Abstract
For the quantification of certain proteins of interest within a complex sample, Western blot analysis is the most widely used method. It enables detection of a target protein based on the use of specific antibodies. However, the whole procedure is often very time-consuming. Nevertheless, with the development of fast blotting systems and further development of immunostaining methods, a reduction of the processing time can be achieved. Major challenges for the reliable protein quantification by Western blotting are adequate data normalization and stable protein detection. Usually, normalization of the target protein signal is performed based on housekeeping proteins (e.g., glyceraldehyde 3-phosphate dehydrogenase, ß-actin) with the assumption that those proteins are expressed constitutively at the same level across experiments. However, several studies have already shown that this is not always the case making this approach suboptimal. Another strategy uses total protein normalization where the abundance of the target protein is related to the total protein amount in each lane. This approach is independent of a single loading control, and precision of quantification and reliability is increased. For Western blotting several detection methods are available, e.g., colorimetric, chemiluminescent, radioactive, fluorescent detection. Conventional colorimetric staining tends to suffer from low sensitivity, limited dynamic range, and low reproducibility. Chemiluminescence-based methods are straightforward, but the detected signal does not linearly correlate to protein abundance (from protein amounts >5μg) and have a relatively narrow dynamic range. Radioactivity is harmful to health. To overcome these limitations, stain-free methods were developed allowing the combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer to the membrane. Here, we present a rapid Western blot protocol, which combines fast blotting using the iBlot system and fast immunostaining utilizing ReadyTector® all-in-one solution with the Smart Protein Layers (SPL) approach.
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References
Bass JJ, Wilkinson DJ, Rankin D et al (2017) An overview of technical considerations for Western blotting applications to physiological research. Scand J Med Sci Sports 27(1):4–25. https://doi.org/10.1111/sms.12702
Ni D, Xu P, Gallagher S (2017) Immunoblotting and Immunodetection. Curr Protoc Protein Sci 88:10.10.11–10.10.37. https://doi.org/10.1002/cpps.32
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(9):4350–4354. https://doi.org/10.1073/pnas.76.9.4350
Sundaram P (2018) Protein stains and applications. Methods Mol Biol 1853:1–14. https://doi.org/10.1007/978-1-4939-8745-0_1
MacPhee DJ (2010) Methodological considerations for improving Western blot analysis. J Pharmacol Toxicol Methods 61(2):171–177. https://doi.org/10.1016/j.vascn.2009.12.001
Fradelizi J, Friederich E, Beckerle MC et al (1999) Quantitative measurement of proteins by western blotting with Cy5-coupled secondary antibodies. Biotechniques 26(3):484–486; 488, 490 passim.
Burnette WN (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate—polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112(2):195–203. https://doi.org/10.1016/0003-2697(81)90281-5
Kricka LJ, Thorpe GH (1986) Photographic detection of chemiluminescent and bioluminescent reactions. Methods Enzymol 133:404–420. https://doi.org/10.1016/0076-6879(86)33082-9
Kurien BT, Scofield RH (2006) Western blotting. Methods 38(4):283–293. https://doi.org/10.1016/j.ymeth.2005.11.007
Taylor SC, Posch A (2014) The design of a quantitative western blot experiment. Biomed Res Int 2014:361,590. https://doi.org/10.1155/2014/361590
Welinder C, Ekblad L (2011) Coomassie staining as loading control in Western blot analysis. J Proteome Res 10(3):1416–1419. https://doi.org/10.1021/pr1011476
Ranganathan V, De PK (1996) Western blot of proteins from Coomassie-stained polyacrylamide gels. Anal Biochem 234(1):102–104. https://doi.org/10.1006/abio.1996.0057
Litovchick L (2020) Staining the blot for total protein with ponceau S. Cold Spring Harb Protoc 2020(3):098459. https://doi.org/10.1101/pdb.prot098459
Wang JL, Zhao L, Li MQ et al (2020) A sensitive and reversible staining of proteins on blot membranes. Anal Biochem 592:113,579. https://doi.org/10.1016/j.ab.2020.113579
Steinberger B, Brem G, Mayrhofer C (2015) Evaluation of SYPRO ruby total protein stain for the normalization of two-dimensional Western blots. Anal Biochem 476:17–19. https://doi.org/10.1016/j.ab.2015.01.015
Goldman A, Harper S, Speicher DW (2016) Detection of proteins on blot membranes. Curr Protoc Protein Sci 86:10.18.11. https://doi.org/10.1002/cpps.15
Ferguson RE, Carroll HP, Harris A et al (2005) Housekeeping proteins: a preliminary study illustrating some limitations as useful references in protein expression studies. Proteomics 5(2):566–571. https://doi.org/10.1002/pmic.200400941
Thellin O, Zorzi W, Lakaye B et al (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75(2–3):291–295. https://doi.org/10.1016/s0168-1656(99)00163-7
Colella AD, Chegenii N, Tea MN et al (2012) Comparison of stain-free gels with traditional immunoblot loading control methodology. Anal Biochem 430(2):108–110. https://doi.org/10.1016/j.ab.2012.08.015
Gürtler A, Kunz N, Gomolka M et al (2013) Stain-free technology as a normalization tool in Western blot analysis. Anal Biochem 433(2):105–111. https://doi.org/10.1016/j.ab.2012.10.010
Gilda JE, Gomes AV (2013) Stain-free total protein staining is a superior loading control to β-actin for Western blots. Anal Biochem 440(2):186–188. https://doi.org/10.1016/j.ab.2013.05.027
Gilda JE, Gomes AV (2015) Western blotting using in-gel protein labeling as a normalization control: stain-free technology. Methods Mol Biol 1295:381–391. https://doi.org/10.1007/978-1-4939-2550-6_27
Faden F, Eschen-Lippold L, Dissmeyer N (2016) Normalized quantitative Western blotting based on standardized fluorescent labeling. Methods Mol Biol 1450:247–258. https://doi.org/10.1007/978-1-4939-3759-2_20
Acknowledgments
A part of this study was funded by P.U.R.E. (Protein Research Unit Ruhr within Europe) and ProDi (Center for protein diagnostics), Ministry of Innovation, Science and Research of North-Rhine Westphalia, Germany, and by the H2020 project NISCI, (GA no. 681094).
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Barkovits, K., Pfeiffer, K., Eggers, B., Marcus, K. (2021). Protein Quantification Using the “Rapid Western Blot” Approach. In: Marcus, K., Eisenacher, M., Sitek, B. (eds) Quantitative Methods in Proteomics. Methods in Molecular Biology, vol 2228. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1024-4_3
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DOI: https://doi.org/10.1007/978-1-0716-1024-4_3
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