| Assay Method Information | |
| | Protein Kinase Assay |
| Description: | This buffer consisted of 50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 1 mg ml−1 bovine serum albumin and 0.1% (vol/vol) 2-mercaptoethanol.10× Concentrated Assay Buffer.An assay buffer of 500 mM Tris-HCl, pH 7.5, 1 mM EGTA, and 100 mM magnesium acetate was used for assay of a wide variety of protein kinases. In some experiments, other cofactors were included (e.g., calcium ions and calmodulin were included for assay of calcium-calmodulin-dependent protein kinases). In some cases, the pH of the buffer was changed (e.g., phosphorylase kinase had an optimum pH of 8.6).1 mM [γ-32P] ATP.The specific activity of the [γ-32P] ATP solution was 1×105 to 1×106 c.p.m. per nmol depending on what was needed to produce an optimal signal/noise ratio. Stocks of nonradioactive ( cold ) ATP were dissolved in 10 mM HEPES, and the pH of the resulting stock solutions was adjusted to 7.4. To measure the concentration of ATP, a sample of such a stock solution was diluted to 20 μM, and the absorbance of the diluted sample was measured at 259 nm. The absorbance of a 20-μM stock solution of ATP at 259 nm was about 0.31. The 1-mM solution of cold ATP was spiked with [γ-32P] ATP to produce a radioactivity of 1×105 to 1×106 c.p.m. per nmol. |
| Affinity data for this assay | |
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