Selective extraction, concentration, and assay of orthophosphate from microliter quantities of cultured mammalian cells.
A colorimetric procedure is described for determination of orthophosphate (0.2-2.5 nmol) in sample volumes up to 400 microliters. Orthophosphate is selectively extracted (in the form of phosphomolybdate) into an organic solvent mixture (2-methylpropan-1-ol and petroleum spirit) leaving interfering substances, such as labile organic phosphates, in the aqueous phase. Orthophosphate is then back-extracted into a small volume of aqueous sodium hydroxide. By keeping this volume small, orthophosphate from large dilute samples can be concentrated into small volumes and assayed colorimetrically in microcuvettes using the dye malachite green. The procedure is highly reproducible and insensitive to interfering substances, as shown by comparison with a conventional malachite green assay without the solvent extraction.
Bevington
A
A
Department of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, United Kingdom.
Angier
C M
CM
Kemp
G J
GJ
Russell
R G
RG
D000818:Animals; D002457:Cell Extracts; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002621:Chemistry; D003124:Colorimetry / Q000379:methods; D008322:Mammals; D010710:Phosphates / Q000302:isolation & purification*; D012394:Rosaniline Dyes; D012972:Sodium Hydroxide
D002457:Cell Extracts; D010710:Phosphates; D012394:Rosaniline Dyes; C005095:malachite green; D012972:Sodium Hydroxide