Enzyme-Linked Immunosorbent Assay for the Determination of p21-Activated Kinase Activity

TL;DRAbstract

An enzyme-linked immunosorbent assay (ELISA) for the measurement of p21-activated kinase (PAK) activity is described. The development of this method takes advantage of the fact that a phospho-epitope-specific antibody against the regulatory autophosphorylation site sequence of PAK was successfully produced, and after being phosphorylated by PAK, a cross-linked peptide containing the autophosphorylation site of PAK could be recognized on immunoblot by this antibody. This procedure involves coating the cross-linked peptide on microtiter plates, phosphorylating the cross-linked peptide by adding active PAK plus ATP.Mg(2+), and detecting peptide phosphorylation using the phospho-epitope-specific antibody and secondary antibody conjugated with alkaline phosphatase followed by reaction with p-nitrophenyl phosphate (for colorimetric detection) or fluorescein diphosphate (for fluorimetric detection). The PAK activity detected by this method was linearly proportional to the amount of kinase use

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