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2024, Number 3

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Investigación en Discapacidad 2024; 10 (3)

Exploration of some proteomic techniques: Western blot, Dot blot and two-dimensional electrophoresis, complemented by mass spectrometry

López-Macay A, Montes-Sánchez D, Narváez-Morales J, Salas-Paniagua M, Barrios-Aguilar S, Zamudio-Cuevas Y, Fernández-Torres J, Martínez K

Language: Spanish
References: 51
Page: 187-202
PDF size: 412.65 Kb.


ABSTRACT

The development of proteomics in biomedicine is crucial because proteins are key to 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 semiquantitative 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 different dyes and other techniques such as mass spectrometry (MS). It is important to note that these methods also require specific means of identifying the proteins of interest, such as a PM marker, stains 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

  1. Sandoval-Usme MC, Umaña-Pérez A, Vallejo-Pulido AF, Arévalo-Ferro C, Sánchez-Gómez M. La proteómica en la era postgenómica. Acta Biol Colomb. 2009; 14 (3): 19-30.

  2. Carbonara K, Andonovski M, Coorssen JR. Proteomes are of proteoforms: embracing the complexity. Proteomes. 2021; 9 (3): 38. doi: 10.3390/proteomes9030038.

  3. Magdeldin S, Enany S, Yoshida Y, Xu B, Zhang Y, Zureena Z et al. Basics and recent advances of two dimensional- polyacrylamide gel electrophoresis. Clin Proteomics. 2014; 11 (1): 16. doi: 10.1186/1559-0275-11-16.

  4. Stott DI. Immunoblotting, dot-blotting, and ELISPOT assays: methods and applications. J Immunoassay. 2000; 21 (2-3): 273-296. doi: 10.1080/01971520009349537.

  5. Gallo G, Scaloni A. Differential proteomics based on 2D-difference in-gel electrophoresis and tandem mass spectrometry for the elucidation of biological processes in antibiotic-producer bacterial strains. Methods Mol Biol. 2018; 1716: 267-289. doi: 10.1007/978-1-4939-7528-0_12.

  6. Mishra M, Tiwari S, Gomes AV. Protein purification and analysis: next generation Western blotting techniques. Expert Rev Proteomics. 2017; 14 (11): 1037-1053. doi: 10.1080/14789450.2017.1388167.

  7. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979; 76 (9): 4350-4354. doi: 10.1073/pnas.76.9.4350.

  8. Taylor SC, Posch A. The design of a quantitative western blot experiment. Biomed Res Int. 2014; 2014: 361590. doi: 10.1155/2014/361590.

  9. Singh KK, Gupta A, Bharti C, Sharma H. Emerging techniques of western blotting for purification and analysis of protein. Futur J Pharm Sci. 2021; 7: 239. Available in: https://doi.org/10.1186/s43094-021-00386-1

  10. 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; 14 (11): 4472-4485. doi: 10.1021/acs.jproteome.5b00654.

  11. Kurien BT. Western blotting for the non-expert. Techniques in life science and biomedicine for the non-expert. 2021. doi: 10.1007/978-3-030-70684-5.

  12. Martínez-Flores K, Salazar-Anzures AT, Fernández-Torres J, Pineda C, Aguilar-González CA, López-Reyes A. Western blot: a tool in the biomedical field. Investigación en Discapacidad. 2024; 6 (3): 128-137.

  13. Kielkopf CL, Bauer W, Urbatsch IL. Bradford assay for determining protein concentration. Cold Spring Harb Protoc. 2020; 2020 (4): 102269. doi: 10.1101/pdb.prot102269.

  14. García-Solaesa V, Abad SC. SDS-polyacrylamide electrophoresis and western blotting applied to the study of asthma. Methods Mol Biol. 2016; 1434: 107-120. doi: 10.1007/978-1-4939-3652-6_8.

  15. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227 (5259): 680-685.

  16. Bass JJ, Wilkinson DJ, Rankin D, Phillips BE, Szewczyk NJ, Smith K et al. An overview of technical considerations for Western blotting applications to physiological research. Scand J Med Sci Sports. 2017; 27 (1): 4-25. doi: 10.1111/sms.12702.

  17. Gibbons J. Western blot: protein transfer overview. N Am J Med Sci. 2014; 6 (3): 158-159. doi: 10.4103/1947-2714.128481.

  18. García Trejo JJ, Ortega R. Inmunotransferencia de geles de proteínas de poliacrilamida tipo Western blot. Mens Bioquim. 2022; 46: 103-116.

  19. Xiang Y, Zheng Y, Liu S, Liu G, Li Z, Dong W. Comparison of the sensitivity of Western blotting between PVDF and NC membranes. Sci Rep. 2021; 11 (1): 12022. doi: 10.1038/s41598-021-91521-8.

  20. Kurien BT, Scofield RH. Western blotting: an introduction. Methods Mol Biol. 2015; 1312: 17-30. doi: 10.1007/978-1-4939-2694-7_5.

  21. Goldman A, Harper S, Speicher DW. Detection of proteins on blot membranes. Curr Protoc Protein Sci. 2016; 86: 10.8.1-10.8.11. doi: 10.1002/cpps.15.

  22. Alegria-Schaffer A, Lodge A, Vattem K. Performing and optimizing Western blots with an emphasis on chemiluminescent detection. Methods Enzymol. 2009; 463: 573-599. doi: 10.1016/S0076-6879(09)63033-0.

  23. Hawkes R, Niday E, Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982; 119 (1): 142-147. doi: 10.1016/0003-2697(82)90677-7.

  24. Guillemin N, Meunier B, Jurie C, Cassar-Malek I, Hocquette JF, Leveziel H et al. Validation of a Dot-Blot quantitative technique for large scale analysis of beef tenderness biomarkers. J Physiol Pharmacol. 2009; 60 Suppl 3: 91-97.

  25. Uritani M, Hamada A. A simple and inexpensive dot-blotter for immunoblotting. Biochemical Education. 1999; 27 (3): 169-170.

  26. Helbing DL, Bohm L, Oraha N, Stabenow LK, Cui Y. A ponceau s staining-based dot blot assay for rapid protein quantification of biological samples. Gels. 2022; 8 (1): 43. doi: 10.3390/gels8010043.

  27. 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.

  28. San Miguel-Hernández A, Martín-Gil FJ, Armentia-Medina A. Metodología y aplicaciones en proteómica clínica. Dial Traspl. 2009; 30 (4): 139-143.

  29. Rozanova S, Barkovits K, Nikolov M, Schmidt C, Urlaub H, Marcus K. Quantitative mass spectrometry-based proteomics: an overview. Methods Mol Biol. 2021; 2228: 85-116. doi: 10.1007/978-1-0716-1024-4_8.

  30. Gorg A. Two-dimensional electrophoresis. Nature. 1991; 349: 545-546. Available in: https://doi.org/10.1038/349545a0

  31. O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975; 250 (10): 4007-4021.

  32. Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R et al. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis. 2000; 21 (6): 1037-1053. doi: 10.1002/(SICI)1522-2683(20000401)21:6<1037::AID-ELPS1037>3.0.CO;2-V.

  33. Wilkins MR, Pasquali C, Appel RD, Ou K, Golaz O, Sanchez JC et al. From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Biotechnology (N Y). 1996; 14 (1): 61-65. doi: 10.1038/nbt0196-61.

  34. Westermeier R, Naven T. Proteomics in practice, a laboratory manual of proteome analysis. Weinheim: Wiley-VCH Verlag GmbH; 2002.

  35. Westermeier R. Electrophoresis in practice. Weinheim: Wiley-VCH Verlag GmbH; 2001.

  36. Stasyk T, Hellman U, Souchelnytskyi S. Optimizing sample preparation for 2-D electrophoresis. Life Sci. 2001; 9: 8-11.

  37. Mackintosh JA, Choi HY, Bae SH, Veal DA, Bell PJ, Ferrari BC et al. A fluorescent natural product for ultra sensitive detection of proteins in one-dimensional and two-dimensional gel electrophoresis. Proteomics. 2003; 3 (12): 2273-2288. doi: 10.1002/pmic.200300578.

  38. Tonge R, Shaw J, Middleton B, Rowlinson R, Rayner S, Young J et al. Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics. 2001; 1 (3): 377-396. doi: 10.1002/1615-9861(200103)1:3<377::AID-PROT377>3.0.CO;2-6.

  39. Yan JX, Harry RA, Spibey C, Dunn MJ. Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes. Electrophoresis. 2000; 21 (17): 3657-3665. doi: 10.1002/1522-2683(200011)21:17<3657::AID-ELPS3657>3.0.CO;2-2.

  40. Villegas-Rivera GA, Torres-Madronero MC, Rothlisberger-Booth S, Delgado-Trejos E. Procesamiento de imágenes de electroforesis bidimensional: una revisión. Scientia Et Technica. 2019; 24 (1): 76-84.

  41. Kendrick N, Darie CC, Hoelter M, Powers G, Johansen J. Correction to: 2D SDS PAGE in combination with western blotting and mass spectrometry is a robust method for protein analysis with many applications. Adv Exp Med Biol. 2019; 1140: C1. doi: 10.1007/978-3-030-15950-4_47. Erratum for: Adv Exp Med Biol. 2019; 1140: 563-574.

  42. Vargas Altamirano R. Sistema informático de análisis de geles de electroforesis bidimensional: de la adquisición de imágenes a la identificación de proteínas. Acta Nova. 2001; 1 (1): 01-25. Disponible en: http://www.scielo.org.bo/scielo.php?script=sci_arttext&pid=S1683-07892001000000007&lng=es&tlng=es

  43. López Farré A, González Armengol J, Mateos-Cáceres PJ, Macaya C. Horizontes de la proteómica en el diagnóstico y el tratamiento de la enfermedad cardiovascular. Clínica e Investigación en Arteriosclerosis. 2008; 20 (4): 164-172.

  44. Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P. In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem. 1992; 203 (1): 173-179. doi: 10.1016/0003-2697(92)90061-b.

  45. Pappin DJ, Hojrup P, Bleasby AJ. Rapid identification of proteins by peptide-mass fingerprinting. Curr Biol. 1993; 3 (6): 327-332. doi: 10.1016/0960-9822(93)90195-t. Erratum in: Curr Biol. 1993; 3 (7): 487.

  46. Birhanu AG. Mass spectrometry-based proteomics as an emerging tool in clinical laboratories. Clin Proteomics. 2023; 20 (1): 32. doi: 10.1186/s12014-023-09424-x.

  47. Abián J, Carrascal M, Gay M. Introducción a la espectrometría de masas para la caracterización de péptidos y proteínas en proteómica. Proteómica. 2008; 2: 16-35.

  48. Curreem SO, Watt RM, Lau SK, Woo PC. Two-dimensional gel electrophoresis in bacterial proteomics. Protein Cell. 2012; 3 (5): 346-363. doi: 10.1007/s13238-012-2034-5.

  49. Begum H, Murugesan P, Tangutur AD. Western blotting: a powerful staple in scientific and biomedical research. Biotechniques. 2022; 73 (1): 58-69. doi: 10.2144/btn-2022-0003.

  50. Villafañez F, Gottifredi V, Soria G. Development and optimization of a miniaturized western blot-based screening platform to identify regulators of post-translational modifications. High Throughput. 2019; 8 (2): 15. doi: 10.3390/ht8020015.

  51. Rodríguez-Vázquez R, Mouzo D, Zapata C. Phosphoproteome analysis using two-dimensional electrophoresis coupled with chemical dephosphorylation. Foods. 2022; 11 (19): 3119. doi: 10.3390/foods11193119




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