Mesowestern Blot: Simultaneous Analysis of Hundreds of Sub-Microliter Lysates

Western blotting is a widely-used technique for molecular-weight-resolved analysis of proteins and their post-translational modifications, but has been refractory to affordable scale-up. Here, we report the Mesowestern blot, which uses a 3D-printable gel-casting mold to enable affordable, high-throughput Western blotting with standard sample preparation and small (<1 uL) sample sizes. The casted polyacrylamide gel contains 336, 0.5 uL micropipette-loadable sample wells arranged within a standard microplate footprint. Polyacrylamide % can be altered to change molecular weight resolution range. Proof-of-concept experiments using both infrared-fluorescent molecular weight protein ladder as well as cell lysate (RIPA buffer) demonstrate protein loaded in Mesowestern gels is amenable to the standard Western blotting steps. The main difference between Mesowestern and traditional Western is that semi-dry horizontal instead of immersed vertical gel electrophoresis is used. The linear range of detection is approximately 2 orders of magnitude, with a limit of detection (for {beta}-actin) of around 30 ng of total protein from mammalian cell lysates (~30-3000 cells). Because the gel mold is 3D-printable, users have significant design freedom for custom layouts, and there are few barriers to adoption by the typical cell and molecular biology laboratory already performing Western blots.