Exploration of some proteomic techniques: Western blot, dot blot and two-dimensional electrophoresis, complemented by mass spectrometry
DOI:
https://doi.org/10.35366/118258Keywords:
Protein, Western blot, Dot blot, Two-dimensional electrophoresis, Immunodetection, semiquantitativeAbstract
The development of proteomics in biomedicine is of great importance because proteins perform most cellular functions, making them essential molecules in research to understand their biological role. To do this, it is necessary to consider both their biochemical and electrophysical properties in order to achieve their separation and determination. To study the expression of proteins, techniques such as Western blot (WB), dot blot (DB) and two-dimensional electrophoresis are used to identify their expression, intensity and important post-translational modifications during biological activity. WB is based on the separation of proteins according to their charge and molecular weight (MW) and their subsequent transfer to a solid membrane so that a specific antibody recognizes the protein of interest. DB does not require electrophoretic separation, but samples are applied directly to a membrane to provide a semi-quantitative estimate of protein concentration. Finally, there is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), which can simultaneously separate more than 1,500 proteins from a single sample and can also be combined with various dyes and other techniques. It is important to note that these methods also require specific means of identifying the proteins of interest, such as a MW marker, dyes or antibodies coupled to enzymes or fluorophores. The application of proteomic techniques ranges from basic research to the clinic, with the aim of discovering new biomarkers of biomedical interest. The aim of this review is to provide an overview of the rationale behind these techniques.
References
Towbin, H., Staehelin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America, 76(9), 4350–4354. https://doi.org/10.1073/pnas.76.9.4350
Taylor, S. C., & Posch, A. (2014). The design of a quantitative western blot experiment. BioMed research international, 2014, 361590. https://doi.org/10.1155/2014/361590
Singh, K.K., Gupta, A., Bharti, C. et al. Emerging techniques of western blotting for purification and analysis of protein. Futur J Pharm Sci 7, 239 (2021). https://doi.org/10.1186/s43094-021-00386-1
Mishra M, Tiwari S, Gomes AV. Protein purification and analysis: next generation Western blotting techniques. Expert Rev Proteomics. 2017 Nov;14(11):1037-1053. doi: 10.1080/14789450.2017.1388167.
Glatter T, Ahrné E, Schmidt A. Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells. J Proteome Res. 2015 Nov 6;14(11):4472-85. doi: 10.1021/acs.jproteome.5b00654.
Kurien, B. T. (2021). Western Blotting for the Non-Expert. Techniques in Life Science and Biomedicine for the Non-Expert. doi:10.1007/978-3-030-70684-5
Martínez-Flores K, Salazar-Anzures ÁT, Fernández-Torres J, et al. Western blot: a tool in the biomedical field. Investigación en Discapacidad. 2017;6(3):128-137.
Kielkopf CL, Bauer W, Urbatsch IL. Bradford Assay for Determining Protein Concentration. Cold Spring Harb Protoc. 2020 Apr 1;2020(4):102269. doi: 10.1101/pdb.prot102269. PMID: 32238597.
García-Solaesa V, Abad SC. SDS-Polyacrylamide Electrophoresis and Western Blotting Applied to the Study of Asthma. Methods Mol Biol. 2016;1434:107-20. doi: 10.1007/978-1-4939-3652-6_8.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophageT4. Nature 227:680–685.
Bass JJ, Wilkinson DJ, Rankin D, Phillips BE, Szewczyk NJ, Smith K, Atherton PJ. An overview of technical considerations for Western blotting applications to physiological research. Scand J Med Sci Sports. 2017 Jan;27(1):4-25. doi: 10.1111/sms.12702. Epub 2016 Jun 5. PMID: 27263489; PMCID: PMC5138151.
Gibbons J. Western blot: protein transfer overview. N Am J Med Sci. 2014 Mar;6(3):158-9. doi: 10.4103/1947-2714.128481. PMID: 24741557; PMCID: PMC3978941.
García TJJ & Ortega R. Inmunotransferencia de geles de proteínas de poliacrilamida tipo Western blot. Mens. Bioquim. 46 (2022): 103-116
Xiang, Y., Zheng, Y., Liu, S. et al. Comparison of the sensitivity of Western blotting between PVDF and NC membranes. Sci Rep 11, 12022 (2021). https://doi.org/10.1038/s41598-021-91521-8
Kurien BT, Scofield RH. Western blotting: an introduction. Methods Mol Biol. 2015;1312:17-30. doi: 10.1007/978-1-4939-2694-7_5. PMID: 26043986; PMCID: PMC7304528.
Goldman A, Harper S, Speicher DW. Detection of Proteins on Blot Membranes. Curr Protoc Protein Sci. 2016 Nov 1;86:10.8.1-10.8.11. doi: 10.1002/cpps.15. PMID: 27801518; PMCID: PMC5646381.
Alegria-Schaffer A, Lodge A, Vattem K. Performing and optimizing Western blots with an emphasis on chemiluminescent detection. Methods Enzymol. 2009;463:573-99. doi: 10.1016/S0076-6879(09)63033-0. PMID: 19892193.
Hawkes R, Niday E, Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142-7. doi: 10.1016/0003-2697(82)90677-7. PMID: 7072935.
Guillemin N, Meunier B, Jurie C, Cassar-Malek I, Hocquette JF, Leveziel H, Picard B. Validation of a Dot-Blot quantitative technique for large scale analysis of beef tenderness biomarkers. J Physiol Pharmacol. 2009 Oct;60 Suppl 3:91-7. PMID: 19996488.
Uritani M., Hamada A. A simple and inexpensive dot-blotter for immunoblotting. Biochemical Education. 1999, 27 (3), pp. 169-170.
Helbing, D. L., Böhm, L., Oraha, N., Stabenow, L. K., & Cui, Y. (2022). A Ponceau S Staining-Based Dot Blot Assay for Rapid Protein Quantification of Biological Samples. Gels (Basel, Switzerland), 8(1), 43. https://doi.org/10.3390/gels8010043
Cabrera-Blanco O, Pisonero-Triana M, Rodríguez-Bejerano M, et al. Dot Blot para determinar la identidad antigénica en vacunas conjugadas contra Streptococcus pneumoniae serotipo 19F. VacciMonitor. 2017;26(1):1-7.
San Miguel-Hernández, Á., Martín-Gil, F. J., & Armentia-Medina, A. (2009). Metodología y aplicaciones en proteómica clínica. Dial Traspl, 30(4), 139-143.
Rozanova, S., Barkovits, K., Nikolov, M., Schmidt, C., Urlaub, H., & Marcus, K. (2021). Quantitative Mass Spectrometry-Based Proteomics: An Overview. Methods in molecular biology (Clifton, N.J.), 2228, 85–116. https://doi.org/10.1007/978-1-0716-1024-4_8
Gorg, A. Two-dimensional electrophoresis. Nature 349, 545–546 (1991). https://doi.org/10.1038/349545a0.
O'Farrell P. H. (1975). High resolution two-dimensional electrophoresis of proteins. The Journal of biological chemistry, 250(10), 4007–4021.
Görg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R., & Weiss, W. (2000). The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 21(6), 1037–1053. https://doi.org/10.1002/(SICI)15222683(20000401)21:6<1037::AIDELPS1037>3.0.CO;2-V
Wilkins, M. R., Pasquali, C., Appel, R. D., Ou, K., Golaz, O., Sanchez, J. C., Yan, J. X., Gooley, A. A., Hughes, G., Humphery-Smith, I., Williams, K. L., & Hochstrasser, D. F. (1996). From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Bio/technology (Nature Publishing Company), 14(1), 61–65. https://doi.org/10.1038/nbt0196-61
Westermeier R and Naven T. Proteomics in Practice, A Laboratory Manual of Proteome Analysis. Weinheim. Wiley-VCH Verlag GmbH. 2002.
Westermeier R. Electrophoresis in Practice. Weinheim. Wiley-VCH Verlag GmbH. 2001.
Stasyk T., Hellman U., Souchelnytskyi S. (2001) Optimizing sample preparation for 2-D electrophoresis. Life Science News 9, 8–11
Mackintosh, J. A., Choi, H. Y., Bae, S. H., Veal, D. A., Bell, P. J., Ferrari, B. C., Van Dyk, D. D., Verrills, N. M., Paik, Y. K., & Karuso, P. (2003). A fluorescent natural product for ultra sensitive detection of proteins in one-dimensional and two-dimensional gel electrophoresis. Proteomics, 3(12), 2273–2288. https://doi.org/10.1002/pmic.200300578
Tonge, R., Shaw, J., Middleton, B., Rowlinson, R., Rayner, S., Young, J., Pognan, F., Hawkins, E., Currie, I., & Davison, M. (2001). Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics, 1(3), 377–396. https://doi.org/10.1002/1615-9861(200103)1:3<377::AID-PROT377>3.0.CO;2-6
Yan, J. X., Harry, R. A., Spibey, C., & Dunn, M. J. (2000). Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes. Electrophoresis, 21(17), 3657–3665. https://doi.org/10.1002/1522-2683(200011)21:17<3657::AID-ELPS3657>3.0.CO;2-2
Aitken, Alastair. (2003). Proteins and Proteomics: A Laboratory Manual. R. J. SIMPSON. Genetics Research . 81. 243-244. 10.1017/S0016672303216323.
Birhanu A. G. (2023). Mass spectrometry-based proteomics as an emerging tool in clinical laboratories. Clinical proteomics, 20(1), 32. https://doi.org/10.1186/s12014-023-09424-x
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2024 Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra
This work is licensed under a Creative Commons Attribution 4.0 International License.
© Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra under a Creative Commons Attribution 4.0 International (CC BY 4.0) license which allows to reproduce and modify the content if appropiate recognition to the original source is given.
