, 168-176 describe a
method in which the measurement of ATP via bioluminescence is
related to the amount of substrate (galactose) which is present in
a urine sample.
[0010] Sala-Newby & Campbell (1992) FEBS Lett 307, 241-244
describe the use of a firefly luciferase which was engineered to
contain a protein kinase A recognition site RRFS and to lack the
C-terminal peroxisomal signal of native luciferase. The mutant
luciferase was expressed in COS cells and used to detect and
quantify protein kinase A activation by cyclic AMP in those
cells.
[0011] It will be appreciated that the above method is extremely
specific, being useful only for protein kinase A activation by
cyclic AMP. Hence, it suffers from the same problems as the protein
kinase detection systems described above which are based on the
specific enzymes and substrates with which they react.
[0012] There are no assays that have the ability to determine
kinase activity irrespective of the kinase family or the amino acid
residues that are phosphorylated. This is due to the fact that all
the methods currently available focus on the specific enzymes and
substrates involved.
[0013] The present invention seeks to provide methods for measuring
protein kinase activity which are not specific for a single protein
kinase, but rather can be used as a general means of measuring the
activity of a wide range of protein kinases.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1: Shows the drop in ATP light output (relative light
units; RLUs) associated with the protein kinase JNK-1. The means of
three separate experiments .+-.standard error of the mean (SEM) are
shown. These are the results from the data generated in the
presence of 200 mM Hepes;
[0015] FIG. 2: Shows the effect of Hepes and stop solution on the
light output (RLUs) obtained in the presence and absence of
activated JNK. The results are expressed as the means of two
separate experiments, performed in triplicate .+-.standard
deviation (SD). As controls 20 .mu.l of distilled water was added
in place of stop solution.
[0016] FIG. 3: Shows the effect of MAPK-1/ERK-1 on an increase in
ADP. The data are presented as the difference in RLUs, pre and post
addition of converting reagent. The results represent the means of
6 different replicates for each condition .+-.SD.
[0017] FIG. 4: Shows the effect of increasing concentrations of
MAPK-1 on the drop in light output. The results are expressed as
the means of three separate experiments performed in triplicate
.+-.SEM.
[0018] FIG. 5: Shows the initial light output and subsequent signal
decay observed with ATP monitoring reagent and 12 .mu.M ATP in
different buffering conditions. The results shown are from one
representative experiment.
[0019] FIG. 6: Shows the effect of JNK-2 activity on the signal
decay measured over the first two minutes, in the presence of ATP
monitoring reagent. The results are from one experiment,
representative of three. The assays were performed in triplicate
wells of a white 96 well microtitre plate.
[0020] FIG. 7: Shows the effect of increasing concentrations of
MAPK-1 on the decay in light signal. This experiment also compared
the effect of the activated form of the enzyme versus the inactive
form. The results are expressed as the means of triplicate
experiments .+-.SD.
[0021] FIG. 8: Shows the effect of different kinase buffers on
light output (RLUs) with 1 .mu.M ATP. The data are shown as the
means of 6 replicate wells .+-.SD. See Table 1 for buffer
details.
[0022] FIG. 9: Shows the time course for the reduction in light
output over time as a result of JNK2.alpha.2 kinase activity with
ATF-2 as substrate. ADP detection was performed to act as a control
confirming cleavage of the phosphate group from ATP. JNK2.alpha.2
was used at 50 mU and ATF-2 at 5.38 .mu.g.
[0023] FIG. 10: Shows a JNK2.alpha.2 concentration curve of the
reduction in ATP with time at 30.degree. C. as an indication of
kinase activity. ADP converting reagent was added to confirm the
presence of ADP in the reaction mixture.
[0024] FIG. 11: Shows a comparison of the drop in light signal
using JNK2.alpha.2 with c-Jun and ATF-2 as substrates.
[0025] FIG. 12: Shows a SAPK3 concentration curve with a
concentration dependent drop in light signal. The SAPK3
concentrations are shown as nanomolar.
[0026] FIG. 13: Shows the concentration dependent effect of SAPK4
on reduced light output (with MBP substrate). The SAPK4
concentrations are shown as nanomolar.
[0027] FIG. 14: Shows the effect of increasing concentrations of
ATP detection of JNK2.alpha.2 activity in the presence of ATF-2 as
substrate. The results are the means of triplicate wells
.+-.SD.
[0028] FIG. 15: Shows the effect of increasing ATP concentrations
on SAPK3 activity in the presence of MBP as substrate. The results
are shown as the means of triplicate wells .+-.SD.
[0029] FIG. 16: Shows the reduction in RLUs in the presence of
increasing concentrations of ATP for SAPK3 and MBP. SAPK3 was used
at 728 nM with MBP at a final concentration of 100 .mu.g/ml.
[0030] FIG. 17: Shows a comparison of performance of the kinase
assay when using 20 .mu.l from a larger reaction volume and when
the assay is performed directly in the wells of a 384 well
microtitre plate.
[0031] FIG. 18: Shows the activation of MAPK2 by MEK-1, followed by
the phosphorylation of MBP by the previously activated MAPK-2.
[0032] FIG. 19: Shows the correlation of a drop in light signal
with the immunostaining of the phosphorylated MBP by western
blotting. The left hand lane on the blot correlates with the no MBP
control, and the right hand lane shows the effect of SAPK3 activity
on Upstate Biotechnology (UBI) MBP.
[0033] FIG. 20: Shows a comparison between the bioluminescent assay
of kinase activity and the results of the Western blotting.
[0034] FIG. 21: Shows the effect of staurosporine on the
bioluminescent detection system. The results are presented as the
means of triplicate wells SD.
[0035] FIG. 22: Shows the effect of two different staurosporine
concentrations on JNK2.alpha.2 activity (with ATF-2 as
substrate).
[0036] FIG. 23: Shows the effect of increasing concentrations of
genistein on MAPK-1 activity (with MBP as substrate).
[0037] FIG. 24: Shows the effect of two different concentrations of
PD098059 on raf-1 activity (with inactive MEK-1 as substrate). The
results are the means of triplicate wells .+-.SD.
DETAILED DESCRIPTION OF THE INVENTION
[0038] According to the invention there is provided a method for
measuring protein kinase activity, said method comprising:
[0039] (a) providing a first solution comprising ATP and a protein
kinase to be tested, and a second solution comprising ATP in the
absence of said protein kinase to be tested;
[0040] (b) adding a substrate capable of being phosphorylated by
the protein kinase to be tested to the first and second solutions
of step (a);
[0041] (c) measuring the concentration of ATP and/or ADP, or the
rate of change thereof with respect to time, in each of the
reaction mixtures formed in step (b) using a bioluminescence
reaction; and
[0042] (d) using the information about the concentration of ATP
and/or ADP to determine the activity of the protein kinase to be
tested.
[0043] The kinase is preferably activated prior to step (a) by
phosphorylation. Kinases are involved in very complex intracellular
signalling cascades. On binding of an agonist to a cell membrane
receptor a number of phosphorylation events are very rapidly set in
motion. In quiescent cells, a number of kinases are in their
inactive form and require phosphorylation in order to allow these
enzymes to then phosphorylate their substrates. By a domino-like
effect, activation of one component of the pathway may provide a
large amplification of the signal. For example, Raf is a
serine/threonine kinase that phosphorylates and activates MEK (MAPK
kinase). MEK, in turn, is a dual tyrosine/threonine kinase which
will activate MAPK (Erk-1 and Erk-2) by phosphorylation of the
tyrosine and threonine residues. The MAPKs, in turn, are then
activated such that they can phosphorylate their substrates, i.e.
myelin basic protein.
[0044] Commercially available kinases can be obtained in their
active form (already phosphorylated by the suppliers) or in their
inactive form. The latter require phosphorylation by another kinase
which would be upstream from them in the signal transduction
pathway.
[0045] Since the methods of the invention are not selective for
particular types of kinases (i.e. serine/threonine vs. tyrosine),
they can be used to monitor the step-wise activation of all the
kinases in a particular pathway, for example by measuring the
reduction in ATP seen when MEK phosphorylates Erk-1, which in turn
phosphorylates myelin basic protein.
[0046] A second aspect the invention provides a method for
identifying a compound which modulates the activity of a protein
kinase, said method comprising:
[0047] (a) providing a first solution comprising ATP, a protein
kinase and a compound to be tested, and a second solution
comprising ATP and the protein kinase in the absence of said
compound to be tested;
[0048] (b) adding a substrate capable of being phosphorylated by
said protein kinase to the first and second solutions of step
(a);
[0049] (c) measuring the concentration of ATP and/or ADP, or the
rate of change thereof with respect to time, in each of the
reaction mixtures formed in step (b) using a bioluminescence
reaction;
[0050] (d) using the information about the concentration of ATP
and/or ADP to determine the activity of the protein kinase in the
first and second solutions;
[0051] (e) comparing the activity of the protein kinase in the
first solution with the activity of the protein kinase in the
second solution to identify compounds which modulate the activity
of a protein kinase, whereby the compound to be tested is
identified as a protein kinase modulator if the activity of the
protein kinase in the first solution is different from the activity
of the protein kinase in the second solution.
[0052] Exemplary kinase/substrate combinations for use in the
methods of the invention include JNK-1/cjun, JNK-2/cjun, MAP
Kinase-1 (ERK-1)/myelin basic protein, MAP Kinase-2 (ERK-2)/myelin
basic protein, PKA/Kemptide, MEK-1/inactive MAP Kinase-2 (ERK-2),
JNK2.alpha.2/ATF-2, JNK2.alpha.2/cjun, SAPK-3/myelin basic protein,
SAPK4/myelin basic protein and raf-1/inactive MEK-1.
[0053] By "modulate" we include the meaning that the activity of
the protein kinase is increased or decreased or prevented/inhibited
in the presence of the test compound. Thus, the methods of the
invention may be used to determine whether a compound is an
inhibitor or activator of a protein kinase.
[0054] The compound to be tested is identified as a protein kinase
inhibitor if the activity of the kinase in the first solution is
lower than the activity of the kinase in the second solution.
Preferably, the compound to be tested is identified as a protein
kinase inhibitor if the activity of the kinase in the first
solution is less than 90% of the activity of the kinase in the
second solution. More preferably, the compound to be tested is
identified as a protein kinase inhibitor if the activity of the
kinase in the first solution is less than 80%, 70%, 60%, 50%, 40%,
30%, 20% or 10% of the activity of the kinase in the second
solution. Most preferably, the compound to be tested is identified
as a protein kinase inhibitor if the activity of the kinase in the
first solution is less than 50% of the activity of the kinase in
the second solution.
[0055] Likewise, the compound to be tested is identified as a
protein kinase activator if the activity of the kinase in the first
solution is higher than the activity of the kinase in the second
solution. Preferably, the compound to be tested is identified as a
protein kinase activator if the activity of the kinase in the first
solution is more than 10% greater that the activity of the kinase
in the second solution. More preferably, the compound to be tested
is identified as a protein kinase inhibitor if the activity of the
kinase in the first solution is more than 20%, 30%, 40%, 50%, 75%,
100% or 200% greater than the activity of the kinase in the second
solution. Most preferably, the compound to be tested is identified
as a protein kinase activator if the kinase in the first solution
at least 50% greater than the activity of the kinase in the second
solution.
[0056] Conveniently, the first and second solutions of step (a) of
the methods of the invention are substantially cell-free.
[0057] Steps (a) to (d) of the method according to the second
aspect of the invention may be repeated one or more times using a
different kinase and its corresponding substrate each time.
[0058] Compounds which increase the activity of a protein kinase
may find utility in medicine, especially the study of cancers and
may be useful as therapeutic agents. Compounds which decrease or
prevent/inhibit the activity of a protein kinase may also find
utility in such applications.
[0059] Preferably, the first and second solutions of step (a)
comprise a buffer, conveniently Hepes buffer.
[0060] Advantageously, steps (a) to (c) are carried out
consecutively.
[0061] It will be appreciated by persons skilled in the art that,
following addition of the substrate in step (b), the reaction may
be allowed to proceed for various durations and at different
temperatures prior to step (c). Advantageously, following addition
of the substrate in step (b), the reaction mixture is allowed to
react for 10 minutes at 30.degree. C. prior to step (c).
Conveniently, following addition of the substrate in step (b), the
reaction mixture is allowed to react for 10 minutes at 30.degree.
C. or for 1 hour at room temperature prior to step (c).
[0062] Preferably, step (c) of the methods of the invention
comprises:
[0063] (i) adding a bioluminescent reagent comprising luciferin or
a derivative thereof and a luciferase to said reaction mixtures,
said luciferin or a derivative thereof emitting light in a
bioluminescent reaction with the luciferase in the presence of ATP;
and
[0064] (ii) measuring the intensity of light emitted by the
resultant bioluminescent reaction, or its change with respect to
time, as a measure of ATP concentration.
[0065] The bioluminescent reagent of step (c) can be any of the
luciferin/luciferase general type. The active substrate is
D-luciferin or a derivative thereof. U.S. Pat. No. 5,374,534
discloses D-luciferin derivatives which may be used with luciferase
in the methods of the invention. Any other derivative can be
used.
[0066] The luciferase enzyme is preferably obtained naturally,
especially from fireflies and most especially Photinus pyralis.
However, the source of the luciferase is not critical, so long as
it reacts with luciferin (or a derivative thereof) and ATP in the
bioluminescent reaction. Examples are luciferases from Luciola
cruciata, Diptera spp. and Coleoptera spp.
[0067] Synthetic, for example, recombinant luciferase can be used
in the invention. It is described by Devine et al., (1993)
Biochemica et Biphysica Acta 1173, 121-132 and in European Patent
No 0 301 541 and U.S. Pat. No. 5,583,024.
[0068] Mutant luciferases may also be used in the methods of the
invention (see below).
[0069] In a preferred embodiment, the method comprises a further
step (b'), carried out after step (b) and before step (c), of
adding a reagent to the reaction mixture formed in step (b) which
stops the reaction of the kinase with the substrate.
[0070] There are a number of acids that are suitable for use as a
stop reagent, for example commonly used laboratory acids such as
phosphoric acid. Alternatively, high concentrations of EDTA or EGTA
may be employed. Luciferase is more resistant to the effects of
EDTA/EGTA than other enzymes, and performance in high
concentrations of these salts is increased by the use of mutant
luciferase enzymes. In addition, any other known buffer for
stopping enzyme reactions may be used.
[0071] The stopping reagent is preferably phosphoric acid, EDTA or
EGTA.
[0072] The use of a stopping reagent is particularly advantageous
as it allows one to make up and store large numbers of samples
prior to testing. This feature is particularly desirable for high
throughput applications of the methods of the invention (see
below).
[0073] The method of this preferred embodiment advantageously
comprises a further step (b"), carried out after step (b') and
before step (c), of adjusting the pH of the mixture formed in step
(b') to a pH at which the luciferase enzyme is active, normally pH
7.0. Preferably, step (b") comprises adding Hepes buffer.
[0074] The step of pH adjustment can be avoided by the use of a
mutant luciferase which retains the required activity at the pH of
the solution following addition of the stopping reagent. It is also
useful to employ a luciferase which is active at 30.degree. C.,
rather than wild type luciferase which is not very active above
25.degree. C. An additional benefit of using thermostable
luciferase mutants is that, in addition to their resistance to
elevated temperatures, is that key amino acids within the enzyme
can be mutated so as to confer other favourable properties that
enhance the performance of the enzyme, including resistance to low
pH and high salt solutions. These properties are therefore helpful
if a stop solution is to be used.
[0075] Suitable mutant luciferases can be obtained from Kikkoman
Biochemicals, Japan.
[0076] Other exemplary mutant luciferases suitable for use in the
methods of the invention are disclosed in White et al. (1996)
Biochem J. 319, 343-350, Squirrel et al. (1997) J. Defence Science
2, 292-297, Karp & Oker-Blom (1999) Biomolecular Engineering
16,101-104, Branchini et al (1999) Biochemistry 38, 13223-13230,
Branchini et al. (2000) Biochemistry 39, 5433-5440, Tatsumi et al.
(1996) Anal. Biochem. 243, 176-180, WO 98/46729, WO 96/22376, WO
99/02697, EP 0 449 621 B, U.S. Pat. No. 5,330,906, U.S. Pat. No.
6,074,859, and WO 95/18853.
[0077] Advantageously, step (c) further comprises the following
steps carried out after the light intensity measured in step (ii)
has reached a substantially constant level:
[0078] (iii) adding a reagent that converts ADP to ATP;
[0079] (iv) adding a bioluminescent reagent comprising luciferin or
a derivative thereof and a luciferase to said reaction mixture of
step (iii); and
[0080] (v) measuring the intensity of light emitted by the
resultant bioluminescent reaction
[0081] wherein the difference in the intensity of light in step (v)
and the steady state intensity of light in step (ii) is a measure
of ADP concentration in the reaction mixture of step (ii).
[0082] By "substantially constant" we include the meaning that the
light intensity does not vary significantly over the same time
period as is taken to carry out the light intensity measurements.
As a non-limiting example, the term is intended to include the
meaning that the rate of change of emitted light intensity is less
than 5% per minute, and preferably less than 3% per minute. In any
event the person skilled in the art will be able to appreciate
whether the level is sufficiently constant to be able to obtain a
valid reading of the ATP produced by adding the ADP-converting
reagent, not significantly affected by any small change in the ATP
baseline.
[0083] In an alternative preferred embodiment, steps (b) and (c)
are carried out simultaneously.
[0084] According to a further aspect, the invention provides a
compound identified using a method of the invention.
[0085] It will be appreciated by persons skilled in the art that
the methods of the invention are suitable for high throughput
screening, i.e. screening of large numbers of chemically generated
and naturally-derived products for generating leads to
pharmaceutical products. In such screening assays, compounds may be
put into groups for screening using microtitre plate
technologies.
[0086] Thus, the methods of the invention can be performed in the
small volumes associated with 384 and 1536 well plates, in addition
to the 96 well plate format. Under these circumstances, where
laboratory robots are used, the assays would be prepared in a large
number of plates. The assays could then be carried out using the
robots to transport the plates into a luminometer with injectors,
and the assay performed as described above.
[0087] Another option arises due to the long half-life of the
`glow` of light from the bioluminescence reaction. Once the
reaction has plateaued, the emitted light intensity remains
substantially constant. This allows for the bioluminescent reagent
to be added to the plates in batches, so the plates can be read
even 3-4 hours after addition of the reagents.
[0088] The invention further provides a kit for use in the method
of the second aspect of the invention, comprising:
[0089] (a) a bioluminescent reagent comprising luciferin or a
derivative thereof and a luciferase, said luciferin or a derivative
thereof emitting light in a bioluminescent reaction with the
luciferase in the presence of ATP;
[0090] (b) a kinase;
[0091] (c) a substrate capable of being phosphorylated by said
kinase; and
[0092] (d) ATP.
[0093] The kit conveniently further comprises one or more buffers
for reconstituting, diluting or dissolving the bioluminescent
reagent, kinase, substrate and/or ATP.
[0094] The kit may also further comprise a reagent capable of
stopping the reaction of said kinase with said substrate, for
example phosphoric acid.
[0095] A kit according to the invention may additionally comprise
one or more reagent(s) which converts ADP to ATP, such as one which
comprises pyruvate kinase and phosphoenol pyruvate.
[0096] In a preferred embodiment, the kit of the invention
comprises two or more different kinases and their substrates. Thus,
there is envisaged a kit suitable for screening compounds to be
tested against a plurality of different kinases to determine
whether the compound modulates kinase activity and the specificity
of such inhibition.
[0097] A kit according to the invention may further comprise a
multi-well microtitre plate. This term is intended to embrace
apparatus which comprises a plurality of reaction vessels or wells
linked together in the form of a plate. Each well has a small
volume, usually 250 to 300 .mu.l in a 96 well plate, 60 to 70 .mu.l
in a 384 well plate and 6-8 .mu.l in a 1536 well plate. At present,
the most common plates have 96 wells, but plates having 384 and
1536 wells are known and useful according to the invention.
Preferably, the kit comprises a multiwell microtitre plate contains
96 wells or more.
[0098] Advantageously, the reagent or reagents in a kit of the
invention is or are provided in lyophilised form.
[0099] Examples which embody certain aspects of the invention will
now be described by way of a non-limiting illustrations which refer
to the figures.
EXAMPLES
[0100] We have performed a series of experiments to show the effect
of protein kinase activity in a cell free system. All the protein
kinases and substrates were supplied by Upstate Biotechnology Inc.,
(UBI) Lake Placid, USA. Any other reagents used in a number of
different formulations are shown in appendix 1.
Example 1
Determination of the Activity of JNK-1
[0101] The first set of experiments was to determine the activity
of JNK-1 after the activation of this enzyme by two other kinases,
MEKK1 and MKK4. The assay buffer used to activate the enzymes was
made up as a 10 times stock (the formulation is shown in appendix
1). The assay was performed as follows, with an initial preparation
of a pre-mix for JNK activation.
[0102] The JNK enzyme was used at a stock concentration of 1.234
mg/ml, MEKK1 at 1 mg/ml and MKK4 at 0.28 mg/ml. The activation
mixture was added to a polypropylene tube (Sarstedt) with 8.7 .mu.l
of JNK, 2.5 .mu.l of MEKK1, 8.4 .mu.l of MKK4, 1 .mu.l of 10 mM ATP
(Calbiochem, UK), 1 .mu.l of dithiothreitol (Sigma, UK), 5 .mu.l of
10.times. assay buffer and 23.4 .mu.l of distilled water. This was
incubated overnight at room temperature. To perform the actual
assay, the 50 .mu.l of activated concentrate was diluted in 9 mls
of JNK assay buffer at the working concentration (i.e. diluted
1:10). The substrate used was GST-c-jun at a concentration of 8.61
mg/ml. Briefly, 12 .mu.l of GST-cjun was diluted in 1545 .mu.l of
working strength assay dilution buffer. Each assay point was
performed in triplicate wells of a white, opaque, 96 well
microtitre plate (Dynex). To each well was added 15 .mu.l of the
substrate mix, followed by 30 .mu.l of the activated JNK enzyme.
The reaction was then allowed to proceed at room temperature for
one hour. The reaction was then stopped by the addition of 20 .mu.l
of 2% (v/v) phosphoric acid (Sigma, UK). To one set of triplicate
wells was added 135 .mu.l of Tris-acetate buffer (pH7.75, see
appendix 1 for formulation), to a second set of triplicate wells
was added 135 .mu.l of 200 mM Hepes buffer (pH 7.75, see appendix 1
for formulation). After this 20 .mu.l of ATP monitoring reagent
(see appendix 1 for formulation) was added to each well. The plate
was then placed immediately into a Microbeta (RTM) Jet luminometer
(Perkin-Elmer Life Sciences), and the light output was determined
over a 1 second integral. FIG. 1 shows the drop in light output
seen in the presence of the kinase.
[0103] Stop Solution
[0104] These experiments were performed with 2% phosphoric acid to
halt the kinase reaction at a set time point. A particular
advantage of the use of a stop solution, that is, any reagent which
can halt the reaction of the kinase and the substrate which it
phosphorylates, is that one can make up and store large numbers of
samples prior to testing in the methods of the invention. This
feature is a particular benefit for high-throughput screening
applications. One of the problems with the stop solution is that a
reduction in the pH adversely affects the luciferase enzyme, so
there must be sufficient buffering capacity when the ATP is
measured. In this first series of experiments Hepes proved to be a
much better buffer for counteracting the effects of the phosphoric
acid. Experiments were performed with increasing concentrations of
Hepes, and showed that the 200 mM buffer brought the pH in the well
back to pH 7.0 allowing the luciferase-luciferin reaction to
proceed without reactivating the kinase. The Tris acetate buffer
was less efficient, and while there were still differences in the
detected RLUs in the presence of the kinase enzyme, however, they
were not as marked as with the Hepes. The results with Hepes showed
lower RLUs than without, but this approach did allow for the kinase
activity to be controlled prior to being detected using the
luciferase-luciferin reaction. As a result of this, most of the
following experiments were performed with the 200 mM Hepes, as a
dilution buffer, unless otherwise stated. FIG. 2 shows the data
from two separate experiments and demonstrates the effects of both
the phosphoric acid stop solution and the Hepes buffer.
[0105] Skilled persons will appreciate that one may avoid the use
of a buffer after addition of the stop solution by using mutant
luciferases which are pH and salt stable. It is also desirable to
use mutant luciferase which are thermostable at 30.degree. C.
rather than wild type luciferase, which is not very active above
25.degree. C. Suitable mutants are available from a variety of
sources. For example, pH, salt stable and thermostable mutants can
be acquired from Kikkoman Biochemicals, Japan (see above).
Example 2
Determination of the Activity of MAPK-1/ERK-1
[0106] To confirm that monitoring of kinase activity by the above
method of the invention would proceed with other enzymes that
cleave the phosphate from ATP, we looked at a number of other
kinases which are important in signal transduction and used as
targets in drug discovery.
[0107] MAP Kinase-1/ERK-1 activity was investigated in the presence
of myelin basic protein as the phospho-acceptor. This enzyme was
supplied in the active form from UBI at a stock concentration of 25
.mu.g in 250 .mu.l. Briefly, 10 .mu.l of assay dilution buffer
(UBI, see appendix 1 for formulation) was added to triplicate wells
of a white-walled 96 well microtitre plate (Dynex).
[0108] To this was added 10 .mu.l of MAPK1 and 10 .mu.l of myelin
basic protein (UBI, 2 mg/nl), plus 10 .mu.l of ATP cocktail (UBI,
for formulation see appendix 1). The plate was sealed and the
reaction was allowed to proceed for 10 minutes at 30.degree. C.
After this time 110 .mu.l of either assay dilution buffer or Hepes
buffer were added to the wells followed by 20 .mu.l of ATP
monitoring reagent reconstituted in 200 mM Hepes buffer. The
results showed a reduction in light output with this enzyme in the
presence of its substrate. With the UBI buffer there was a drop in
RLUs from 77367 to 35578, with the Hepes buffer there was a
reduction from 91256 to 73424.
[0109] Detection Based on Increase in ADP
[0110] With this experiment we also determined whether it would be
possible to detect any resultant increase in ADP by the conversion
of ADP to ATP, through the addition of 20 .mu.l of an ADP
converting reagent containing pyruvate kinase (for formulation see
appendix 1). To determine the amount of ADP, a reading was taken
after the initial ATP light signal had been allowed to decay for 10
minutes. The converting reagent was then added and a further
reading taken after 5 minutes. All of these readings were integrals
taken after 1 second using the Microbeta (RTM) Jet (Perkin-Elmer
Life Sciences). The amount of ADP present correlated with the
difference in light output between the final reading and the
reading taken prior to the addition of converting reagent. The data
showed that it was possible to determine an increase in ADP, as
shown in FIG. 3.
Example 3
Determination of the Activity of MAPK-2/ERK-2
[0111] The above protocol was also used for determining the effect
of MAPK-2/ERK-2 in the presence of the same substrate, myelin basic
protein. The MAPK-2 was supplied by UBI at a concentration of 2.5
.mu.g of enzyme in 25 .mu.l of buffer (for formulation see appendix
1), with a specific activity of 662.5 U/mg, where 1 U=1 nmole of
phosphate incorporated into myelin basic protein. This enzyme was
compared with the inactive form, also supplied by UBI at a
concentration of 12.5 .mu.g in 50 .mu.l. Both enzymes were diluted
in UBI assay dilution buffer to allow for the addition of 25 ng of
protein in 10 .mu.l to each well. The myelin basic protein was
added as described in the above example, and 200 mM Hepes was used
as the buffer added to the wells prior to the addition of the ATP
monitoring reagent. The assay was performed in triplicate wells and
showed RLUs of 10075.+-.339 for the inactive enzyme, which was very
similar to the light output from the no enzyme controls
(11440.+-.1372). The RLUs for the active enzyme showed a drop in
ATP, after the 10 minutes incubation, to 7008.+-.430. It was also
possible to detect an increase in RLUs after addition of ADP
converting reagent in the active sample of 5272, compared with 441
with the inactive enzyme.
Example 4
Effect of Kinase Concentration on Reduction in ATP
[0112] To determine whether there was a concentration dependent
effect of kinase activity on reduction in ATP, MAPK-1 was used at
concentrations ranging from 1.56 ng/10 .mu.l to 100 ng/10 .mu.l,
with serial doubling dilutions from the highest concentrations. The
concentration of myelin basic protein used was the same as in the
previous experiments. The assays were performed as in the previous
two experiments, i.e. using UBI reagents, but with the addition of
110 .mu.l of 200 mM Hepes buffer prior to determination of ATP
readings. The results showed a concentration dependent reduction in
ATP levels detected with increasing concentrations of the MAPK-1.
There was no effect of the enzyme when 1.56 ng was added to each
well, however, there was a significant effect at concentrations of
3.13 ng and above (see FIG. 4).
Example 5
Effect of ATP Concentration on Light Decay: Influence of Buffer
Type
[0113] The luciferase enzyme itself is an ATPase that converts ATP
to AMP and inorganic phosphate. After the initial increase in light
output as a result of the luciferin-luciferase reaction, the light
signal begins to decay over time. We examined the light decay with
increasing concentrations of ATP up to the 200 .mu.M used in the
above experiments. The ATP standards (Sigma, UK) were diluted at
serial doubling dilutions from 200 .mu.M down to 3.125 .mu.M per 10
.mu.l added to each well. The standards were diluted in three
different buffers, assay dilution buffer from UBI, Tris acetate
buffer (pH 7.75) and 200 mM Hepes pH 7.7. 10 .mu.l of the
standards, plus a no ATP buffer control, were added in to
triplicate wells of the opaque-white 96 well microtitre plates. The
experiments were run with duplicate plates, one where 1401 .mu.l of
the Tris-acetate buffer was added to all wells, and the other where
the same volume of 200 mM Hepes buffer. Immediately after the
addition of these reagents 20 .mu.l of ATP monitoring reagent was
added to all wells. The plate was then placed in a Berthold (RTM)
Detection Systems MPL2 luminometer, and the program was set to take
1 second integral readings for each well every 2 minutes. The FIG.
5 graph shows the initial light output (at time 0) and the decay in
the light signal observed with the different buffer conditions and
the 200 .mu.M stock ATP (final concentration in the well of 12
.mu.M).
Example 6
Effect of Luciferin-Luciferase (ADR) Reagent on Kinase Assay:
Detection of Kinase Activity as a Drop in Light Signal
[0114] Experiments were also carried out to investigate the effect
of performing the kinase assay in the presence of
luciferin-luciferase reagent (ADR), and its effect upon the rate of
signal decay. The enzyme used was JNK-2 (Upstate Biotechnology Inc,
USA) at 1 .mu.g per 10 .mu.l added to each well, the c-jun
(1-169)-GST substrate (Upstate Biotechnology Inc, USA) was also
added at the same concentration. Into each well was added 10 .mu.l
of 200 .mu.M ATP standard and 10 .mu.l of Hepes buffer (200 mM).
Into control wells the 10 .mu.l of enzyme was replaced by 10 .mu.l
of Hepes buffer, once all the active reagents were in the wells and
an additional 120 .mu.l Hepes dilution buffer had been added, then
20 .mu.l of ADR was added and the signal decay monitored every 20
seconds over a 2 minute period, using a Berthold (RTM) Detection
Systems MPL-2 luminometer. A 1 second integral reading was taken at
each time point. The data showed an increase in the rate of signal
decay in the presence of the JNK-2 enzyme, as shown in FIG. 6. This
shows that kinase activity can also be detected as an accelerated
drop in light signal, in the absence of a stop solution.
Example 7
Comparison of Active and Inactive Forms of a Kinase
[0115] We also compared activated and inactive forms of MAPK-1 and
MAPK-2, and showed reduced kinase activity with the inactive forms
of the enzymes, although in some cases there did appear to be a
certain amount of autophosphorylation, this was not significantly
different from the no enzyme controls. A concentration curve for
inactive MAPK-1 versus the activated form, showed clearly how the
kinase activity of the enzyme reduced the amount of ATP and hence
increased the signal decay. The experiment was performed as
detailed in Example 3, however in this case the kinetics of the
reaction were investigated over the first 6 minutes of the
reaction. From FIG. 7, it can be seen that at 100 ng of MAPK-1 per
10 .mu.l (588 ng/ml final concentration) gave a significant
increase in the % signal decay by 6 minutes. The assay was
performed in triplicate in 3 different experiments. As soon as the
ADR had been added the plate was read for 1-second integrals every
minute for 6 minutes, using a Labsystems Luminoskan (RTM)
luminometer.
[0116] A similar effect was also seen with the phosphorylation of
MAPK-2 by MEK-1. In this series of experiments the MEK-1 was used
at final concentrations in the well of 75 .mu.g/ml through to 375
.mu.g/ml by the addition of 1-5 .mu.L of stock enzyme at 5 U/50
.mu.l. The activity quoted from Upstate Biotechnology Inc was 7850
U/mg where 1 unit will maximally activate 1 unit of inactive
MAPK-2. The inactive MAPK-2 was added to each well in 10 .mu.l
volumes to give a final concentration of 5.88 mg/ml. Again, in the
presence of 200 .mu.M ATP and Hepes buffer, there was a
concentration dependent increase in the signal decay from 14% in
the control to 36% for the highest concentration used.
Example 8
Effect of Different Kinase Buffers
[0117] In the literature there are a number of different reaction
buffers that are used to perform kinase assays, we therefore
decided to test the ATP detection reagent with these buffers, to
ensure that the assay would perform irrespective of the
constituents of the buffer. The reason these buffers are used is to
supply optimal conditions for the kinase reaction in the presence
of the ATP and protein/peptide substrates. FIG. 8 shows the effect
of 13 different buffers commonly used in protein kinase assays. The
buffer constituents are shown in Table 1.
1TABLE 1 Used for Buffer i.d. Buffer contents Kinases 0 100 mM Tris
Hcl pH 7.4 containing 20 mM DTT, 20 mM MnCl.sub.2, 10% Glycerol SBE
Mix (Astrazeneca) and 0.004% Brij-35 1 8 mM MOPs pH7.0 containing
0.2 mM EDTA GSK3.beta., S6k1, MAPKAP- K1b/RSK2, PKA, CHK1 CHK2,
MSK1 and SGK 2 50 mM Tris-Hcl pH7.75 containing 0.05%
2-Mercaptoethanol PKB.alpha. 3 25 mM Tris-Hcl pH 7.5 containing 0.1
mM EGTA ERK-2 SAPk2.alpha./p38, SAPK2b/p38/p32, SAPk3 and SAPk4 4
50 mM Sodium .beta. glycerophosphate pH7.5 containing 0.1 mM EGTA
MAPKAP-K2 and PRAK 5 50 mM Tris-Hcl pH 7.5 containing 0.1 mM EGTA
and 0.1% 2-Mercaptoethanol. JNK-1, ROCKII and PRK-2 6 50 mM Hepes
pH7.4 containing 1 mM DTT, 0.02% Brij-35, and 0.2 mM AMP. AMPK
Respiratory 7 20 mM Hepes pH 7.4 containing 0.03% Triton X-100, 0.1
mM Cacl.sub.2, 0.1 mg.ml PKC.alpha. phosphatidylserine and 10
.mu.g.ml 1,2-dioleoyl-sn-glycerol. 8 20 mM Hepes pH7.4 containing
0.03% Triton X-100, 0.1 mM EGTA, 0.1 mg.ml PKC.delta.
phosphatidylserine, 10 .mu.g.ml 1,2-dioleoyl-sn-glycerol 9 20 mM
Hepes pH7.6 containing 150 mM NaCl, 0.1 mM EDTA, 5 mM DTT and CK2
0.1% Triton X-100 10 50 mM Tris-Hcl pH7.5 containing sodium
.beta.-glycerophosphate, and 0.04 mM PHK Cacl.sub.2 11 25 mM
Tris-Hcl pH7.5 containing 0.1 mM EGTA, 0.1% 2-Mercaptoethanol and
ERK2 0.01% Brij-35 12 50 mM Tris-Hcl pH7.5 containing 0.1%
2-Mercaptoethanol PDK-1 13 50 mM Tris-Hcl pH7.5 containing 0.1 mM
CaCl.sub.2, 10 units per ml calmodulin MLCK and 0.1% 2
Mercaptoethanol. 14 50 mM Tris-Hcl pH7.5 containing 0.1 mM EGTA,
0.1 mM Na.sub.4Vo.sub.3 LCK 14
[0118] The ATP was diluted in each of the buffers and 100 .mu.L was
added to each well of a 96 well microtitre plate, 20 .mu.l of ATP
detection reagent was added to the wells, and the light output was
detected after a 1 second integral reading using a Berthold (RTM)
Detection Systems Orion Luminometer.
[0119] The results in FIG. 8 show that buffers 0 and 9 compromise
the light output, however the sensitivity of the assay was
unaffected. These assays are normally performed with ATP
concentrations from tens to hundreds of micromolar, i.e. greater
than the 1 .mu.M concentration used in this example, where a
significant light output was still detectable with the buffers that
reduced the signal.
Example 9
Use of Tris Acetate Buffer in Bioluminescence Assay
[0120] We have compared the performance of the bioluminescent assay
in Tris buffer as well as Hepes. We have shown that it is possible
to reconstitute the ATP Detection Reagent in Tris Acetate Buffer at
pH 7.75. However, when using an acid stop solution, for example
phosphoric acid, it is preferable to use the Hepes buffer
reconstitution system described above.
[0121] The assays were performed using a number of different
kinases and substrates. The experiments were carried out using
methods similar to that described for Hepes buffer. The assays were
performed in 100 .mu.l volumes in wells of a 96 well microtitre
plate, the appropriate concentrations of each enzyme and substrate
were added to the wells in the most suitable reaction buffer for
that enzyme. The reaction was then initiated by the addition of ATP
at the appropriate concentration, the reaction was allowed to
proceed at 30.degree. C. for 10 minutes prior to addition of 20
.mu.l of ATP detection reagent (reconstituted in Tris acetate
buffer), light output was detected over a 1 second integral. In the
following examples, the ATP detection reagent was reconstituted in
Tris-acetate buffer pH 7.75, rather than a Hepes.
[0122] JNK2.alpha.2 with ATF-2 and c-Jun as Substrates
[0123] This enzyme was tested with both the ATF-2 and c-Jun
substrate peptides from Upstate Discovery.
2 Assay Buffer: 100 mM Tris-HCl pH 7.4 20 mM Dithiothreitol 20 mM
MgCl.sub.2 10% glycerol 0.004% Brij 35
[0124] Time course experiments were performed in 100 .mu.l (or 200
.mu.l) volumes in clear plastic test tubes. The reaction was
carried out in either a 30.degree. C. waterbath or in an
incubator.
[0125] Upstate Discovery recommend use of the ATF-2 substrate at 5
.mu.g per assay point and the JNK2.alpha.2 at 20 mU per assay
point.
[0126] Stock ATF-2 (LB018LFp107) at 4.3 mg/ml in 10 .mu.l aliquots
(43 .mu.g in 10 .mu.l). The substrate was diluted 1:8 from stock
and then 10 .mu.l added to the tube to give a final amount in the
reaction mixture of 5.38 .mu.g.
[0127] Stock JNK2.alpha.2 (LB021SDp81) is at 77 U/mg (100 U/ml) and
1.3 mg/ml and aliquoted into 10 .mu.l aliquots (1 U). Concentration
curves were performed with the addition of 5 .mu.l of neat enzyme
stock (50 mU), 5 .mu.l of 1:2 (25 mU), 1:4 (12.5 mU) and 1:8 (6.25
mU), per 100 .mu.l of final reaction volume.
[0128] Stock ATP at 1M in 10 .mu.l aliquots. The ATP was diluted
1:40 with Tris-HCl buffer (addition of 390 .mu.l) and then a
further 1:100 to give a working solution of 250 .mu.M. Then 10
.mu.L was added to each 10011 of final reaction volume, this gave a
final concentration in the tubes of 25 .mu.M.
3 The tubes therefore contained: 10 .mu.l substrate 5 .mu.l enzyme
10 .mu.l ATP 75 .mu.l assay buffer
[0129] To determine kinase activity 20 .mu.l samples were removed
from the tubes at 5 minute intervals and added to the wells of a 96
well white opaque microtitre plate. Then 20 .mu.l of ADR
reconstituted in Tris-Ac buffer was added to the wells, and the
plate read in the luminometer over a 1 second integral.
[0130] To determine reproducibility a larger volume was prepared
and triplicate samples taken over reduced time points.
[0131] FIG. 9 shows the drop in light output with time in the
presence of enzyme and substrate compared with the substrate only
control.
[0132] FIG. 9 also shows the effect of adding ADP converting
reagent (20 .mu.l), this converted the ADP formed as a result of
kinase activity back to ATP, for detection with the ATP detection
reagent. The drop in light signal after pipetting of the ADP
converting reagent was due to a drop in pH in the well after
addition. FIG. 9 clearly shows a marked drop in light output in the
presence of enzyme and substrate compared with the no enzyme
control.
[0133] As described in the above methods, the assay was repeated
with decreasing concentrations of JNK2.alpha.2, against the same
ATP and peptide concentrations. FIG. 10 shows the data obtained
with 2.55 .mu.M (50 mU) down to 320 nM JNK2.alpha.2.
[0134] In addition to ATF-2 we also studied another JNK substrate,
namely the peptide c-Jun. For determining JNK2.alpha.2 activity
with the c-Jun peptide the experiments were performed using the
same protocol as above, but with the addition of 10 .mu.l of c-Jun
rather than the ATF-2.
[0135] Stock c-Jun (LB023JTp72) at 4.83 mg/ml in 80511 in 10
.mu.l=48.3 .mu.g, to use approximately the same amount of peptide
as ATF-2 again dilute 1:9 and then add 10 .mu.l per tube.
[0136] FIG. 11 compares the drop in ATP as a result of kinase
activity when the two different substrates were compared. The data
confirmed information received from Upstate Discovery that the
ATF-2 was a more efficient substrate than the c-Jun, for
JNK2.alpha.2 activity.
[0137] SAPK-3 and Myelin Basic Protein
[0138] SAPK3 is a member of the mitogen activated protein kinase
(MAPK) family, which can be activated by a variety of extracellular
agonists. These stress activated protein kinases can utilise myelin
basic protein (MBP) as the phospho-acceptor in kinase reactions. We
have shown that this kinase activity can be determined with ADP
detection reagent reconstituted in Tris acetate buffer (pH 7.75) in
addition to Hepes buffer. The assays were performed as follows.
[0139] This assay was performed using the same buffer as for
MAPK-2/ERK-2, with a 30.degree. C. incubation. ATP was used at the
same concentration as for JNK2.alpha.2.
4 Assay Buffer: 25 mM Tris-HCl pH 7.5 10 mM Mg acetate 0.1 mM
EGTA
[0140] As with the previous enzyme the assay was set up in tubes in
order to perform a time course. After the kinase reaction was
completed 20 .mu.l of reaction mixture was placed in the wells of a
96 well opaque white microplate, followed by addition of 20 .mu.l
of ATP detection reagent. The light output was again determined
over a 1 second integral.
[0141] Tubes contained: 10 .mu.l enzyme, 10 .mu.l substrate, 10
.mu.l ATP and 70 .mu.l assay buffer.
[0142] SAPK3 stock solution (LB012SDp174; 220 .mu.l) was provided
at a concentration of 1.55 mg/ml and 87.3 U/mg. The enzyme was
aliquoted into 10 .mu.l aliquots (15.5 .mu.g). The enzyme was then
diluted 1:3 (=5.17 .mu.g/10 .mu.l).
[0143] For enzyme concentration curves the enzyme was diluted a
further 1:5 (1.03 .mu.g) and 1:10 (0.52 .mu.g). The assay was
performed by the addition of 10 .mu.l per 100 .mu.l final reaction
volume. The final enzyme concentrations were 0, 0.517, 1.03 and
5.17 .mu.g/ml (corresponding to nanomolar concentrations of 0,
72.8, 145.6 and 728.0 nM, respectively; see legend to FIG. 12).
[0144] MBP from Calbiochem (0.5 mg/ml) used 10 .mu.l of the neat
stock per 100 .mu.l final reaction volume (2.72 .mu.M final
concentration).
[0145] Results of the SAPK3/MBP experiment are shown in FIG. 12, in
which a concentration dependent drop in ATP (as measured by light
output) is evident.
[0146] SAPK4 and Myelin Basic Protein
[0147] The above experiment was also repeated with SAPK4 and MBP.
These assays were performed using the same assay buffer as for
MAPK2/ERK-2 and SAPK3.
[0148] Stock SAPK4 (LB012SDp176) at 1.64 mg/ml and 144.1 U/mg in
1271 .mu.l. This was aliquoted into 5 .mu.l samples (8.2
.mu.g/aliquot). To generate a working stock the enzyme was diluted
1:4 (2.05 .mu.g/5 .mu.l), then further 1:5 (410 ng/5 .mu.l) and
1:10 (205 ng/5 .mu.l). Then 5 .mu.l was added to each tube, this
gave final concentrations in the tubes, for a 100 .mu.l reaction
volume of 0, 0.103, 0.205 and 1.25 .mu.g/ml (corresponding
nanomolar concentrations are shown in the FIG. 13).
[0149] Stock MBP, Life Technologies at 2.5 mg/ml. 10 .mu.l was
added to each 100 .mu.l final reaction volume (250 .mu.g/ml or 13.6
.mu.M).
[0150] The higher concentration of MBP showed a rapid decrease in
the light signal at time 0, this was in fact approximately 2
minutes after the addition of the reagents, as it took this long to
remove samples from the tubes, plate them out and then add the ATP
detection reagent (see FIG. 13).
[0151] The work performed with the SAP kinases also showed that it
was possible to use MBP from a number of different suppliers
(although the performance of the assay was found to depend upon the
quality of the protein provided).
[0152] These data confirm that Tris-acetate buffer could be used
for ATP detection reagent reconstitution as well as the Hepes
buffer described previously.
Example 10
Effect of ATP Concentration
[0153] All of the above assays were performed with ATP at a final
concentration of 25 .mu.M. We went on to investigate whether the
bioluminescent system could detect kinase activity with higher and
lower ATP concentrations. The assays were performed with the same
buffers as described above, and with the same volumes. However, in
the following examples the experiments were performed in the wells
of white-walled 96 well microtitre plates, rather than in tubes.
After 30 minutes at 30.degree. C., the plates were removed from the
incubator and 2011 of ATP detection reagent was added to each well
and the light out put read over a 1 second integral.
[0154] JNK2.alpha.2
[0155] FIG. 14 shows the data obtained with ATP at 3 different
final concentrations 1, 10 and 100 .mu.M (results are shown as
means of triplicate wells .+-.SD). The substrate used was ATF-2 at
a final concentration of 2.1 .mu.M, with enzyme at 1.25 .mu.M. The
assay buffer was the same as that described for JNK2.alpha.2
above.
[0156] SAPK3
[0157] The ATP concentration curve experiments were repeated with
SAPK3 and MBP as substrate. FIG. 15 shows the effect of different.
ATP concentrations on the change in light output (results are shown
as the means of triplicate wells +SD). The SAPK3 was used at a
concentration of 728 nM, with myelin basic protein (MBP) at a final
concentration of 2.72 .mu.M in the wells. At 100 .mu.M there was an
effect of the enzyme in the presence of the substrate where the
light signal dropped by 693,234 RLUs, from 5,267,900.+-.133,688 to
4,574,666.+-.283,204. Th is was a significant decrease in RLUs and
indicated that the amount of enzyme and substrate was limiting at
this high concentration of ATP.
[0158] The differences in RLUs correlate directly with ATP
concentrations this drop in light out put relates to the amount of
ATP dephosphorylated by the SAPK3. This is further demonstrated in
FIG. 16, where the differences in RLUs are shown, which indicates
that the same amount of ATP was consumed at 100 .mu.M ATP as 10
.mu.M.
[0159] Although it is not clear from FIG. 16, there was also a
significant difference in RLUs with the lowest concentration of ATP
used, where the RLUs dropped from 818.+-.18 to 300.+-.17
(means.+-.SD).
Example 11
Assay Performance in 384 Well Microtitre Plates
[0160] The time course experiments described above confirmed that
it was possible to perform the assay in tubes in large volumes, and
then at each time point sample 20 .mu.l for addition to a white
opaque microtitre plate (96 wells), with light output being
measured after the addition of 20 .mu.l of ATP detection reagent.
We have also shown that it is possible to perform the assay in 100
.mu.l volumes in the wells of a 96 well plate.
[0161] FIG. 17 shows a comparison of performance of the assay when
using 20 .mu.l from a larger reaction volume and when the assay is
performed directly in the wells of a 384 well microtitre plate. In
384 wells, the kinase reaction is performed in 20 .mu.l volumes
with the addition of 20 .mu.l of ATP detection reagent. The data
show that there was no difference in the performance of the assay
when carried out in tubes (100 .mu.l) or 384 well plates (20
.mu.l).
Example 12
Study of Kinase Cascades
[0162] The activation of kinases is most often the result of
phosphorylation by other kinases upstream in the signal
transduction pathways. An example of this is the activation of
MAPK-2 by MEK-1, followed by MAPK-2's ability to phosphorylate MBP.
We used this system to confirm that MAPK-2 had been phosphorylated
and activated by MEK-1. If the protein had been activated, there
would be a reduction in ATP when MAPK-2 was subsequently exposed to
MBP in the presence of ATP. The assay was performed in tubes in 100
.mu.l volumes, the initial activation of inactive MAPK2 was
performed at 30.degree. C. with 10 .mu.M ATP. The assay buffer
comprised 25 mM Tris acetate pH 7.75 with 0.1 mM EGTA and 10 mM
magnesium acetate. MEK-1 was used at a final concentration of 114
nM with inactive MAPK-2 at a final concentration of 516 nM.
[0163] To determine kinase activity, 20 .mu.l was then added to
duplicate wells of a 96 well plate, and 20 .mu.l of ATP detection
reagent added and the light signal determined over a 1 second
integral.
[0164] A second plate had been set up containing triplicate wells
with 10 .mu.l of MBP (Calbiochem) at 0.25 mg/ml, 10 .mu.l of 10
.mu.M ATP and 60 .mu.l of assay buffer, to this 20 .mu.l of the
MEK-1/MAPK2 reaction mixture from the tubes was added, and the
reaction allowed to proceed in an incubator at 30.degree. C. for 30
minutes. After this time 20 .mu.l of ATP detection reagent was
added to the wells and the light output was determined.
[0165] The results are shown in the following FIG. 18 wherein an
increase in the RLUs in the substrate only controls is observed,
which relates to addition of ATP from the original MEK-1/MAPK-2
reaction mixture. The data clearly show that the MAPK-2 had been
phosphorylated and was therefore able to exhibit kinase activity
itself in the presence of MBP. An additional control was run with
the inactive MAPK-2 and MBP which showed no drop in light
signal.
Example 13
Western Blotting Studies of Substrate Phosphorylation
[0166] In addition to showing that we could induce functional
activity of kinases, and detect this using the bioluminescent
assay, we confirmed that the drop in light output was associated
with phosphorylation of amino acids on peptide/protein substrates
by Western Blotting.
[0167] Methods
[0168] Sample Preparation: After completion of the kinase reaction
in 100 .mu.l volumes, 20 .mu.l of reaction mixture was added to 20
.mu.l of 2.times. Laemmli sample buffer (Amersham, Bucks, UK) and
heated at 100.degree. C. for 4 minutes, before being placed
immediately on ice until required.
[0169] A molecular weight HRP protein marker (New England BioLabs)
was prepared by adding 10 .mu.l of marker to 10 .mu.l of Western
blotting sample buffer.
[0170] Gel Electrophoresis (SDS PAGE) and Transfer Blot: 20 .mu.l
of each of the prepared samples (equivalent to 10 .mu.l of kinase
reaction mixture) was added into the lane wells of a 12% SDS Ready
Gel (BioRad, Herts, UK) and run with a standard Tris-Glycine
running buffer for 45 minutes at 180 V.
[0171] The gel was equilibrated in standard Tris-Glycine-Methanol
transfer buffer for 5 minutes at room temperature, before transfer
to nitrocellulose membrane using the BioRad mini blot apparatus at
100 V for 60 minutes.
[0172] Membrane Probing: The membrane was blocked with Pierce
Superblock.RTM. (IL, USA) blocking buffer for 1 hour at room
temperature. Primary antibodies used were supplied by Promega
(Wisconsin, USA) for MAPK, p38 and JNK, and by UBI for
anti-phosphorylated MBP. Antibody dilutions were made in
Superblock.RTM. diluted 10 fold in distilled water. The primary
antibody was used at 1:10000, and where appropriate secondary
antibodies at 1:20000. The HRP conjugated detection reagent was
used at 1:10000 for detection of the HRP protein marker.
[0173] Chemiluminescent Detection: Probed membranes were incubated
with 10 mls of SuperSignal.RTM. West Pico (Peirce, Ill. USA) for 2
minutes with slight agitation. The probed blots were then exposed
to Hyperfilm (Amersham, Bucks, UK) for 20 seconds. Phosphorylated
targets were compared against the molecular weight markers for
identification.
[0174] Results
[0175] FIG. 19 shows the effect of SAPK3 activity on MBP after 30
minutes at 30.degree. C. In this experiment, the assays were run in
tubes as described previously. For determination of ATP levels, a
201 aliquot of sample was removed into duplicate wells of a 96 well
luminescent compatible plate, and the remainder used for Western
blotting analysis. The blot was probed using an antibody to
phosphorylated MBP (Upstate Biotechnology).
[0176] The above experiment was repeated using SAPK4 and a number
of different supplies of MBP. The results are shown in FIG. 20, and
highlight that some proteins, even when used at the same
concentrations gave different results with both the bioluminescent
assay and Western blotting. The blots were again probed with the
same anti-phosphorylated MBP. As the results show, one of the
batches of MBP used (CB) had no effect in either detection assay.
Two different samples of MBP were used from Upstate Biotechnology,
one was the dephosphorylated form (DePhos) and the other standard
MBP (UBI). The bioluminescent data suggested that for the same
amount of protein used (2.72 .mu.M), the dephosphorylated form was
more efficient in the SAPK4 assay. This could not be determined
from the immunoblotting, confirming that bioluminescence is a more
sensitive and quantitative assay for kinase activity.
[0177] Summary
[0178] The studies described in Examples 1 to 13 above demonstrate
the versatility of the methods and kits of the present
invention.
[0179] In particular, the methods of the invention may be used to
study a number of different protein kinase/substrate combinations.
The protein kinase enzymes used in the above examples included:
[0180] (i) JNK-land JNK-2 in the presence of c-jun peptide as
substrate;
[0181] (ii) MAPK-1/ERK-1 and MAPK-2/ERK-2 with myelin basic protein
as substrate;
[0182] (iii) MEK-1 with inactive MAPK-2 as substrate;
[0183] (iv) JNK2.alpha.2 with ATF-2 and c-jun as substrates;
[0184] (v) SAPK-3 with myelin basic protein as substrate; and
[0185] (vi) SAPK-4 with myelin basic protein as substrate;
[0186] In addition, comparisons were made between the activated and
inactive forms of both MAPK-1/ERK-1 and MAPK-2/ERK-2 in the
presence of myelin basic protein.
[0187] The results from these experiments show that it was possible
to detect kinase activity in the presence of ATP and the
appropriate enzyme substrate, through a decrease in detectable ATP,
an increase in measured ADP, and also accelerated signal decay in
the presence of luciferase.
[0188] Several advantages of the assays of the invention are
summarised in the following points.
[0189] The assay can be applied to any protein kinase that cleaves
a phosphate from ATP.
[0190] The protein kinase activity could be allowed to go to
completion, prior to detection of ATP and/or ADP.
[0191] The assay could be performed in the presence of the ATP
monitoring reagent, with protein kinase activity being determined
as a drop in light output, together with an increase in signal
decay.
[0192] Changes in adenylate nucleotides showed a concentration
dependent effect with variations in enzyme, substrate and ATP
concentrations.
[0193] The drop in light signal correlates with protein
phosphorylation.
[0194] The methods can be used to detect and study protein kinase
inhibitors.
[0195] The methods can be used to study protein kinase cascade
systems.
[0196] The assays could be performed at room temperature or
30.degree. C.
[0197] Changes in adenylate nucleotides could be detected in the
presence or absence of a stop solution, permitting mass screening
of samples. By using an appropriate buffer (e.g. Hepes), it was
also possible to stop the protein kinase reaction with 2%
phosphoric acid and detect the reduced amount of ATP as a result of
protein kinase activity.
[0198] The methods can be used with a number of different protein
kinase buffers
[0199] The ATP detection reagent can be used in either Tris acetate
or Hepes buffers.
[0200] The assay can be supplied as a kit. Different kits could be
supplied for measurement of drop in ATP, with or without the use of
a stop reagent. The kits could also contain the ADP converting
reagent (as outlined in appendix 1), for detecting an increase in
ADP as a result of protein kinase activity.
[0201] Applications of the Methods of the Invention
[0202] The methods and assays of the present invention, as
described in the above examples can be used as a measure of kinase
activity in cell free systems. This is of particular importance in
the pharmaceutical industry since it enables the methods and assays
to be used in high throughput screening laboratories, e.g. for
identification of drugs that can act as kinase modulators,
especially inhibitors. For this application, the assays can be
carried out exactly as described in the above examples.
[0203] The methods and assays of the invention may also be used to
determine kinase activity in cellular extracts, and to determine
the effect of modulators of cellular kinase activity. To perform
these experiments, it is possible to substitute the known kinase in
the above examples for the cell extract/supernatant. The substrate
is added as described, and any changes in kinase activity in cells
treated with inhibitors/activators can be detected.
[0204] There is an increasing number of protein kinases that are
being targeted for the development of new tumour therapeutics. As
this list increases it will become impractical and extremely
expensive to use specific tests for each kinase. The assays and
kits of the present invention allow for the detection of a wide
spectrum of kinases and provide a common end point detection system
for all kinases. This allows for greater ease of use, particularly
in high throughput screening laboratories, where robots can be set
up with all the detection reagents and the various kinases and
inhibitors, of interest, and with opaque white (or black)
luminometer microtitre plates.
[0205] Moreover, the use of a stop solution allows for a large
number of plates to be batched up (i.e. stored) prior to analysis
of ATP and/or ADP levels.
Example 14
Study of Kinase Inhibitors
[0206] Staurosporine
[0207] We initially chose to look at a broad spectrum kinase
inhibitor staurosporine (Calbiochem). Firstly, we tested the
inhibitor (in DMSO) on the ATP detection reagent to ensure that the
inhibitor would not affect the luciferase-luciferin reaction.
[0208] FIG. 21 shows the effect of staurosporine on the
bioluminescent detection system. The results are presented as the
means of triplicate wells .+-.SD.
[0209] Data show that, even at the highest concentration of the
inhibitor, there was no significant effect upon light output. The
experiment was performed using 10 .mu.M ATP in 100 .mu.l volumes in
a 96 well plate with the addition of 20 .mu.l of ATP detection
reagent.
[0210] The effect of staurosporine was tested on the JNK2.alpha.2
enzyme with ATF-2 as substrate, and as the following figure shows
the expected inhibitory activity could be detected using the
bioluminescent protein kinase assay system.
[0211] FIG. 22 shows the effect of two different staurosporine
concentrations on JNK2.alpha.2 activity. The lower concentration
had little effect, however 5 .mu.M caused approximately 50%
inhibition after 30 minutes at 30.degree. C.
[0212] The above assay was performed in 200 .mu.l volumes in
plastic tubes in a 30.degree. C. waterbath. At each time point 20
.mu.l samples were removed from the tubes and added to wells of a
96 well plate, followed by the addition of 20 .mu.l of ATP
detection reagent. The final concentration of ATP used was 12.5
.mu.M, with JNK2.alpha.2 at 1.25 .mu.M and ATF-2 at 2.55 .mu.M.
[0213] Genistein
[0214] We also investigated the effect of genistein (Calbiochem) on
MAPK-1 activity with MBP as substrate.
[0215] The assay buffer used was the same as for the SAP kinases
(see above). The assay was performed in 100 .mu.l volumes in a 96
well microtitre plate at 30.degree. C. for 30 minutes. MBP
(Calbiochem) was used at the same concentration as previously
described (2.72 .mu.M), with the activated MAPK1/ERK1 (UBI) used at
a final concentration of 2.5 U per 100 .mu.l reaction volume. The
inhibitor was added at concentrations of 0, 0.1, 0.25, 0.5 and 1.0
.mu.M, with 10 .mu.l being added per well in 0.1% (v/v) DMSO.
[0216] For completeness, control samples with and without DMSO
(0+DMSO and 0-DMSO, respectively) were also analysed to confirm
that there was no effect of DMSO on the performance of the
assay.
[0217] FIG. 23 shows the effect of increasing concentrations of
Genistein (in .mu.M) on MAPK-1 activity with MBP as substrate
(results are presented as the means of duplicate wells .+-.SD).
Specifically, the data showed an effect of genistein at 500 nM,
with increased inhibitory activity at 1 .mu.M.
[0218] PD098059
[0219] We also investigated the effect of the selective inhibitor
PD098059 on the raf-1 activation of inactive MEK-1.
[0220] With PD098059, there was a concentration dependent increase
in the light output in the presence of the inhibitor indicating
reduced kinase activity.
[0221] FIG. 24 shows the effect of two different concentrations of
PD098059 on raf-1 activity (results are the means of triplicate
wells .+-.SD). These data confirmed the suitability of the assay
for determination of kinase inhibitory activity.
[0222] This experiment also provides evidence of the versatility of
methods and assays of the present methods since it was possible to
detect activity of another kinase/substrate system, namely
raf-1/inactive MEK-1.
[0223] Appendix 1
[0224] ATP Detection Reagent (ADR) Formulation
5 Reconstituted ADR Magnesium acetate 20 mM Sigma Tetrasodium
pyrophosphate 8 .mu.M Sigma Bovine Serum Albumin 0.32% w/v Sigma
D-Luciferin 712 .mu.M ConCell L-Luciferin 17.8 .mu.M ConCell
Luciferase 17 nM Europa Bioproducts Dextran 3 mg ml.sup.-1 Sigma
Tris 40 mM Sigma EDTA 800 .mu.M Sigma
[0225]
6 Final Reaction Concentrations Magnesium acetate 2.36 mM
Tetrasodium pyrophosphate 236 nM Bovine Serum Albumin 0.009% w/v
D-Luciferin 21 .mu.M L-Luciferin 525 nM Luciferase 500 pM Dextran
88.5 g ml.sup.-1 Tris 1.18 mM EDTA 23.6 .mu.M
[0226]
7 Tris Acetate (TA) buffer (sufficient for 1 liter) Tris 12.1 g
Sigma EDTA 0.744 g Sigma
[0227] 0.1M Tris, 2 mM EDTA adjust to pH 7.75 with glacial acetic
acid.
8 Hepes Buffer 200 mM (sufficient for 1 liter) EDTA 0.744 g Sigma
Hepes 47.6 g Sigma
[0228] Adjust to pH 7.75 with glacial acetic acid
[0229] UBI Buffer (as per UBI data sheet)
[0230] 20 mM MOPS pH7.2
[0231] 25 mM .beta.-glycerol phosphate
[0232] 5 mM EGTA
[0233] 1 mM sodium orthovanadate
[0234] 1 mM dithiothreitol
9 JNK Assay Buffer (formulation for .times. 10 concentrate) 250 mM
Hepes, pH 7.5 Sigma 1.5M Sodium Chloride Sigma 200 mM Magnesium
Chloride Sigma 0.01% Tween 20 Sigma
[0235] At time of use there is the addition of 20 mM dithiothreitol
(Sigma) and 150 .mu.M ATP (Sigma).
10 ADP Converting Reagent (sufficient for 600 mls) Pyruvate kinase
(50 000 Units) 20 ml Calbiochem 1M Phosphoenol pyruvate (monosodium
salt) 10 ml Sigma 2M Potassium Acetate 100 m Sigma Tris acetate
buffer pH7.75 470 ml
[0236]
11 Final Concentrations: Reaction Stock Mixture PK 7.6 U/ml 0.8
U/ml PEP 1.67 mM 175 nM Potassium acetate 33 mM 3.5 mM
[0237] Appendix 2: Suppliers
[0238] Berhold Detection systems GmbH
[0239] Bleichstrasse 56-58
[0240] D-75173 Pforzheim
[0241] Germany
[0242] Biltrace Ltd
[0243] The Science Park
[0244] Bridgend
[0245] CF31 3NA
[0246] Calbiochem-Nobabiochem (UK) Ltd
[0247] Boulevard Industrial Park
[0248] Padge Road
[0249] Beeston
[0250] Nottingham NG9 2JR
[0251] ConCell BV
[0252] Wevelinghoven 26
[0253] Nettetal
[0254] D-41334
[0255] Germany
[0256] Europa Bioproducts Ltd
[0257] Europa House
[0258] 15-17 North Street, Wicken
[0259] Ely, Cambridge
[0260] CB7 5XW
[0261] Fahrenheit Lab Supplies
[0262] Northfield Road
[0263] Rotherham
[0264] Dynex Labsystems
[0265] Action Court
[0266] Ashford Road
[0267] Ashford
[0268] Middlesex TW15 1XB
[0269] Labsystems Oy
[0270] Sorvaajankatu 15
[0271] Helsinki
[0272] Finland
[0273] 00810
[0274] Perkin Elmer Life Sciences
[0275] Perkin Elmer House
[0276] 204 Cambridge Science Park
[0277] Cambridge CB4 0GZ
[0278] Sarstedt
[0279] 68 Boston Road
[0280] Beaumont Leys
[0281] Leicester LE4 1AW
[0282] Sigma-Aldrich Co Ltd
[0283] Fancy Road
[0284] Poole
[0285] Dorset BH12 4QH
[0286] Upstate Biotechnology Inc. (UBI)
[0287] 199 Saranac Avenue
[0288] Lake Placid
[0289] NY 12946
* * * * *