I plated my cells on a 96 well plate and stimulated them with BMPs. After 5 days I aim to detect the Alkaline Phosphatase by using Sigma's Leukocyte Alkaline Phosphatase Assay Kit. I get a beautiful dose titration of ALP by visualizing the purple coloration. My question is - Is there a way to quantify the ALP in my assay? If yes how do I do it?
Thanks in advance for your suggestions
If you buy recombinant alkaline phosphatase, you can make a standard curve, by having increasing amounts of a known concentration of ALP and then measuring the rate of purple color formation or if it is an endpoint assay, measuring total purple color formation.
Hi Vineeth, the easiest way to quantify the AP activity is to normalize the activity to cell protein concentrations.
After your AP assay, you have V. Solubilize the cells and measure the protein conc. Then you have V/mg cell protein. If you know the cell number then you can correlate mg/ml cell protein to cell number and get V/cell.
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