Tyrosine Kinase Activity Assay Kit

For 96 Assays

Catalog No. SGT410

FOR RESEARCH USE ONLY Not for use in diagnostic procedures

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Introduction

Protein Tyrosine Kinases (PTKs) perform a critical role in signal transduction pathways that control cell proliferation, differentiation, metabolism and apoptosis (1, 2). Phosphorylation of proteins by PTKs is essential for the regulation of these biological mechanisms and defects in these pathways may result in a number of human diseases, including cancer (1, 2). The important aspect of these enzymes in cellular regulation is accentuated by the fact that for a large number of kinases a corresponding viral oncogene product has been identified.

Protein tyrosine kinase activity is often associated with membrane receptor protein tyrosine kinases (e.g. EGF-, PDGF-, CFS-, IGF-1 and insulin receptor) and soluble non-receptor tyrosine kinases (e.g. p60c-Src, yes, lck, lyn, fyn) (3, 4).

Assaying of PTK activity allows for purification and characterization of protein tyrosine kinases, elucidation of their biological functions as well as aiding in development of specific PTK inhibitors. CHEMICON’s non-radioactive Tyrosine Kinase Activity Assay provides a simple, convenient and specific method for quantification and comparative determination of a wide range of PTKs. Testing of soluble and receptor tyrosine kinases, in vitro inhibitor screening and the study of PTK regulation can be performed with this assay. Our assay is immunoprecipitation compatible and will not cross-react with serine/threonine kinases.

Test Principle

CHEMICON’s non-radioactive Tyrosine Kinase Activity Assay Kit consists of a synthetic Biotinylated Peptide Substrate, a Biotinylated Phosphopeptide, purified Phosphotyrosine specific monoclonal antibody conjugated to horseradish peroxidase (HRP) and other components required to perform 96 ELISA-based assays.

The synthetic Biotinylated Substrate, poly [Glu:Tyr], 4:1 contains multiple tyrosine residues and can be phosphorylated by a wide range of PTKs. After quenching the enzyme reaction with an inhibitor, both the phosphorylated and dephosphorylated substrates are immobilized by binding to the streptavidin-coated plate. The fraction of phosphorylated substrate is visualized using a phosphotyrosine monoclonal antibody conjugated to HRP and an ensuing chromagenic substrate reaction. The quantity of phosphate incorporated into the tyrosine kinase substrate is determined utilizing the phosphopeptide standard curve.

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Figure 1: Test Principle

The CHEMICON Tyrosine Kinase Activity Assay Kit is intended for research use only; not for diagnostic or therapeutic applications. Reagents and materials supplied in the Tyrosine Kinase Activity Assay Kit are sufficient for 96 reactions (including STD curve and controls).

Kit Components

1. Streptavidin Coated Plate (Part No. 90190): One 96-well plate with 12 strips.

2. Protein Tyrosine Kinase Substrate (Part No. 90191): One vial contains 10 µg of biotinylated poly [Glu:Tyr] 4:1, MW: 3884.8 (lyophilized).

3. Phosphopeptide Standard (Part No. 90192): One lyophilized vial contains 10 µg of biotinylated phosphopeptide, one phosphate group per molecule, MW: 1826 (lyophilized).

4. ATP/MgCl2 Solution (Part No. 90194): One 1.5 mL vial containing 5 mM ATP, 50 mM MgCl2 in TBS (25 mM Tris, 0.15 M NaCl) pH 7.2, and 0.01% thimerosal added as preservative.

5. HRP-conjugated Mouse anti-Phosphotyrosine, clone PY20 (Part No. 90193): One 50 µL vial.

6. Blocking Buffer (Part No. 90195): One 50 mL bottle (Ready to Use).

7. Wash Buffer (Part No. 60245): One 100 mL bottle (10X).

8. TMB Substrate Solution (Part No: 90028): One 12 mL bottle (Ready to Use).

9. Stop Solution (Part No: 60193): One 12 mL bottle (Ready to Use).

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Materials Required But Not Supplied

1. Tyrosine Kinase Assay Buffer: Each individual tyrosine kinase requires different conditions for it to be optimally active. Therefore, the composition of the assay buffer (buffer substance, pH, stabilizers) depends largely on the specific tyrosine kinase of interest (refer to literature) (5, 6). In all assay protocols, a reducing agent (e.g. β-mercaptoethanol, DTT) is essential. To prevent phosphatase activity, 10-100 µM sodium orthovanadate is recommended (7).

2. Tyrosine Kinase Inhibitor: Kinase inhibitors are required for stopping the kinase reaction after incubation with the peptide substrate. Several kinase inhibitors are available, for example EDTA and Piceattanol.

3. Lysis Buffer: Such as RIPA buffer: 50 mM Tris, pH 8.0, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, 100 µg/mL PMSF*, 1 µg/mL Aprotinin*, 2 µg/mL Leupeptin*, 100 µM Sodium Vanadate.

Note: Protease Inhibitors (PMSF*, Aprotinin*, Leupeptin*) should be added freshly from stock solution.

4. Microcentrifuge and tubes

5. 30°C Water Bath

6. 37°C Incubator

7. 96-well Microplate Reader

Preparation of Reagents

1. Protein Tyrosine Kinase Substrate. Reconstitute contents of PTK substrate vial with 1 mL of deionized water for a concentration of 10 µg/mL. Reconstituted substrate should be stored at -20°C in small aliquots. Avoid freeze-thaw cycle of the reconstituted substrate.

2. Phosphopeptide Standard. Reconstitute contents of Phosphopeptide Substrate vial with 1 mL of deionized water for a concentration of 10 µg/mL. Reconstituted substrate should be stored at -20°C in small aliquots. Avoid freeze-thaw cycle of the reconstituted phosphopeptide standard.

3. HRP-conjugated Mouse anti-Phosphotyrosine. Immediately before use dilute the HRP conjugated antibody 1:500 with Blocking Buffer. Do not store diluted solutions.

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4. Wash Buffer (10X). Dilute wash buffer 1:10 with distilled water for a 1X Wash Buffer.

Sample Preparation

Various extraction and purification methods can be utilized to isolate protein tyrosine kinases from blood, solid tissue or cell culture. The following protocols are provided as EXAMPLES of suitable methods. It is strongly advised that an initial experiment should be performed to determine the proper dilution of the crude sample or purified tyrosine kinase to be used in subsequent experiments.

Solid Tissue Extracts:

1. Homogenize fresh tissue (brain, liver, etc.) one to four volumes of a pre-chilled detergent lysis buffer (RIPA buffer).

2. Centrifuge the homogenate at 12,000 x g for 10 minutes at 4°C to pellet the insoluble fraction.

3. Remove clear supernatant carefully from pellet. This supernatant is your total protein extract. It should be stored at –70°C.

Note: Every freeze-thaw cycle will decrease the enzymatic activity of protein extract.

Cell Culture Lysates:

1. Remove cell media and wash once with pre-chilled PBS.

2. Adherent cells: Add 1 mL/100 mm dish of the pre-chilled detergent lysis buffer. Place culture dish on ice for 10 minutes. Harvest cells with rubber policeman and collect cell lysate by centrifugation at 12,000 x g for 10 minutes.

Suspension cells: Place cells on ice. Add 1 mL/107 cells of pre-chilled detergent lysis buffer and lyse resuspended cells using either a homogenizer, sonication, or three cycles of freezing and thawing.

3. Transfer extracts to microcentrifuge tubes and centrifuge at 12,000 x g for 10 minutes.

4. Collect clear lysate and store at –70°C. This supernatant is your cytosolic fraction.

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Immunoprecipitation:

1. Add 10-20 µL of the PTK antibody to 0.5-1 mL of cell lysate. Incubate 1-12 hours at 4°C on a shaking or rocking platform.

2. Add 50-100 µL of protein A and/or G agarose beads (50% slurry) and incubate for 1-4 hours at 4°C on a rocking/shaking platform.

3. Collect protein A/G - antigen - antibody complexes by centrifugation at 2000 x g for 1 minute at 4°C.

4. Remove supernatant carefully and add 1 mL cell lysis buffer to the beads, resuspend and incubate for 10 minutes at 4°C on a rocking/shaking platform.

5. Collect protein A/G – antigen - antibody complexes by centrifugation at 2000 x g for 1 minute at 4°C.

6. Repeat steps 4 and 5 once more.

7. Repeat steps 4 and 5 once more, using the appropriate kinase assay buffer instead of cell lysis buffer.

8. Remove supernatant carefully. The precipitated protein can be used directly for the kinase assay (see Assay Protocol).

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Assay Protocol

I. Phosphopeptide Standard Curve:

Table 1: Preparation of Phosphopeptide Dose Curve

Phosphopeptide

Substrate (10 µg/mL)

Peptide Substrate

(10 µg/mL) H2O Total

Volume

1 8µL 2µL 50µL 60µL

2 6µL 4µL 50µL 60µL

3 4µL 6µL 50µL 60µL

4 2µL 8µL 50µL 60µL

5 1µL 9µL 50µL 60µL

6 0.5µL 9.5µL 50µL 60µL

7 0.25µL 9.75µL 50µL 60µL

8 0µL 10µL 50µL 60µL

1. Label 8 eppendorf tubes and pipette of each diluted component the specific volume into the specifically labeled tube, according to Table 1.

2. Vortex the tubes well and transfer 50 µL of each tube to a well on the streptavidin plate and incubate at 37°C for 30 minutes.

3. Proceed to step 5 in the below protocol of Tyrosine Kinase Assay.

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II. Tyrosine Kinase Assay:

Table 2: Composition of reaction mix

Peptide Substrate (10

µg/mL

)

5X A

ssay B

uffer

PTK

(Solution)

PTK

(Imm

uno-com

plex)

Inhibitor

DI

Water

Total V

olume

Negative Control 10 µL 10 µL _ _ _ 20 µL 40 µL

Kinase Auto-phosphorylation Control

_ 10 µL 1 part* _ _

Add up to 40 µL

40 µL

Kinase Assay (PTK in solution)

10 µL 10 µL 1

part* _ _ Add up to 40 µL

40 µL

Kinase Assay (PTK in immunocomplex)

10 µL 10 µL _ 1

part* _ 20 µL 40 µL

Protein Kinase Inhibitor

10 µL 10 µL 1

part* _ 1 part*

Add up to 40 µL

40 µL

*(1 part) is stated because the exact microliter volume added will depend upon the activity of the enzyme or inhibitor or the amount of material used in IP, and this amount should be optimized for the best signals.

1. Prepare the assay mixture according to Table 2 and start the reaction by adding 10 µL of 5X ATP/MgCl2 solution.

2. Following the composition of the reaction mix is an incubation period at 30°C. The phosphorylation reaction time will vary from a few minutes to 60 minutes depending on the individual tyrosine kinase, a recommended starting reaction time is 30-45 minutes.

3. Terminate enzyme reaction by adding 10 µL of kinase inhibitor, such as 120 mM EDTA.

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4. Transfer 50 µL of reaction mix to Streptavidin-coated strip wells and incubate at 37°C for 30 minutes.

5. Wash four times with 1X Wash Buffer. A thorough washing of the plate is important to reduce background. We recommend using a multi-channel pipette to fill each well with 250 µL of 1X Wash Buffer. Fluid removal from the wells is best accomplished by inverting the plate over a sink and flicking the fluid out of the wells and then blotting the plate on clean paper towels.

6. Add 200 µL of Blocking Buffer Solution to each well and incubate at 37°C for 30 minutes.

7. Discard Blocking Buffer Solution and add 100 µL of diluted mouse anti-PY20-HRP to each reaction well. Incubate for 1 hour at room temperature on a shaking platform.

8. Wash four times with 1X Wash Buffer according to step 5 above. 9. Warm TMB substrate to room temperature. Add 100 µL of TMB

Substrate to each well. Incubate at room temperature for 5 – 15 minutes (time determined by end user).

10. Stop the enzyme reaction by adding 100 µL of Stop Solution into each well, including the control wells. Results should be read immediately (color will fade over time).

11. Read absorbance of each microwell on a standard microplate reader using 450 nm as the primary wave length.

Note: Background reading of control samples should be subtracted from the readings of the kinase reaction samples before calculating the tyrosine kinase activity.

Storage

Store kit materials at 2-8°C for up to their expiration date.

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Calculation of Results

Relative optical density (OD) values obtained with the CHEMICON Tyrosine Kinase Activity Assay Kit may be compared with known standards or other test samples to obtain relative activities. All of the following charts illustrate typical results upon using either purified or immunoprecipitated p60c-Src tyrosine kinase. Incubation time of TMB substrate is 5 minutes. It is always recommended to run a PTK standard curve.

One should use the data below for reference only. This data should not be used to interpret actual assay results.

Figure 1: Phosphopeptide Standard Curve

0

0.5

1

1.5

2

2.5

0 20 40 60 80

Phosphopeptide (ng)

OD

450

nm

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Figure 2: Example of Protein Tyrosine Kinase Activity Assay

Activity of p60c-Src Kinase was determined as described in the Assay Protocol. Kinase reaction time is 30 minutes.

Unit definition: One unit is the amount of p60c-Src tyrosine kinase that will transfer 1 pmole phosphate/min to the biotinylated poly [Glu:Tyr] peptide at saturated substrate concentrations, pH 7.4 at 30 °C.

Purified Src Tyrosine Kinase Activity Assay

0

0.5

1

1.5

0.01 0.1 1 10

Src Kinase (Units)

OD

450

nm

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Figure 3: Time dependence of Protein Tyrosine Kinase Assay.

p60 c-Src (3 Units) was incubated with the biotinylated poly [Glu:Tyr] peptide substrate for different time periods at 30°C and activity was detected as described in the Assay Protocol.

0

0.4

0.8

1.2

1.6

0 20 40 60

Time (min)

OD

450

nm

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Figure 4: p60 c-Src kinase activity in A431 cell lysate

Serum-starved A431 cells were stimulated with 100 ng/ml EGF for 1 minute. Cells were lysed in RIPA buffer and cell lysates were immunoprecipitated with an anti-p60 c-Src antibody. Activity of c-Src Kinase was determined as described in the Assay Protocol. Kinase reaction time is 60 minutes.

Activity of Src Immunoprecipitated from A431 Lysate

0

0.5

1

1.5

2

2.5

3

Buffer Ctrl A431 Ctrl A431, EGFTreated

OD

450

nm

13

Figure 5: Inhibition of Protein Tyrosine Kinase Activity

p60 c-Src (3 Units) was preincubated with different concentrations of the PTK inhibitor, Genistein for 10minutes at room temperature, then assayed as described in the Assay Protocol. Kinase reaction time is 30 minutes.

0

25

50

75

100

0 52 104 208 417

Genistein (uM)

Rel

ativ

e A

ctiv

ity (%

)

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References

1. Hunter, T. (1995) Cell. 80, 225-236.

2. Pawson, T. (1995) Nature. 363, 573-580.

3. Cheng H-C et al. (1992) J Biol Chem. 267, 9248.

4. Borowski P et al .(1994) J Biochem. 115, 825-829.

5. Hunter T and Sefton BM. (Eds.) (1991). Methods in Enzymology. Volume 200.

6. Hunter T and Sefton BM. (Eds.) (1991). Methods in Enzymology. Volume 201.

7. Gordon JA. 1991. Methods Enzymol. 201, 477.

Warranty

These products are warranted to perform as described in their labeling and in CHEMICON literature when used in accordance with their instructions. THERE ARE NO WARRANTIES, WHICH EXTEND BEYOND THIS EXPRESSED WARRANTY AND CHEMICON DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CHEMICON’s sole obligation and purchaser’s exclusive remedy for breach of this warranty shall be, at the option of CHEMICON, to repair or replace the products. In no event shall CHEMICON be liable for any proximate, incidental or consequential damages in connection with the products.

2002 – 2003: CHEMICON International, Inc. - By CHEMICON International, Inc. All rights reserved. No part of these works may be reproduced in any form without permissions in writing.

Cat No. SGT410 July 2003

Revision C: 41290