The kit suggests, 'tissues or cells can be extracted with 4 volumes of the assay buffer'. This statement appears quite cryptic as it doesn't tell me 4 volumes of what? For instance, I'm going to use the kit to measure pyruvate kinase activity in my cells, and I have plated approximately 2 million cells in a 6 well plate with 3 ml of media. And with such a vague statement, I'm clueless as to what volume of assay buffer should I use to lyse my cells?
Any suggestion would be highly appreciated.
I believe they mean volumes of tissue or cell pellet. There is a phrase about homogenization in their troubleshooting section, so I think you're supposed to get a cell pellet, and add 4 volumes of buffer to that pellet.
But yeah, horribly cryptic protocol!
You should have 1ug/ul protein for getting optimum enzyme activity. So use lysis buffer accordingly. I use to do enzyme activity manually, so I use 100-200 ul of lysis buffer depending upon cell types.
It basically refers that if you have 100 microliters of cells suspension, add 400 microliters of assay buffer, mix or flick and perform further steps or enzyme assay.
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