Journal of Lipid Research, Vol 24, 324-331, Copyright © 1983 by Lipid Research, Inc.
Quantitation of hepatic fatty acid-binding proteins by post- chromatographic ligand binding assay
FD Morrow and RJ Martin
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.
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