| Popis: |
A new procedure for the detection and quantitation of small molecular weight cytosolic fatty acid-binding proteins (FABP) in chromatographic fractions is described. Aliquots of the fractionated cytosol are incubated with radiolabeled palmitate and the unbound fatty acid ligand is quickly removed by addition of a dextran-gelatin-coated charcoal suspension. Quantitation of the FABP is accomplished by counting the protein-bound radioactivity in the supernatant fraction after a brief centrifugation step. Validation studies have shown the assay to be linear over a range of 10-40 micrograms or 20-80 micrograms of FABP depending on specific activity of [14C]palmitate used. Bovine serum albumin can be included as an external binding protein to correct for the nominal day-to-day variation in the assay system. The assay has been found to give consistent results with a wide variety of buffer salts, ionic strength, and pH, and therefore is compatible with the usual conditions of gel filtration, ion exchange, and affinity chromatography. Compared to detection methods involving prechromatographic addition of bromosulfophthalein or radiolabeled fatty acids to cytosolic proteins, the post-chromatographic binding assay offers the advantage of leaving the bulk of the FABP preparation free of these marker ligands. |
