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1 - Does the kinase buffer contain protease inhibitors or phophotase inhibitors? If not, should they be added and how do they effect the experiment?
2 - Are there any modifications for this protocol for tissue samples?
Asked on Jan 17 2014
The kinase buffer contains PMSF, Na-vanadate and Na-pyrophosphate and EDTA. Typically nothing more is required to protect the Akt enzyme activity. Additional protease inhibitors might be added if desired. This should not affect the assay, but addition of more protease inhibitors and phosphatase inhibitors has not yet been tested.
Tissue samples should be homogenized in the extraction buffer and then centrifuged to obtain a clarified homogenate which can be used in the assay.
Jeremy KasanovAbcam Scientific Support
Answered on Jan 17 2014