The primary goal of kinase activity assays is to determine the catalytic efficiency of kinases, which is typically expressed as the kinetic constant kcat/Km, where kcat is the turnover number and Km is the Michaelis-Menten constant. This measure provides insights into the enzyme's substrate specificity and catalytic efficiency.
1. Radiometric assays: These methods use radioactive substrates and measure the incorporation of radioactive phosphate into the product. The most common radiometric assay is the [gamma-32P]ATP assay, where [gamma-32P]ATP is used as the radioactive substrate.
2. Luminescent assays: These assays use non-radioactive substrates that emit light upon phosphorylation. The most common luminescent assay is the Amplex Red assay, which uses Amplex Red as the substrate and measures the light emitted upon phosphorylation.
3. Colorimetric assays: These assays use substrates that change color upon phosphorylation. The most common colorimetric assay is the Biotium Kinase-Glo assay, which uses Kinase-Glo substrate and measures the color change using a plate reader.
4. Mass spectrometry-based assays: These assays use mass spectrometry to detect and quantify the phosphorylated product. This method provides high sensitivity and specificity but is more complex and expensive than other methods.
Kinase activity assays are subject to various factors that can affect the results, including the concentration of kinase, substrate, and ATP, the incubation time, and the presence of inhibitors or activators. Therefore, it is essential to optimize the assay conditions for each specific kinase and substrate.