| 摘要 |
The present invention is a simple, selective, rapid and a non-radioactive procedure for estimating the activity of protein kinases and protein phosphatases. The method involves attaching a suitable substrate to a 96-well plate surface and then measuring the activity of the specific enzymes using suitable incubating conditions. The PKC assay involves immobilizing histone (type III-SS), the substrate for PKC, and then determining the extent of phosphorylation by using ammonium molybdate and 1-amino-2-naphthol-4-sulfonic acid based reaction for inorganic phosphate quantitation. The PP-1 assay involves immobilizing phosphorylase-b substrate to the assay plate and then phosphorylating the substrate using phosphorylase kinase. Finally, the phosphate is hydrolyzed from the substrate by protein phosphatase. The assay methods are suitable for estimating PKC and PP-1 activity in tissue and cell samples without using any radiolabeled substrate.
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