Human adipose tissue protein analyses using capillary western blot technology.

A capillary western blot (Wes®) technology has recently been validated for analyses of cell culture lysate proteins, but whether it is reliable for human tissue proteins is unknown. We compared traditional western blotting to the Wes® capillary western method to quantitate the relative amount of human adipose tissue CD36, the ratio of phosphorylated Erk1/2 (pErk1/2) to total Erk1/2 during insulin clamp or after niacin treatment and the fold increase in pAktS473 (Akt phosphorylation on Ser473) in response to feeding. The results from these two methods were highly correlated (r = 0.932 for CD36, r = 0.905 for pErk1/2:Erk1/2, r = 0.923 for the change in pAkt/Akt, P < 0.001). On Wes® we observed the distinct peaks around the expected molecular weights for these proteins with decreasing peak areas with serial dilutions of loading protein amount. Wes® and traditional western blot both had linear dynamic ranges for CD36, Erk1/2 and Akt. Due to differences in signal responsiveness for pAkt/Akt, we employed a calibrator sample and log transformation of data to allow proper comparisons. The Wes® approach required less sample than the traditional western blot and less technician/assay time, while achieving high sensitivity and good reproducibility. Capillary western technology (Wes®) provides a satisfactory alternative for analyses of human adipose tissue proteins.

A novel ELISA for measuring CD36 protein in human adipose tissue.

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.