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A nonradioactive method for detecting phosphates and polyphosphates separated by PAGE

Omelon, Sidney, Grynpas, Marc
Electrophoresis 2007 v.28 no.16 pp. 2808-2811
hydrochloric acid, molybdates, orthophosphates, polyacrylamide gel electrophoresis, polyphosphates, sodium chloride
Nonradioactive polyphosphate (poly(P); (PO₃⁻)n) species resolved by PAGE can be detected by hydrolytic degradation of the polyphosphates into orthophosphates (Pi) with a 5 M HCl solution saturated with NaCl, followed by staining the Pi degradation products in a 1 M HCl solution of 0.25% w/v methyl green and 1% w/v ammonium molybdate. This method detects down to 0.5 nmol of phosphate as Pi, linear poly(P) (condensed phosphate), pyrophosphate (P₂O₇⁴⁻), or cyclic trimetaphosphate ion (P₃O₉)³⁻ species. This method improves the current method of staining linear poly(P) longer than four phosphate units with Toludine blue-O after PAGE. This study also shows that Stains-All can visualize resolved linear poly(P) shorter than those visualized by Toluidine blue-O. It is hoped that this sequential hydrolytic degradation and phosphate visualization method for detecting ortho-, linear, and cyclic poly(P) species will be a useful tool, as poly(P) are being discovered in a wide variety of biological systems, and their biochemical roles are still largely unknown.