Abstract
Western blotting is an important analytical technique used in cell and molecular biology for last four decades. It involves separation of proteins in SDS-PAGE and then transfer of proteins to a membrane followed by detection. By using a western blot, one can identify specific protein from a complex mixture of proteins. Along with its use as a diagnostic aid, it can also be used to verify proteins of interest in exploratory proteomic studies to identify different disease mechanisms. The ease of performing the technique, low cost, and accessibility further support the use of western blot in proteomic research. However, a good understanding, initial training, and optimization are of utmost importance because being a multi-step technique, it is prone to false results and incorrect interpretation. This chapter attempts to explain the technique and theory behind western blot along with some ways to troubleshoot.
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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(9):4350
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
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98(3):503–517
Alwine JC, Kemp DJ, Stark GR (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A 74(12):5350–5354
Gwozdz T, Dorey K (2017) Western blot. In: Basic science methods for clinical researchers. Academic, Boca Raton, pp 99–117
Counts SE (2010) Western blot. In: Encyclopedia of movement disorders, vol 2010. Academic, pp 323–326 . ISBN 9780123741059. https://doi.org/10.1016/B978-0-12-374105-9.00297-5
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1):76–85
Kurien BT, Scofield RH (2015) Western blotting: an introduction. Methods Mol Biol 1312:17–30. https://doi.org/10.1007/978-1-4939-2694-7_5. PMID: 26043986; PMCID: PMC7304528
Wilson K, Walker JM, Hofmann A, Clokie S (2018) Wilson and Walker’s principles and techniques of biochemistry and molecular biology. Cambridge University Press, New York, NY
Kurien BT, Scofield RH (2009) Protein blotting and detection: methods and protocols. Humana Press, New York
Kattoor JJ, Saurabh S, Malik YS, Sircar S, Dhama K, Ghosh S, Bányai K, Kobayashi N, Singh RK (2017) Unexpected detection of porcine rotavirus C strains carrying human origin VP6 gene. Vet Q 37(1):252–261. https://doi.org/10.1080/01652176.2017.1346849
Todd D, McNulty MS, Allan GM (1984) The use of polyacrylamide gel electrophoresis of virus RNA in the study of rotavirus infections. In: McNulty MS, McFerran JB (eds) Recent advances in virus diagnosis. Current topics in veterinary medicine and animal science, vol 29. Springer, Dordrecht. https://doi.org/10.1007/978-94-009-6039-8_11
Urzainqui A, Tabarés E, Carrasco L (1987) Proteins synthesized in African swine fever virus-infected cells analyzed by two-dimensional gel electrophoresis. Virology 160(1):286–291. https://doi.org/10.1016/0042-6822(87)90076-6
Bhatt M, Mohapatra JK, Pandey LK, Mohanty NN, Das B, Prusty BR, Pattnaik B (2018) Mutational analysis of foot and mouth disease virus nonstructural polyprotein 3AB-coding region to design a negative marker virus. Virus Res 243:36–43. https://doi.org/10.1016/j.virusres.2017.10.010
Greenlee JJ, Kunkle RA, Smith JD, Greenlee MHW (2016) Scrapie in swine: a diagnostic challenge. Food Saf 4(4):110–114. https://doi.org/10.14252/foodsafetyfscj.2016019. PMID: 32231914; PMCID: PMC6989210
Hedman C, Otero A, Douet JY, Lacroux C, Lugan S, Filali H, Corbière F, Aron N, Badiola JJ, Andréoletti O, Bolea R (2018) Detection of PrPres in peripheral tissue in pigs with clinical disease induced by intracerebral challenge with sheep-passaged bovine spongiform encephalopathy agent. PLoS One 13(7):e0199914. https://doi.org/10.1371/journal.pone.0199914
Ameri M, Zhou EM, Hsu WH (2006) Western blot immunoassay as a confirmatory test for the presence of anti-mycoplasma hyopneumoniae antibodies in swine serum. J Vet Diagn Investig 18(2):198–201. https://doi.org/10.1177/104063870601800210
Plotzki E, Keller M, Ivanusic D, Denner J (2016) A new western blot assay for the detection of porcine cytomegalovirus (PCMV). J Immunol Methods 437:37–42. https://doi.org/10.1016/j.jim.2016.08.001
Yang S, Li L, Yin S et al (2018) Single-domain antibodies as promising experimental tools in imaging and isolation of porcine epidemic diarrhea virus. Appl Microbiol Biotechnol 102:8931–8942. https://doi.org/10.1007/s00253-018-9324-7
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Bhatt, M. et al. (2022). SDS-PAGE and Western Blotting: Basic Principles and Protocol. In: Deb, R., Yadav, A.K., Rajkhowa, S., Malik, Y.S. (eds) Protocols for the Diagnosis of Pig Viral Diseases. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2043-4_23
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DOI: https://doi.org/10.1007/978-1-0716-2043-4_23
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