Sensitive fluorescent quantitation of myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate by high-performance thin-layer chromatography

A non-radioactive micro-assay for the cyclic phosphodiesterase reaction cat alyzed by Bacillus cereus phosphatidylinositol-specific phospholipase C is described. The assay involves high-performance thin-layer chromatography on silica gel to resolve the substrate (myo-inositol 1,2-cyclic phosphate) an d the product (myo-inositol 1-phosphate), followed by detection with a lead tetraacetate-fluorescein stain. The quantitation of these inositol phospha tes in sample spots relative to a series of standards is accomplished by an alysis of the fluorescent plate image with a commercial phosphoimager and a ssociated software. The experimental considerations for reliable quantitati on of inositol monophosphates in the range of 0.1 to 50 nmol are presented. (C) 2001 Elsevier Science B.V. All rights reserved.