A 96-Well PolyacrylamideGel for Electrophoresis andWestern Blotting

Western blotting is a stalwart technique for analyzing specific proteins and their post-translational modifications. However, it remains challenging to accommodate more than ∼10 samples per experiment without a substantial departure from trusted, established protocols involving accessible instrumentation. Here, we describe a 96-sample Western blot that conforms to standard 96-well plate dimensional constraints and has little operational deviation from standard Western blotting. The main differences are that (i) submerged polyacrylamide gel electrophoresis is operated horizontally (similar to agarose gels) as opposed to vertically and (ii) a 6 mm thick gel is used, with 2 mm most relevant for membrane transfer (vs ∼1 mm typical). Results demonstrate that both wet and semidry transfer are compatible with this gel thickness. The major trade-off is reduced molecular weight resolution, due primarily to less available migration distance per sample. We demonstrate proof-of-principle using gels loaded with a molecular weight ladder, recombinant protein, and cell lysates. We expect that the 96-well Western blot will increase reproducibility, efficiency (cost and time ∼8-fold), and capacity for biological characterization relative to established Western blots.