The assay kit is designed to measure the phosphotransferase activity of S6 kinase in immunoprecipitates and column fractions. Crude cell lysates may also be used but detergents/biochemicals contained in the cell lysis buffer may inhibit S6 kinase activity. Furthermore, although three inhibitors are included with the kit, editors may suggest other unknown kinases found in crude lysates are responsible for phosphorylation of the substrate peptide. The assay kit is based on phosphorylation of a specific substrate (AKRRRLSSLRA; modeled after the major phosphorylation sites in S6 kinase) using the transfer of the gamma- phosphate of adenosine-5'-[32P] Triphosphate ([ gamma-32P]ATP) by p70 S6 kinase. The phosphorylated substrate is then separated from the residual [ gamma-32P]ATP using P81 phosphocellulose paper and quantitated by using a scintillation counter. The assay is linear for incubation times of up to 30 minutes and incorporation of up to 20% of total ATP. Further incubation or incorporation may not be linear and may therefore not be a true indication of p70 S6 kinase activity in the sample extract. The enzyme assay is rapid, convenient and fairly specific for p70 S6 kinase.
Capacity:
~200 assays
Kit Components:
S0050-40A: Assay Dilution Buffer I (ADBI)
S0050-40B: Substrate Peptide
S0050-40C: p81 Squares
S0050-40D: Inhibitor Cocktail
S0050-40E: MgAc/ATP Cocktail
Storage and Stability:
Stable for 1 year at-20 degrees C from date of shipment.
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