Question (54689) | Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369)

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Question

I want to perform a pull- down assay using protein A/G agarose and the proteins are to be eluted using Glycine pH2.8. I have not done the actual assay yet, but I added the elution buffer to the cell culture medium first to see if it affected the assay and indeed the reading dropped by half. I want to know if its the pH that affects the assay, since I have tried adding Tris pH7.5 and pH9 to try to raise the pH but got no luck with it.

Answer

This kit has been tested and characterized with different types of samples such as urine, serum, plasma, tissue extracts, cell lysate, cell culture media. However, we have never used IP pull- down samples. As for all the enzymes, pH is very crucial. The optimal pH for the ALP activity is around at 8- 8.5 depending on the species (E.coli, human, bovine etc). Once the pH has changed from alkalic to acidic range, it may well be that the enzyme activity has been changed irreversibly.

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