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Mesowestern blot: Simultaneous analysis of hundreds of submicroliter lysates
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2022 (English)In: ACS Omega, E-ISSN 2470-1343, Vol. 7, no 33, p. 28912-28923Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2022. Vol. 7, no 33, p. 28912-28923
Keywords [en]
Biopolymers, Electrophoresis, Immunology, Membranes, Peptides and proteins
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-218254DOI: 10.1021/acsomega.2c02201ISI: 000846755100001OAI: oai:DiVA.org:umu-218254DiVA, id: diva2:1820879
Available from: 2023-12-19 Created: 2023-12-19 Last updated: 2023-12-19Bibliographically approved

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Erdem, Cemal

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