Abstract
Background/Objectives: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.Subjects: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.Results: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.Conclusions:Capillary Western technology (Wes) provides a satisfactory alternative for analyses of human adipose tissue proteins.
Highlights
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’
Because capillary Western approaches offer investigators the ability to measure multiple proteins and require much smaller amounts of tissue, we wished to test whether this method compares favorably to traditional western blotting for human adipose tissue proteins
We found that the ProteinSimple Wes, a more sensitive and automated approach for protein detection and characterization, provided comparable results to traditional western blotting
Summary
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’. A novel capillary electrophoresis-based western blot technology has been developed by ProteinSimple Inc.[1] 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. This method has been validated for analyses of cell lysate proteins,[2] but whether it can be applied to adipose tissue extracts is unknown. If the two approaches give similar results we and other investigators can be confident that data generated from Wes are comparable to standard methods
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