Question (36822) | Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369)

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Question

In the protocol for this ALP assay kit, there are directions for deterimining intracellular ALP levels. I see that I am suppose to lyse 100,000 cellswith Assay Buffer. But no volume of Assay Buffer is given. As well, what is the actual protocol for lysing the cells with Assay Buffer?

Answer

The procedure is as follows:

1. Wash tissue: 10 mg or cells 1X106 cell in ice cold PBS.
2. Spin at 1000 g for 5 mins at 4C.
3. Resuspend and homogenize in ˜2-3 volumes of the assay buffer (use Dounce homogenizer, if needed to ensure cell lysis) on ice.
4. Spin at 13,000 g for 10 min at 4C.
5. Collect the supernatant and test different volumes for the assay.

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