Hello, I am an undergrad trying to follow the methodology of the paper attached. I'm working with 50 mM phosphate buffer and 250 mM MgCl2 for hexokinase phosphorylation of CNF. I don't entirely understand why that concentration of buffer was used, and I keep getting Mg(OH)2 precipitates upon trying to get to pH 7.6. What could I be doing wrong?
I wonder if the 250mM MgCl2 concentration is an error in that paper, that is a really high Mg concentration to use. You might ask someone in your lab if that could be correct, or review some other papers on hexokinase.
Having said that, if you need to work at that concentration, I would create separate stock solutions of the phosphate buffer and the MgCl2 and adjust each to the proper pH. Then you you can mix at the appropriate concentrations afterwards.
Often it is magnesium phosphate that is the precipitate. Usually you formulate your phosphate buffer to be the right pH based on the mix of mono- and di-basic phosphate that you add and then autoclave. You then add the magnesium when cold from a separate stock solution.
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