Abstract
The Western blot is widely used in the study of protein biochemistry, but it is notoriously labor-intensive, and it is limited in its reproducibility and quantification, among many other challenges. By contrast, capillary-based protein separation and immunodetection, known as Simple Western™, overcomes many of the challenges associated with the traditional Western blot, and it is quickly gaining traction as a replacement for traditional Western blot analysis. The advantages that capillary-based immunoassay offers include ease of use, automation, reproducibility, quantification, and even built-in total protein normalization. In this chapter, we describe protocols for the two basic types of capillary-based immunodetection assays, one by molecular weight separation and the other by charge separation. In both methods, protein samples are separated in the capillary followed seamlessly by immunodetection with chemiluminescent or fluorescent antibodies for highly sensitive and specific detection of target proteins.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354. https://doi.org/10.1073/pnas.76.9.4350
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517. https://doi.org/10.1016/S0022-2836(75)80083-0
Fosang AJ, Colbran RJ (2015) Transparency is the key to quality. J Biol Chem 290:29692–29694. https://doi.org/10.1074/jbc.E115.000002
Hantelys F, Godet A-C, David F et al (2019) Vasohibin1, a new mouse cardiomyocyte IRES trans-acting factor that regulates translation in early hypoxia. Elife 8:1–26. https://doi.org/10.7554/eLife.50094
Varkey R, Du Q, Karnell JL et al (2019) Discovery and characterization of potent IL-21 neutralizing antibodies via a novel alternating antigen immunization and humanization strategy. PLoS One 14:1–26. https://doi.org/10.1371/journal.pone.0211236
Du Y, Walsh A, Ehrick R et al (2012) Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. MAbs 4:578–585. https://doi.org/10.4161/mabs.21328
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Lück, C., Haitjema, C., Heger, C. (2021). Simple Western: Bringing the Western Blot into the Twenty-First Century. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 2261. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1186-9_30
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1186-9_30
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1185-2
Online ISBN: 978-1-0716-1186-9
eBook Packages: Springer Protocols