Question (75976) | Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369)

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Question

My sample for use with this assay will be a cell extract. However the protocol does not provide details on how to create this extract. Specifically, how should washed cells be homogenized in Assay Buffer?

Answer

To measure intracellular ALP, washed cells (˜100,000 cells) can be homogenized, preferably using a dounce homogenizer in the Assay Buffer (2-3 times the volume of the cells). Then centrifuge to remove insoluble material at 13,000g for 3 minutes at 4 deg C. ITs recommended to use different volumes of the supernatant to determine optimum sample volume per well for each sample. Bring the total volume to 80 ul with Assay Buffer. The optimized volume should give numbers within the linear range of the standard curve.

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