To test for activity of a poorly characterized cytosolic phosphatase expressed in e. coli, I am looking for a suitable buffer and substrate (p-Nitrophenylphosphate?) to run activity tests. Any suggestions?
Thanks in advance for any help,
Peter
I would suggest trying various buffers at 50 mM, covering a range of pH between 6 and 9, overlapping the ranges of the buffers +/- 0.5 pH unit from their pKas to try to find the best one. Tris-HCl, HEPES-NaOH, and MOPS-NaOH are good examples. Here is a table of buffers:
https://en.wikipedia.org/wiki/Buffer_solution#Common_buffer_compounds_used_in_biology
pNPP is a good substrate to start with because its hydrolysis can be easily monitored by absorbance increase. The fluorogenic substrates 4-methylumbelliferyl phosphate may be useful if the phosphatase activity is too low to measure by absorbance.
You should also optimize the metal ion concentration (probably Mg2+, but try other divalent cations such as Ca2+, Mn2+, Co2+, Zn2+ and Ni2+) at concentrations up to about 20 mM, and the salt concentration. I usually test a range of concentrations of ammonium, potassium, and sodium salts of acetate, sulfate, chloride, and glutamate at concentrations up to a few hundred mM.
I totally agree with the answer of Adam. I am sending you a chapter of a book that will let you perform simple calculations for various buffers at different pH values. But I recomiento use software "Buffer Maker", this allows even adjustment of the values of ionic strength with neutral salts. Regards
I agree with Adam. I would add that magnesium may be a necessary component, and I would stay away from phosphate buffer, as phosphate may inhibit the phosphatase.
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