Rapid assays for protein kinase activity

Details Rapid assays for protein kinase activity Rapid assays for protein kinase activity

1994-04-08

1996-06-18

Wityshyn, Michael G.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving transferase

435 4, 435 21, 435194, 436164, 436172, 424 103, C12Q 148

Patent

active

055276881

ABSTRACT:
A method of measuring enzymatic activity of a protein kinase is disclosed. The method is an improvement to existing methodology which involves phosphorylating a peptide substrate, adsorbing the phosphorylated peptide to a solid phase, washing the phase to remove non-adsorbed constituents, and measuring the amount of phosphorylated peptide adsorbed to the phase. The disclosed improvement uses a membrane as the solid phase and positions the membrane within a chamber to separate the chamber into a first and a second region. Washing is accomplished with centrifugal force; the washed solution being forced through the membrane from the first region into the second region. Radioactive and non-radioactive assays are disclosed. The latter uses a support containing the Fe.sup.+3 ion chelated to the support through imminodiacetic acid groups. The peptide contains a dye and measurement is spectrophotometric or fluorometric.

REFERENCES:
patent: 5120644 (1992-06-01), Ikenaka et al.
Yano et al, Biochemical and Biophysical Research Communications, vol. 175, No. 3, Mar. 29, 1991, pp. 1144-1151.
Toomik et al, Analytical Biochemistry, vol. 209, pp. 348-353, 1993.
Casnellie, Methods in Enzymology, vol. 200, pp. 115-120, 1991.
Sigma catalog, Biochemicals and Organic Compounds for Research and Diagnostic Reagerts, pp. 1072-1073 and pp. 854-857, 1993.

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