Rapid Protein Analysis and Western Blotting by Capillary Gel Electrophoresis

Capillary gel electrophoresis has been used since the early 1980s to separate analytes based upon their mass to charge ratio. However, optical limitations prevented high sensitivity instruments from being developed, limiting functionality. As manufacturing capabilities have improved the high‐throughput, hands‐off nature of parallel capillary gel electrophoresis has made it a desirable technique for analysis of DNA, RNA, and protein. Traditionally, protein analysis and Western blotting have been accomplished using SDS‐PAGE followed by staining of proteins in the gel or by downstream transfer of proteins to a blotting membrane, respectively. The trademark feature of these methods is their labor‐intensive nature with many hours or days passing between sample loading and results. Additionally, manual analysis prevents processing of large numbers of samples. Advanced Analytical Technologies, Inc, (AATI) has developed a high‐throughput capillary electrophoresis instrument capable of running 12 samples in parallel with analysis of a complete 96‐well plate without user intervention. This method relies on the rapid fluorescent labeling (~5 min hands on, 15 min total) of total protein sample, with a linear labeling dynamic range of 1 ng − 1000 ng protein input, followed by heat denaturing of the sample in an SDS buffer. The samples are then separated electrophoretically through a proprietary gel matrix (~30 min run time). Sizing of samples is provided using internal markers and comparison to a standardized ladder, allowing for sizing of samples from 14 kDa to greater than 200 kDa with an accuracy of ≤5% and precision of less ≤5%. Relative quantification of proteins is accomplished by comparing the corrected peak area of sample peaks with a precision of ≤10%. Absolute quantification of proteins can be accomplished through a calibration curve using the same protein as the unknown, as each unique protein species will label at varying efficiencies. Capillary gel electrophoresis coupled with total protein labeling can be used to replace traditional Western blots by analyzing and quantifying immunoprecipitations. Analysis of immunoprecipitations by SDS‐PAGE and Coomassie staining are limited due to sensitivity and the copious amounts of antibody present in the sample. By using an unlabeled antibody coupled with labeled total protein, the capillary electrophoresis instrument will only detect the labeled protein of interest isolated by the immunoprecipitation. This provides relative concentration and size information analogous to that provided by the traditional western in a fraction of the time. The AATI protocol eliminates signal bleeding which allows for a greater dynamic range than film. A pull down of endogenous GAPDH demonstrated a 3‐fold dynamic response (0.01 μg – 10 μg protein input). By harnessing capillary gel electrophoresis, AATI has developed a high sensitivity SDS‐PAGE/Western blot replacement with a 3‐fold dynamic range, allowing for rapid analysis of total and purified protein. Please contact the authors at AATI‐us.com if you have interest in this technology. Support or Funding Information This work was funded by Advanced Analytical Technologies, Inc. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .