Solid-phase tyrosine-specific protein kinase assay in multiwell substrate-immobilized polyacrylamide gel

Journal Article · Sun Oct 01 04:00:00 EDT 1989 · Analytical Biochemistry; (USA)

DOI:https://doi.org/10.1016/0003-2697(89)90714-8· OSTI ID:6983997

Sahal, D; Fujita-Yamaguchi, Y ^[1]

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1. Beckman Research Institute of the City of Hope, Duarte, CA (USA)

Since tyrosine-specific protein kinase (TPK) is much less abundant than Ser/Thr-specific kinases in cells, determination of TPK activity in crude cell extracts or column chromatography eluates has been difficult. This is compounded by the absence of a rapid, economical method for the separation of high endogenous protein phosphorylation background from exogenously added tyrosine-containing substrates. We have developed a new solid-phase assay, which provides high sensitivity and efficiency at a low cost for assaying the TPK activity of crude enzyme preparations. This assay utilizes immobilized tyrosine-containing synthetic polymers such as (Glu:Tyr, 4:1)n in polyacrylamide gels. The kinase reaction is started by adding crude enzyme solutions and (tau-{sup 32}P)ATP-metal ion mixtures into microtiter-size wells made in the gels. After the phosphorylation reaction, the reaction mixtures are removed and the gels are prewashed in water followed by electrophoresis to completely remove free radioactive ATP. {sup 32}P incorporation into the immobilized TPK-specific substrate can be detected by autoradiography and quantitated by cutting the gel pieces and counting them with a liquid scintillation counter. The simple, rapid method should facilitate screening of TPK inhibitors and activators as well as examining the substrate specificity of TPKs. Other enzymes, including Ser/Thr-specific protein kinases, can also be analyzed by this technique.

• OSTI ID: 6983997
• Journal Information: Analytical Biochemistry; (USA), Journal Name: Analytical Biochemistry; (USA) Vol. 182:1; ISSN ANBCA; ISSN 0003-2697
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
ATP
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CATTLE
CHEMICAL REACTIONS
CHROMATOGRAPHY
COUNTING TECHNIQUES
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ELECTROPHORESIS
ENZYME ACTIVITY
ENZYMES
IMMOBILIZED ENZYMES
ISOTOPES
LIGHT NUCLEI
MAMMALS
MAN
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PRIMATES
RADIOISOTOPES
RUMINANTS
SCINTILLATION COUNTING
SEPARATION PROCESSES
SUBSTRATES
TRANSFERASES
VERTEBRATES