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Alkaline phosphatase assay

SO Shane O’Grady
MM Maria P. Morgan
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To measure alkaline phosphatase (ALP) activity, cells were washed once with cold PBS, scraped into 1 mL PBS, pelleted by 5 minutes centrifugation at 2,000 RPM and resuspended in 100 µL ALP buffer (100 mM Tris-HCL pH 9.5, 100 mM NaCl, 5 mM MgCl, 1% Triton X-100). For analysis, ALP samples were diluted with ALP buffer (typically a 1:3 to 1:50 dilution). 50 µL of each sample was added to a flat bottom, clear plastic 96-well plate along with 50 µL of p-nitrophenyl phosphate (pNPP) chromogenic substrate (Sigma). A standard curve of purified bovine ALP (Sigma) was prepared in the range of 0–100 mU/mL using the batch information sheet to calculate units of enzymatic activity. Samples were incubated at 37 °C for 30 minutes and analysed by absorbance at 405 nm. All results were normalised to protein content as determined by BCA assay (Sigma).

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