Human adipose tissue protein analyses using capillary western blot technology

Abstract

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 pAkt S473 (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.