Kk. Hedberg et al., Sensitive fluorescent quantitation of myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate by high-performance thin-layer chromatography, J CHROMAT B, 757(2), 2001, pp. 317-324
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.