loading protein ladder for western blot transfer

#LOADING PROTEIN LADDER FOR WESTERN BLOT TRANSFER SERIAL#

This capillary western blot assay incorporates sample separation performed in glass capillaries and immobilization of the proteins directly onto the capillary walls, followed by immuno-probing and chemiluminescent detection. Recently, a novel capillary electrophoresis-based western blot technology has been developed by ProteinSimple, Inc. The traditional western blot is one of the most widely used analytical techniques to quantitate specific proteins in tissues and is considered the “gold standard”. Capillary western technology (Wes®) provides a satisfactory alternative for analyses of human adipose tissue proteins.

The Wes® approach required less sample than the traditional western blot and less technician/assay time, while achieving high sensitivity and good reproducibility. Due to differences in signal responsiveness for pAkt/Akt, we employed a calibrator sample and log transformation of data to allow proper comparisons. Wes® and traditional western blot both had linear dynamic ranges for CD36, Erk1/2 and Akt.

#LOADING PROTEIN LADDER FOR WESTERN BLOT TRANSFER SERIAL#

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.

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). 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. 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.

Loading protein ladder for western blot transfer