CY-1162

CY-1162
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
Non-Radioisotopic Kit for Measuring Chk1, Chk2 and C-TAK1 Activities
CycLex Checkpoint Kinase Assay/Inhibitor
Screening Kit-1
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Intended Use................................................ 1
Storage......................................................... 1
Introduction ................................................. 2
Principle of the Assay.................................. 3
Materials Provided ...................................... 4
Materials Required but not Provided .......... 4
Precautions and Recommendations............. 5
Detailed Protocol......................................... 6-9
Evaluation of Results .................................. 10
Assay Characteristics .................................. 10
Troubleshooting .......................................... 10
Reagent Stability ......................................... 10
Example of Test Results.............................. 11-16
References................................................... 17
Related Products...........................................17
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Intended Use
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The CycLex Research Product CycLex Checkpoint Kinase Assay / Inhibitor Screening Kit-1 is
designed to measure the activities of purified checkpoint kinase enzyme such as Chk1, Chk2 and
C-TAK1 for the rapid and sensitive evaluation of checkpoint kinase inhibitors using recombinant
checkpoint kinases. The phospho-specific monoclonal antibody used in this assay kit has been
demonstrated to recognize the phospho-serine 216 residue in Cdc25C, which is phosphorylated by
checkpoint kinases.
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Applications of this kit include:
1) Screening inhibitors or activators of checkpoint kinases.
2) Detecting the effects of pharmacological agents on checkpoint kinases.
This assay kit is for research use only and not for use in diagnostic or therapeutic procedures.
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Storage
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• Upon receipt store all components at 4°C.
• Don’t expose reagents to excessive light.
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
Introduction
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Three different human Cdc25 family members exist with Cdc25A regulating the G1/S transition and
Cdc25B and Cdc25C involved in G2/M progression. Evidence suggests that two critical amino acids,
threonine 14 and tyrosine 15, located within the cyclin-dependent kinases represent the major target for
the Cdc25 family of protein phosphatases. Dephosphorylation of these two critical amino acid residues is
essential for proper cell cycle progression and the subsequent association of cyclin-dependent kinases
with their associated cyclins (1).
Given their crucial role in cell cycle progression and checkpoint control, the regulation of the activity
of the various Cdc25 family members has been the subject of numerous investigations. For the case of
Cdc25C, enzymatic activity has been demonstrated to be low during interphase, in part because the
phosphatase is phosphorylated on serine 216. In response to DNA damage, various intracellular kinases
including Chk1, Chk2 and C-TAK1 appear to phosphorylate Cdc25C on this residue (2–6).
One of the important functional consequences of phosphorylation of Ser-216 is to create a consensus
binding site for 14-3-3 protein binding (4). A variety of evidence suggests that in human cells, the
binding of 14-3-3 increases the cytoplasmic localization of the protein (7–9). In addition to 14-3-3
binding, Cdc25C is also actively transported from the nucleus through a leptomycin B-sensitive pathway
that requires an N-terminal nuclear export sequence (9).
Measurement of checkpoint kinases (Chk1, Chk2 and C-TAK1) activity
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The protocol generally regarded as most sensitive for the quantitative measurement of checkpoint
kinase activity involves incubation of the checkpoint kinase sample with substrate, either a natural or
synthetic polypeptide (such as Chktide substrate peptide), in the presence of Mg2+and 32P-labeled ATP.
The reaction is terminated by "spotting" a sample onto a filter paper disc, followed by immersion in acid
to precipitate the radiolabeled product. The filter papers are then washed extensively to remove
unincorporated radiolabel and the radioactivity counted. While sensitive, this method is labor-intensive,
generates hazardous radioactive waste and depends on a radioisotope of short half-life. It is particularly
unsuitable when kinase assays are only performed on an infrequent basis. The CycLex Checkpoint
Kinase Assay / Inhibitor Screening Kit-1 uses a peroxidase coupled anti-phospho-Cdc25C S216
monoclonal antibody as a reporter molecule in a 96-well ELISA format. This assay provides a
non-isotopic, sensitive and specific method to measure the activities of checkpoint kinases.
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
Principle of the Assay
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The CycLex Research Products CycLex Checkpoint Kinase Assay / Inhibitor Screening Kit-1 is a
single-site, semi-quantitative immunoassay for checkpoint kinase activity. Plates are pre-coated with a
substrate corresponding to recombinant Cdc25C, which contains serine residues that can be
phosphorylated by checkpoint kinases, including Chk1, Chk2 and C-TAK1.
The detector antibody specifically detects only the phosphorylated form of serine 216 residue on
Cdc25C. The CycLex Checkpoint Kinase Assay / Inhibitor Screening Kit-1 may be used to study the
kinetics of a purified or partially purified individual checkpoint kinase (Chk1, Chk2 and C-TAK1) as
well as to screening individual checkpoint kinase inhibitor. To perform the test, the sample is diluted in
Kinase Buffer, pipetted into the wells and allowed to phosphorylate the bound substrate following the
addition of Mg2+ and ATP. The amount of phosphorylated substrate is measured by binding it with a
horseradish peroxidase conjugate of 1F1, a anti-phospho-Cdc25C serine 216 specific antibody, which
then catalyzes the conversion of the chromogenic substrate tetra-methylbenzidine (TMB) from a
colorless solution to a blue solution (or yellow after the addition of stopping reagent). The color is
quantitated by spectrophotometry and reflects the relative amount of checkpoint kinases activity in the
sample. For kinetic analysis, the checkpoint kinase containing sample is added to the wells in a similar
fashion and at varying times the reaction is stopped by the addition of the chelator, sodium
ethylenediaminetetraacetate (EDTA) and the amount of phosphorylated substrate determined as before.
Summary of Procedure
Add 100 µL of sample to the wells
Incubate for 1hr at 30°C
Wash the wells
Add 100 µL of HRP conjugated anti-phospho-specific antibody
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Incubate for 1hr at room temp.
Wash the wells
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Add 100 µL of Substrate Reagent
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Add 100 µL of Stop Solution
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Measure absorbance at 450 nm
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
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Materials Provided
All samples and standards should be assayed in duplicate. The following components are supplied and
are sufficient for the one 96-well microtiter plate kit.
Microplate: One microplate supplied ready to use, with 96 wells (12 strips of 8-wells) in a foil, zip-lock
bag with a desiccant pack. Wells are coated with recombinant Cdc25C as Checkpoint kinases substrate.
10X Wash Buffer: One bottle containing 100 mL of 10X buffer containing 2 %Tween®-20
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Kinase Buffer: One bottle containing 20 mL of 1X buffer; used for Kinase Reaction Buffer and sample
dilution.
20X ATP: One vial of lyophilized ATP Na2 salt.
HRP conjugated Detection Antibody: One vial containing 20 mL of HRP (horseradish peroxidase)
conjugated anti-phospho-Cdc25C S216 (1F1) antibody. Ready to use.
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Substrate Reagent: One bottle containing 20 mL of the chromogenic substrate, tetra-methylbenzidine
(TMB). Ready to use.
Stop Solution: One bottle containing 20 mL of 1 N H2SO4. Ready to use.
Materials Required but not Provided
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• Recombinant Chk1, Chk2 and C-TAK1 positive control: Available from CycLex (Chk1 positive
control: Cat# CY-E1162-1, Chk2 positive control: Cat# CY-E1162-2 and C-TAK1: Cat#
CY-E1162-3); One vial containing 2 Units checkpoint kinase enzyme. Positive control should be
added to the first well at 10 m units/well. For instance, diluted positive control 1 m unit/µL, use 10 µL
for 1 assay. (Unused checkpoint kinase enzyme should be stored in aliquots at below -70°C.)
• 10X Staurosporine (10 µM): Staurosporine is available from Sigma, Cat#.S-4400. 1 mM stock
solution (DMSO) diluted 1:100 in Kinase Buffer
• Pipettors: 2-20 µL, 20-200 µL and 200-1000 µL precision pipettors with disposable tips.
• Precision repeating pipettor
• Wash bottle or multichannel dispenser for plate washing.
• Microcentrifuge and tubes for sample preparation.
• Vortex mixer
• Plate reader capable of measuring absorbance in 96-well plates at dual wavelengths of 450 nm/540
nm. Dual wavelengths of 450/550 or 450/595 nm can also be used. The plate can also be read at a
single wavelength of 450 nm, which will give a somewhat higher reading.
• 500 or 1000 mL graduated cylinder
• Reagent reservoirs
• Deionized water of the highest quality
• Disposable paper towels
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Precautions and Recommendations
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
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• Store the checkpoint kinase enzyme at below -70°C and the ATP at -20°C when not in use Store all
other components at 4°C. Do not expose reagents to excessive light. Avoid freeze/thaw cycles.
• Allow all the components to come to room temperature before use.
• All microplate strips that are not immediately required should be returned to the zip-lock pouch, which
must be carefully resealed to avoid moisture absorption.
• Use only the microtiter wells provided with the kit.
• Rinse all detergent residue from glassware.
• Do not mix reagents from different kits.
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• Use deionized water of the highest quality.
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• Do not use kit components beyond the indicated kit expiration date.
• The buffers and reagents in this kit may contain preservatives or other chemicals. Care should be taken
to avoid direct contact with these reagents.
• Do not mouth pipette or ingest any of the reagents.
• Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are
handled.
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• Dispose of tetra-methylbenzidine (TMB) containing solutions in compliance with local regulations.
• Avoid contact with Substrate Solution which contains hydrogen peroxide.
• Avoid contact with Stop Solution which contains Sulfuric Acid.
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• In case of contact with the Stop Solution and the Substrate Solution, wash skin thoroughly with water
and seek medical attention, when necessary.
• Biological samples may be contaminated with infectious agents. Do not ingest, expose to open
wounds or breathe aerosols. Wear protective gloves and dispose of biological samples properly.
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• CAUTION: Sulfuric Acid is a strong acid. Wear disposable gloves and eye protection when
handling Stop Solution.
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
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Detailed Protocol
The CycLex Checkpoint Kinase Assay / Inhibitor Screening Kit-1 is provided with removable
strips of wells so the assay can be carried out on separate occasions using only the number of strips
required for the particular determination. Since conditions may vary, running an aliquot of the
appropriate checkpoint kinase positive control (Cat# CY-E1162-1-3), separately available from CycLex,
should be included in each assay. Disposable pipette tips and reagent troughs should be used for all
transfers to avoid cross-contamination of reagents or samples.
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Preparation of Working Solution
1. Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer (provided) to
900 mL of ddH2O. Mix well. Store at 4°C for two weeks or -20°C for long-term storage.
2. Prepare 20X ATP Solution by adding 1.6 mL of ddH2O to the vial of 20X ATP (provided,
lyophilized). Mix gently until dissolved. The Final concentration of the 20X ATP Solution should be
1.25 mM. Store the solution in small aliquots (e.g. 100 µL) at -20°C.
Kinase Buffer (provided)
20X ATP Solution
Total
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3. Prepare Kinase Reaction Buffer by mixing following reagents.
96 assays
10 assays
1 assay
9.5 mL
0.5 mL
950 µL
50 µL
95 µL
5 µL
10 mL
1000 µL
100 µL
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You will need 80-90 µL of Kinase Reaction Buffer per assay well. Mix well. Discard any
unused Kinase Reaction Buffer after use.
Standard Assay
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1. Remove the appropriate number of microtiter wells from the foil pouch and place them into the well
holder. Return any unused wells to the foil pouch, refold, seal with tape and store at 4°C.
2. Prepare all samples (diluted with Kinase Buffer. as needed).
3. Duplicate wells containing 10 µL of checkpoint kinase positive control (10 m units) should be
included in each assay as a positive control for phosphorylation.
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4. Begin the kinase reaction by addition of 90 µL Kinase Reaction Buffer per well, cover with plate
sealer or lid, and incubate at 30°C for 60 minutes.
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5. Wash wells five times with Wash Buffer making sure each well is filled completely. Remove
residual Wash Buffer by gentle tapping or aspiration.
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6. Pipette 100 µL of HRP conjugated Detection Antibody into each well, cover with plate sealer or
lid, and incubate at room temperature (ca.25°C) for 60 minutes. Discard any unused conjugate
after use.
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
7. Wash wells five times with Wash Buffer making sure each well is filled completely. Remove
residual Wash Buffer by gentle tapping or aspiration.
8. Add 100 µL of Substrate Reagent to each well and incubate at room temperature (ca.25°C) for
5–15 minutes.
9. Add 100 µL of Stop Solution to each well in the same order as the previously added Substrate
Reagent.
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10. Measure absorbance in each well using a spectrophotometric plate reader at dual wavelengths of
450/540 nm. Dual wavelengths of 450/550 or 450/595 nm can also be used. Read the plate at 450
nm if only a single wavelength can be used. Wells must be read within 30 minutes of adding the
Stop Solution.
Kinetic Assays
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1. Remove the appropriate number of microtiter wells from the foil pouch and place them into the well
holder. Return any unused wells to the foil pouch, refold, seal with tape and store at 4°C.
2. Prepare all samples (diluted with Kinase Buffer. as needed).
3. Duplicate wells containing 10 µL of checkpoint kinase positive control (10 m units) should be
included in each assay as a positive control for phosphorylation.
4. Begin the kinase reaction by addition of 90 µL Kinase Reaction Buffer in duplicate per well in
timed intervals (suggested interval is 5 minutes but should be individually determined for each
system). After the final addition, cover with plate sealer or lid, and incubate at 30°C for 20
minutes.
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5. Stop the reaction by flicking out the contents. (Alternatively, the reaction may be terminated by the
addition of 150 µL 0.1 M Na EDTA, pH 8.0 to each well).
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6. Wash wells five times with Wash Buffer making sure each well is filled completely. Remove
residual Wash Buffer by gentle tapping or aspiration.
7. Pipette 100 µL of HRP conjugated Detection Antibody into each well, cover with plate sealer or
lid, and incubate at room temperature (ca.25°C) for 60 minutes. Discard any unused conjugate.
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8. Wash wells five times with Wash Buffer making sure each well is filled completely. Remove
residual Wash Buffer by gentle tapping or aspiration.
9. Add 100 µL of Substrate Reagent to each well and incubate at room temperature (ca.25°C) for
5-15 minutes.
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10. Add 100 µL of Stop Solution to each well in the same order as the previously added Substrate
Reagent.
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11. Measure absorbance in each well using a spectorphotometric plate reader at dual wavelengths of
450/540 nm. Dual wavelengths of 450/550 or 450/595 nm can also be used. Read the plate at 450
nm if only a single wavelength can be used. Wells must be read within 30 minutes of adding the
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
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Stop Solution.
Recommendations
Assay reagents
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Special considerations when screening activators and inhibitors
In order to estimate the inhibitory effect on individual checkpoint kinase activity in the test chemicals
correctly, it is necessary to conduct the control experiment of “Solvent control” at least once for every
experiment and “Inhibitor control” at least once for the first experiment, in addition to “Test sample”, as
indicated in the following table. When test chemicals cause an inhibitory effect on individual checkpoint
kinase activity, the level of A450 is weakened as compared with “Solvent control”. The high level of
A450 is not observed in “Inhibitor control” (usually A450<0.3).
Test sample
Kinase Reaction buffer
80 µL
10X Inhibitor or equivalent
10 µL
-
10X Staurosporine* (10 µM)
-
Inhibitor
control
80 µL
-
-
10 µL
-
-
10 µL
10 µL
10 µL
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Solvent for Inhibitor
Solvent
control
80 µL
CycLex checkpoint kinase (1 m unit/µL) **
or your enzyme fraction
10 µL
* Cat# S-4400: See Page 4, section “Materials Required but not Provided”
** Chk1 positive control: Cat# CY-E1162-1, Chk2 positive control: Cat# CY-E1162-2 and C-TAK1: Cat# CY-E1162-3:
See Page 4, section “Materials Required but not Provided”
1. Following the above table, add the Reagents to each well of the microplate. Finally, initiate reaction
by adding 10 µL of “CycLex checkpoint kinase” to each well and mixing thoroughly at room
temperature. Cover with plate sealer or lid, and incubate at 30°C for 60 minutes.
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2. Follow the Standard Assay steps 5-10, page 6-7.
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
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Special considerations when measuring precise Checkpoint kinase activity
In order to measure the activity of checkpoint kinase family correctly, it is necessary to conduct the
control experiment of “Inhibitor control” at least once for every experiment and “ATP minus control” at
least once for the first experiment, in addition to “No enzyme control” as indicated in the following table.
Although the level of A450 increases in “Test sample” when checkpoint kinase family enzyme activity is
in the sample, the high level of A450 is not observed in “Inhibitor control”, “ATP minus control” and
“No enzyme control”.
Inhibitor
control
80 µL
Kinase Buffer (provided)
-
-
10X Staurosporine (10 µM)*
-
10 µL
10 µL
-
10 µL
-
Your enzyme fraction
CycLex checkpoint kinase (1 m unit/µL) **
Buffer
ATP minus
control
-
Positive
control
90 µL
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Kinase Reaction buffer
Test
Sample
90 µL
Assay reagents
No enzyme
control
90 µL
90 µL
-
-
-
-
-
10 µL
-
10 µL
-
10 µL
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* Cat# S-4400: See Page 4, section “Materials Required but not Provided”
** Chk1 positive control: Cat# CY-E1162-1, Chk2 positive control: Cat# CY-E1162-2 and C-TAK1: Cat# CY-E1162-3:
See Page 4, section “Materials Required but not Provided”
1. Following the above table, add the Reagents to each well of the microplate. Finally, initiate the
reaction by adding 10 µL of “Your enzyme fraction” or “Buffer” to each well and mixing thoroughly
at room temperature. Cover with plate sealer or lid, and incubate at 30°C for 60 minutes.
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2. Follow the Standard Assay steps 5-10, page 6-7.
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
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Evaluation of Results
1. Average the absorbance values for the checkpoint kinase sample duplicates (positive control) and all
experimental sample duplicate values (when applicable). When checkpoint kinase positive control (10
m units/assay) is included as an internal control for the phosphorylation reaction, the absorbance value
should be greater than 1.0 with a background less than 0.15.
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2. For screening of purification/chromatography fractions, on graph paper, plot the mean absorbance
values for each of the samples on the Y-axis versus the fraction number on the X-axis to determine the
location of the eluted, purified individual checkpoint kinase.
3. For kinetic analysis, on graph paper, plot the mean absorbance values for each of the time points on
the Y-axis versus the time of each reaction (minutes) on the X-axis.
Assay Characteristics
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The CycLex Research Product Checkpoint Kinase Assay / Inhibitor Screening Kit-1 has been
shown to detect the activity of indicated checkpoint kinases in the column fractions of mammalian cell
lysates. The assay may be used to follow the purification of individual checkpoint kinase.
Troubleshooting
1. The CycLex checkpoint kinase should be run in duplicate, when a standard assay is being performed,
using the protocol described in the “Detailed Protocol”. Incubation times or temperatures
significantly different from those specified may give erroneous results.
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2. The reaction curve is nearly a straight line if the kinetics of the assay is of the first order. Variations in
the protocol can lead to non-linearity of the curve, as can assay kinetics of other than first order. For a
non-linear curve, point to point or quadratic curve fit methods should be used.
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3. Poor duplicates, accompanied by elevated values for wells containing no sample, indicate insufficient
washing. If all instructions in the “Detailed Protocol” were followed accurately, such results indicate
a need for washer maintenance.
4. Overall low signal may indicate that desiccation of the plate has occurred between the final wash and
addition of Substrate Reagent. Do not allow the plate to dry out. Add Substrate Reagent immediately
after wash.
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Reagent Stability
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All of the reagents included in the CycLex Research Product CycLex Checkpoint Kinase Assay /
Inhibitor Screening Kit-1 have been tested for stability. Reagents should not be used beyond the stated
expiration date. Upon receipt kit reagents should be stored at 4°C, except the ATP and checkpoint kinase
component must be stored at below -70°C. Coated assay plates should be stored in the original foil bag
sealed by the zip lock and containing a desiccant pack.
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For research use only, not for use in diagnostic or therapeutic procedures
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
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Example of Test Results
Fig.1-1 Dose dependency of recombinant Chk1 enzyme reaction
2.0
1.5
A450
A TP+
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1.0
A TP-
0.5
0.0
10
20
30
Chk1 dose (mUnit)
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Fig.1-2 Dose dependency of recombinant Chk2 enzyme reaction
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A450
1.00
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1.50
A TP+
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0.50
A TP-
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10
15
Chk2 dose (mUnit)
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
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Fig.1-3 Dose dependency of recombinant C-TAK1 enzyme reaction
2.0
1.0
A TP+
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A450
1.5
A TP-
0.5
0.0
5
10
15
20
25
30
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C-TAK dose (mUnit)
Fig.2-1 Time course of recombinant Chk1 enzyme reaction
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1.25
0.75
0.50
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A450
1.00
0.25
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0.00
15
30
45
60
Reaction Time (min.)
75
90
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
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Fig.2-2 Time course of recombinant Chk2 enzyme reaction
1.5
1.3
0.8
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A450
1.0
0.5
0.3
0.0
15
30
45
60
Reaction time (min.)
75
90
75
90
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Fig.2-3 Time course of recombinant C-TAK1 enzyme reaction
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1.50
1.25
0.75
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A450
1.00
0.50
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0.25
0.00
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30
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60
Reaction time (min.)
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
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Fig.3-1 Km for ATP (recombinant Chk1)
y = 51.643x + 70.388
3,000
2
R = 0.9981
2,500
1,500
1,000
Km for ATP : 1.36uM
500
0
0
10
20
30
Fig.3-2 Km for ATP (recombinant Chk2)
6,000
y = 46.186x + 153.2
5,000
R = 0.996
50
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2
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[S]
4,000
3,000
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[S/V]
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[S/V]
2,000
2,000
Km of ATP : 3.3uM
1,000
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0
20
40
[S]
60
80
100
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Fig.3-3 Km for ATP (recombinant C-TAK1)
6,000
y = 53.501x + 20.514
5,000
R = 0.9999
2
3,000
2,000
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[S/V]
[V/S]
4,000
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
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Km of ATP : 0.38uM
3.3uM
1,000
0
0
20
40
60
80
100
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[S]
Fig.4-1 Effect of broad-spectrum kinase inhibitor staurosporine on Chk1 activity
80.0
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60.0
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100.0
40.0
20.0
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Relative Intensity (% control)
120.0
0.0
20
40
60
80
100
Staurosporine conc. (nM)
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
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Fig.4-2 Effect of broad-spectrum kinase inhibitor staurosporine on Chk2 activity
100.0
80
60
40
20
0
0
0.25
0.5
0.75
80.0
60.0
40.0
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Relative Intens ity (% control)
Relative Intensity (% control)
100
20.0
0.0
0
1
Staurosporine conc. (µM)
5
10
15
20
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Stauros porine conc.(µM )
Fig.4-3 Effect of broad-spectrum kinase inhibitor staurosporine on C-TAK1 activity
100
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80
60
40
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Relative Intensity (% control)
120
20
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0
20
40
60
80
100
Staurosporine conc. (nM)
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References
1. Obaya, A. J., and Sedivy, J. M. Cell. Mol. Life Sci. 59, 126-142, 2002.
2. Walworth, N. C., Davey, S., and Beach, D. Nature 363, 368-371, 1993.
3. Walworth, N., and Bernards, R. Science 271, 353-356, 1996.
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Checkpoint Kinase Assay/Inhibitor Screening Kit-1
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
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4. Peng, C. Y., Graves, P. R., Thoma, R. S., Wu, Z., Shaw, A. S., and Piwnica-Worms, H. Science 277,
1501-1505, 1997.
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6. Sanchez, Y., Wong, C., Thoma, R. S., Richman, R., Wu, Z., Piwnica-Worms, H., and Elledge, S. J.
Science 277, 1497-501. 1997.
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8. Graves, P. R., Yu, L., Schwartz, J. K., Sausville, E. A., O'Conner, P. M., and Piwnica-Worms, H. J.
Biol. Chem. 275, 5600-5605, 2000.
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Ina, Nagano 396-0002
Japan
Fax: +81-265-76-7618
e-mail: [email protected]
URL: http://www.cyclex.co.jp
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CycLex/CircuLex products are supplied for research use only. CycLex/CircuLex products and
components thereof may not be resold, modified for resale, or used to manufacture commercial
products without prior written approval from CycLex Co., Ltd.. To inquire about licensing for
such commercial use, please contact us via email.
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Cat#: CY-1162
17
Version#: 140318
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