Keywords
antibodies
Western Blot
immunodetection
tubulin
protein isolation
electrophoresis
proteínas
anticuerpos
Western Blot
inmunodetección
tubulina
aislamiento
electroforesis
Abstract
Western Blot technique was described for the first time at the end of the seventies and since then, it has been widely used in Molecular and Cellular Biology experiments related to protein studies. This method allows not only the immunodetection of proteins in complex and raw protein extracts and samples, but also the relative amount of a specific protein in a sample. Up to date, there has not been any report of the use and implementation of the Western Blot technique in any laboratory from the Occident Region
of Universidad de Costa Rica. This article aims to become a methodological reference not only for Western Blot implementation but also for preparation and quantification of maize total protein extracts at the Laboratorio de Biotecnología, Sede de Occidente from Universidad de Costa Rica. It is described in detail, from obtaining and preparing maize protein extracts from fresh tissue, to the detection of control protein α-tubulin and the quantification of the signal detected in the immunological assay. Under the
conditions here stablished, the Western Blot was semiquantitative for protein loads between 5 and 45μg.
References
Bernard, L., Ciceri, P., y Viotti, A. (1994). Molecular analysis of wild-type and mutant alleles at the
Opaque-2 regulatory locus of maize reveals different mutations and types of O2 products. Plant Molecular Biology, 24(6), 949–959. https:// doi.org/10.1007/BF00014448
Bradford, M. (1976). A Rapid and Sensitive Method for the Quantitation Microgram Quantities of Protein
Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 72(1–2), 248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Carpentier, S. C., Witters, E., Laukens, K., Deckers, P., Swennen, R., y Panis, B. (2005). Preparation of protein extracts from recalcitrant plant tissues: An evaluation of different methods for two-dimensional gel electrophoresis analysis.
Proteomics, 5(10), 2497–2507. https://doi.org/10.1002/pmic.200401222
Correa Navarro, Y. M., Buriticá Salazar, L. M., Rivera Giraldo, J. D., Penagos González, J. P., y Torres Osorio, J. I. (2017). Optimización del protocolo para la extracción y la cuantificación de proteínas totales en semillas germinadas de maíz (Zea
mays L.). Revista Facultad de Ciencias Básicas, 13(1), 65–68. https://doi.org/10.18359/rfcb.2756
Ford, P., y Southern, E. (1973). Different Sequences for 5S RNA in Kidney Cells and Ovaries of Xenopus laevis. Nature New Biology, 241(105), 7–12. https://doi.org/10.1038/newbio241007a0
Huis, R., Hawkins, S., y Neutelings, G. (2010). Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biology, 10(1), 71. https://doi.org/10.1186/1471-2229-10-71
Kärkönen, A., Meisrimler, C.-N., Takahashi, J., Väisänen, E., Laitinen, T., Jiménez Barboza, L. A., … Lüthje, S. (2014). Isolation of cellular membranes from lignin-producing tissues of Norway spruce and analysis of redox enzymes. Physiologia Plantarum, 152(4), 599–616. https://doi. org/10.1111/ppl.12209
Kurien, B. T., y Scofield, R. H. (2006). Western blotting. Methods, 38(4), 283–293. https://doi.org/10.1016/j. ymeth.2005.11.007
Kurien, B. T., y Scofield, R. H. (2015). Western Blotting. (B. T. Kurien y R. H. Scofield, Eds.), Methods in Molecular Biology (Vol. 1312). New York, NY: Springer New York. https://doi.org/10.1007/978- 1-4939-2694-7
Lara-Núñez, A., García-Ayala, B. B., Garza-Aguilar, S. M., Flores-Sánchez, J., Sánchez-Camargo, V. A.,
Bravo-Alberto, C. E., … Vázquez-Ramos, J. M. (2017). Glucose and sucrose differentially modify cell proliferation in maize during germination. Plant Physiology and Biochemistry, 113, 20–31. https://doi.org/10.1016/j.plaphy.2017.01.018
López Quimbayo, L. D. (2012). Extracción de proteínas a partir de hojas y semillas de Pentacalia nítida y evaluación de la actividad antimicrobiana del extracto proteico acuoso. Pontificia Universidad Javeriana. Retrieved from https://repository.
javeriana.edu.co/bitstream/handle/10554/11802/LopezQuimbayoLinaDaniela2012.pdf?sequence=1
Mahmood, T., y Yang, P. C. (2012). Western blot: Technique, theory, and trouble shooting. North American Journal of Medical Sciences, 4(9), 429–434. https://doi.org/10.4103/1947-2714.100998
Moritz, C. P. (2017). Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control
in Western Blots. Proteomics, 17(20), 1600189. https://doi.org/10.1002/pmic.201600189
Mróz, T. L., Havey, M. J., y Bartoszewski, G. (2015). Cucumber Possesses a Single Terminal Alternative Oxidase Gene That is Upregulated by Cold Stress and in the Mosaic (MSC) Mitochondrial Mutants. Plant Molecular Biology Reporter, 33(6), 1893–
https://doi.org/10.1007/s11105-015-0883-9
Shan, G. (Ed.). (2011). Immunoassays in Agricultural Biotechnology. Hoboken, NJ, USA: John Wiley & Sons, Inc. https://doi.org/10.1002/9780470909935
Taylor, S. C., Berkelman, T., Yadav, G., & Hammond, M. (2013). A Defined Methodology for Reliable Quantification of Western Blot Data. Molecular Biotechnology, 55(3), 217–226. https://doi. org/10.1007/s12033-013-9672-6
Towbin, H., Staehelin, T., y Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. PNAS, 76(9), 4350–4354. https:// doi.org/10.1073/pnas.76.9.4350
Varagona, M. J., Schmidt, R. J., y Raikhel, N. V. (1991). Monocot regulatory protein opaque-2 is localized in the nucleus of maize endosperm and transformed tobacco plants. The Plant Cell,
(2), 105–113.
Wang, W., Vignani, R., Scali, M., y Cresti, M. (2006). A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis. Electrophoresis, 27(13), 2782–2786. https://doi.org/10.1002/elps.200500722
Comments
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Copyright (c) 2021 Pensamiento Actual