Abstract
Western Blot is the most important and powerful technique frequently used in laboratory research. It is used to identify specific proteins in biological samples isolated from cells or tissues. Similar to the Southern Blot for DNA and Northern Blot for RNA, the Western Blot procedures rely upon three key elements: the separation of protein mixtures by size utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the efficient transfer of separated proteins from the gel to a nitrocellulose or polyvinylidene difluoride membrane, and identification of a target protein via conjugation with specific primary and enzyme-labeled secondary antibodies. This specificity of the antibody–antigen interaction enables a target protein to be identified in the midst of a complex protein mixture. Once detected, an appropriate substrate is then added to the enzyme, and together, they produce a detectable band visible on a blotting membrane, X-ray file, or imaging system. Western Blot is rapid, and simple, enabling easy to interpret, unique, and unambiguous results. Along with other immunoassays, Western Blot is routinely used in research and clinical settings.
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References
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
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 112:195–203
Davis BJ (1964) Disc electrophoresis. II. Method and application to human serum proteins. Ann N Y Acad Sci 121:404–427
Gajovic O, Todorovic Z, Nesic L, Lazic Z (2010) [Lyme borreliosis—diagnostic difficulties in interpreting serological results]. Med Pregl 63:839–843
Gershoni JM, Palade GE (1982) Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter. Anal Biochem 124:396–405
Gershoni JM, Palade GE (1983) Protein blotting: principles and applications. Anal Biochem 131:1–15
Kruger NJ (1994) The Bradford method for protein quantitation. Methods Mol Biol 32:9–15
Kurien BT, Scofield RH (1997) Multiple immunoblots after non-electrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens. J Immunol Methods 205:91–94
Kurien BT, Scofield RH (2002) Heat-mediated, ultra-rapid electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes. J Immunol Methods 266:127–133
Kurien BT, Scofield RH (2006) Western blotting. Methods 38:283–293
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
LeGendre N (1990) Immobilon-P transfer membrane: applications and utility in protein biochemical analysis. Biotechniques 9:788–805
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Masciotra S, Livellara B, Belloso W, Clara L, Tanuri A, Ramos AC, Baggs J, Lal R, Pieniazek D (2000) Evidence of a high frequency of HIV-1 subtype F infections in a heterosexual population in Buenos Aires, Argentina. AIDS Res Hum Retroviruses 16:1007–1014
Masciotra S, McDougal JS, Feldman J, Sprinkle P, Wesolowski L, Owen SM (2011) Evaluation of an alternative HIV diagnostic algorithm using specimens from seroconversion panels and persons with established HIV infections. J Clin Virol 52(Suppl 1):S17–22
Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038
Noble JE, Bailey MJ (2009) Quantitation of protein. Methods Enzymol 463:73–95
Ornstein L (1964) Disc electrophoresis. I. Background and theory. Ann N Y Acad Sci 121:321–349
Pappaioanou M, Kashamuka M, Behets F, Mbala S, Biyela K, Davachi F, George JR, Green TA, Dondero TJ, Heyward WL et al (1993) Accurate detection of maternal antibodies to HIV in newborn whole blood dried on filter paper. AIDS 7:483–488
Peferoen M, Fransen P, De Loof A (1982) Influence of procaine HCl on larval development, adult lifespan and acid phosphatase activity in Musca domestica L. Arch Int Physiol Biochim 90:309–315
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Torian LV, Forgione LA, Punsalang AE, Pirillo RE, Oleszko WR (2011) Comparison of Multispot EIA with Western blot for confirmatory serodiagnosis of HIV. J Clin Virol 52(Suppl 1):S41–4
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 76:4350–4354
Wiechelman KJ, Braun RD, Fitzpatrick JD (1988) Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Anal Biochem 175:231–237
Zoller L, Cremer J, Faulde M (1993) Western blot as a tool in the diagnosis of Lyme borreliosis. Electrophoresis 14:937–944
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Liu, J., Haorah, J., Xiong, H. (2014). Western Blotting Technique in Biomedical Research. In: Xiong, H., Gendelman, H.E. (eds) Current Laboratory Methods in Neuroscience Research. Springer Protocols Handbooks. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-8794-4_14
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DOI: https://doi.org/10.1007/978-1-4614-8794-4_14
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