A nonradioactive method for detecting phosphates and polyphosphates separated by PAGE.
Nonradioactive polyphosphate (poly(P); (PO(3) (-))(n)) species resolved by PAGE can be detected by hydrolytic degradation of the polyphosphates into orthophosphates (P(i)) with a 5 M HCl solution saturated with NaCl, followed by staining the P(i) 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 P(i), linear poly(P) (condensed phosphate), pyrophosphate (P(2)O(7) (4) (-)), or cyclic trimetaphosphate ion (P(3)O(9))(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.
Omelon
Sidney
S
Department of Materials Science and Engineering, University of Toronto, Ontario, Canada.
Grynpas
Marc
M
D003720:Densitometry / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006868:Hydrolysis; D008982:Molybdenum; D010710:Phosphates / Q000737:chemistry / Q000302:isolation & purification*; D011122:Polyphosphates / Q000737:chemistry / Q000302:isolation & purification*; D013194:Staining and Labeling / Q000379:methods; D014048:Tolonium Chloride
D010710:Phosphates; D011122:Polyphosphates; D014048:Tolonium Chloride; D008982:Molybdenum; C022175:ammonium molybdate