蛋白酪氨酸激酶活性测定

Protein Tyrosine Kinase Assay Kit

Cat. No. 539700

Introduction

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32P-ATP followed by separation of the 323 SHSWLGH SURGXFW IURP WKH XQUHDFWHG

32P-ATP via the streptavidin membrane still demands an intensive and inconvenient washing procedure.

CALBIOCHEM®’s Protein Tyrosine Kinase Assay Kit is based on a process that combines affinity binding and ultrafiltration separation to analyze a mixture sample.

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32P-ATP. Separation by such affinity ultrafiltration process is easy, gentle, and efficient. Separation occurs in solution relatively free of nonspecific background. In addition, the ultrafiltration separation is performed in a contained and compact system that eases the handling of the radioactive sample and minimizes the radioactive waste. The sample reservoir of the centrifugal ultrafiltration unit containing the 32P-peptide product as retentate can be directly placed into a liquid scintillation vial for counting.

CALBIOCHEM®’s Protein Tyrosine Kinase Assay Kit has been carefully formatted to ensure its assay performance and user-friendliness. This kit uses biotinylated EGF receptor peptide (RENAEYLRVAP) as a substrate (Pearson, et al. 1991, Methods in Enzymology200, 62-81). The Protein Tyrosine Kinase Assay Kit should be used to assay activity of EGF receptor or related PTKs that can phosphorylate the peptide substrate, or potency of an activator or inhibitor of the PTKs. Users who wish to assay other PTKs or other protein kinases with specific peptide substrates and kinase reaction buffers should choose our Universal Protein Kinase Assay Kit (Cat. No. 539551).

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DRUG USE. MSDS AVAILABLE UPON REQUEST.

Kit Components and Storage

Materials Provided Quantity

Store at –20º C - Ship on Dry Ice

10X (0.15 mM) ATP Solution

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10X PTK Reaction Buffer: 200 mM magnesium chloride,

10 mM manganese chloride, 2 mM EGTA, 80 mM beta

glycerophosphate, and 80 mM imidazole hydrochloride,

pH 7.3

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50% Trichloroacetic Acid Solution

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Neutralization Solution

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Wash Solution50 ml

Store at Room Temperature

Centrifugal Ultrafiltration Units100 units

All the kit components are stable for one year from the date of purchase if they are handled and stored correctly.

Items Needed but not Provided with the Kit

(1)PTK Sample: This can be a purified enzyme preparation or cellular/tissue extract. It is advisable to avoid or limit other

PTKs in the sample that may act on the peptide substrate. Caution should also be exercised with regards to the effects of impure proteases and phosphatases in the sample. Purified PTKs that may serve as positive controls for the assay are also available from CALBIOCHEM® such as Epidermal Growth Factor Receptor tyrosine Kinase, Human A431 Cells (Cat. No. 324858).

(2)Test Substance: This is an optional item depending on whether an activator or inhibitor is to be tested. The

concentration of Test Substance should be made as appropriate according to its potency in modulating the activity of PTK.

(3)32P-ATP should have a specific activity of about 3,000

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(4)Deionized Water

(5)Microcentrifuge

(6)Scintillation vials with an inner diameter larger than 17 mm to accommodate the sample reservoir of Centrifugal

Ultrafiltration Unit.

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DRUG USE. MSDS AVAILABLE UPON REQUEST.