Mesowestern Blot: Simultaneous Analysis of Hundreds of Submicroliter Lysates

Abstract

Western blotting is a widely used technique for molecular-weight-resolved analysis of proteins and their posttranslational modifications, but high-throughput implementations of the standard slab gel arrangement are scarce. The previously developed Microwestern requires a piezoelectric pipetting instrument, which is not available for many labs. Here, we report the Mesowestern blot, which uses a 3D-printable gel casting mold to enable high-throughput Western blotting without piezoelectric pipetting and is compatible with the standard sample preparation and small (∼1 μL) sample sizes. The main tradeoffs are reduced molecular weight resolution and higher sample-to-sample CV, making it suitable for qualitative screening applications. The casted polyacrylamide gel contains 336, ∼0.5 μL micropipette-loadable sample wells arranged within a standard microplate footprint. Polyacrylamide % can be altered to change molecular weight resolution profiles. Proof-of-concept experiments using both infrared-fluorescent molecular weight protein ladder and cell lysate (RIPA buffer) demonstrate that the protein loaded in Mesowestern gels is amenable to the standard Western blotting steps. The main difference between Mesowestern and traditional Western is that semidry horizontal instead of immersed vertical gel electrophoresis is used. The linear range of detection is at least 32-fold, and at least ∼500 attomols of β-actin can be detected (∼29 ng of total protein from mammalian cell lysates: ∼100–300 cells). Because the gel mold is 3D-printable, users with access to additive manufacturing cores have significant design freedom for custom layouts. We expect that the technique could be easily adopted by any typical cell and molecular biology laboratory already performing Western blots.

A 96-Well Polyacrylamide Gel for Electrophoresis and Western Blotting

“…This would allow for reliable detection (~90% confidence, ~80% power) of ~1.5 fold changes with triplicate measurements. Previous western blotting work reports CV from 15% to 70% (or more) 13,[22][23][24][25] , so this value is typical or even low for western blots. Importantly, the CV reported for this particular 96-well western is artificially high since there is by definition no loading control, which is generally known to improve CV 21 by about a factor of 1.25 13 , down to ~12-20% 24,25 .…”

“…Previous western blotting work reports CV from 15% to 70% (or more) 13,[22][23][24][25] , so this value is typical or even low for western blots. Importantly, the CV reported for this particular 96-well western is artificially high since there is by definition no loading control, which is generally known to improve CV 21 by about a factor of 1.25 13 , down to ~12-20% 24,25 . Conveniently, one potential use of these 96-well blots is to include replication in the gel loading design to improve statistical performance.…”

“…The microwestern [10][11][12] and mesowestern 13 blot technologies use a horizontal polyacrylamide gel and electrophoresis arrangement to achieve higher sample throughput, and we built on the same approach here. However, these previous techniques required small sample sizes, which are undesirable for medium-to-low abundance targets, and semi-dry electrophoresis, which requires somewhat expensive equipment that is not typically used in western blotting.…”

“…Obstacles to microwestern adoption include the piezoelectric pipetting apparatus (expensive, difficult to use, sample loss in tubing, restricted to non-standard lysis buffers, flat gel with no wells), or the use of semi-dry electrophoresis versus the typical immersed tank (expense and difficulty of use). The mesowestern blot 13 , in part developed by us, has the same scale as the microwestern but eliminates the need for piezoelectric pipetting. Yet, it still uses semi-dry electrophoresis, and requires small (~1 μL) sample sizes, which although can be a benefit, is also a detriment if a low abundance target is of interest.…”