Abstract
CD36 is a transmembrane protein present in many tissues that is believed to facilitate inward fatty acid transport. Western blotting is the most widely used method to measure tissue CD36 protein content, but it is time consuming, technically demanding, and semiquantitative. To more precisely measure adipose tissue CD36 content we developed an enzyme linked immunosorbent assay (ELISA) after establishing that: 1) the anti-CD36 antibodies gave a single distinct band on traditional Western blots, and 2) the vast majority of adipocyte CD36 resides in the plasma membrane. By using serial dilutions of each sample and including a calibrator sample and quality control sample on each plate, we could achieve inter- and intra-assay variability of ∼ 10%. We found that CD36 content in omental and abdominal subcutaneous adipose tissue varied over a 2-5-fold range depending upon the means of data expression (per units of tissue protein, weight, or lipid). Omental CD36 content in women decreased markedly (P = 0.01) as a function of fat cell size. For the most part, tissue CD36 content was not correlated with CD36 mRNA. This ELISA method for tissue CD36 content should enhance research into the role of this protein on tissue fatty acid uptake.
Highlights
CD36 is a transmembrane protein present in many tissues that is believed to facilitate inward fatty acid transport
To extract the sample, ف200 mg of tissue was homogenized in 4 μL/mg tissue standard homogenization buffer with anti-proteases (SHB-P: 20 mM Tris-HCl, 1 mM EDTA, 255 mM sucrose, pH 7.4, anti-protease tablets from Roche) on ice using a rotor-stator for three 10 s cycles of homogenization interspersed with 10 s cycles of rest
Examining the CD36 bands from the total homogenate, cytosolic proteins, total membrane proteins, which is a relatively complete recovery of membranes, the purified mitochondrial/peroxisomal membrane proteins, and purified plasma membrane proteins we consistently found that virtually all the CD36 present in membrane fractions was in the plasma membrane
Summary
CD36 is a transmembrane protein present in many tissues that is believed to facilitate inward fatty acid transport. To more precisely measure adipose tissue CD36 content we developed an enzyme linked immunosorbent assay (ELISA) after establishing that: 1) the anti-CD36 antibodies gave a single distinct band on traditional Western blots, and 2) the vast majority of adipocyte CD36 resides in the plasma membrane. Tissue CD36 content was not correlated with CD36 mRNA This ELISA method for tissue CD36 content should enhance research into the role of this protein on tissue fatty acid uptake.—Allred, C. The expression of adipocyte CD36 in patients may differ with total body fat, fat cell size, and fatty acid metabolism [7,8,9]. Because of the somewhat tedious nature of Western blotting to accomplish these objectives, we developed an ELISA that we believe will enhance research on this important protein
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