Skip to main content
SpringerLink
Log in

Western Blotting as a Diagnostic Method

  • Protocol
Medical Biomethods Handbook

Part of the book series: Springer Protocols Handbooks ((SPH))

Abstract

Protein blotting, the transfer of proteins from a separating gel onto a thin uniform support matrix, first appeared in 1979. Continuing the geographic theme following Southern's publication of his method for the identification of specific DNA fragments (1) in 1975 and the introduction of Northern blotting (2) not long after, the technique became known as Western blotting. Today, the original article by Towbin et al. (3) is cited many thousands of times a year. The technique itself has been modified and extended over the years (4). Once on a solid support, procedures that would otherwise proved difficult or impossible in the gel can be undertaken. A blot allows for rapid staining and destaining protocols of the separated proteins. Low concentrations of sample are more easily detected because they are not spread throughout the thickness of the gel but are “concentrated” on the surface; also, membranes are easier to handle and manipulate.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517.

    Article  PubMed  CAS  Google Scholar 

  2. Alwin, J. C., Kemp, D.J., and Stark, G. R. (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. USA 74(12), 5350–5354.

    Article  Google Scholar 

  3. Towbin, H., Straelin, T., and Gordin, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 7(9), 4350–4354.

    Article  Google Scholar 

  4. Dunbar, B.S. (ed.) (1994) Protein Blotting: A Practical Approach, IRL, Oxford.

    Google Scholar 

  5. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(259), 680–685.

    Article  PubMed  CAS  Google Scholar 

  6. Gershoni, J.M. and Palade, G. E. (1982) Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter. Anal. Biochem. 124(2), 396–405.

    Article  PubMed  CAS  Google Scholar 

  7. Timmons, T. and Dunbar, B. S. (1990) Methods Enzymol. 128, 679–689.

    Article  Google Scholar 

  8. Bers, G. and Garfin, D. (1985) Protein and nucleic acid blotting and immunobiochemical detection. BioTechniques 3(4), 276–288.

    CAS  Google Scholar 

  9. Gershoni, J.M. and Palade, G. E. (1983) Protein blotting: principles and applications. Anal. Biochem. 131(1), 1–15.

    Article  PubMed  CAS  Google Scholar 

  10. Dunn, S.D. (1986) Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Anal. Biochem. 157(1), 144–153.

    Article  PubMed  CAS  Google Scholar 

  11. Van Dam, A. P., et al. (1986) Technical problems concerning the use of immunoblots for the detection of antinuclear antibodies. J. Immunol. Methods 129(1), 63–70.

    Google Scholar 

  12. Matsudaira, P. (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262(21), 10,035–10,038.

    PubMed  CAS  Google Scholar 

  13. Miribel, L. and Arnaud, P.(1988) Electrotransfer of proteins following polyacrylamide gel electrophoresis; nitrocellulose versus nylon membranes. J. Immunol. Methods 107, 253–259.

    Article  PubMed  CAS  Google Scholar 

  14. Gershoni, J.M. and Palade, G. E. (1982) Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter. Anal. Biochem. 124(2), 396–405.

    Article  PubMed  CAS  Google Scholar 

  15. Lin, W and Kasamatsu H. (1983) On the electrotransfer of polypeptides from gels to nitrocellulose membranes. Anal. Biochem. 128(2), 302–311.

    Article  PubMed  CAS  Google Scholar 

  16. Tovey, E. R., Ford, S.A., and Baldo, B. A. (1987) Protein blotting on nitrocellulose: some important aspects of the resolution and detection of antigens in complex extracts. J. Biochem. Biophys. Methods 14, 1–17.

    Article  PubMed  CAS  Google Scholar 

  17. Hewick, R. M., Hunkapiller, M. W., Wood, L.E., and Dreyer, W. J. (1981) A gas-liquid solid phase peptide and protein sequenator. J. Biol. Chem. 256(15), 7990–7997.

    PubMed  CAS  Google Scholar 

  18. Aebersold, R. H., Leavitt, J., Saavedra, R. A., Hood, L.E., and Kent, S. B. (1987) Internal amino acid sequence analysis of proteins separated by one-or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc. Natl. Acad. Sci USA 84(20), 6970–6974.

    Article  PubMed  CAS  Google Scholar 

  19. Moos, M., Jr., Nguyen, N. Y., and Liu, T. Y. (1988) Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support. J. Biol. Chem. 263(13), 6005–6008.

    PubMed  CAS  Google Scholar 

  20. Matsudaira, P. (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262(21), 10,035–10,038.

    PubMed  CAS  Google Scholar 

  21. Walsh, M. J., McDougall, J., and Wittmann-Liebold, B. (1988) Extended N-terminal sequencing of proteins of archaebacterial ribosomes blotted from two-dimensional gels onto glass fiber and poly(vinylidene difluoride) membrane. Biochemistry 27, 6876–6879.

    Article  Google Scholar 

  22. Whitehead, T. P., Thorpe, G. H. C., Carter, T. J. N, Groncutt, C., and Kiricka L. J. (1983) Enhanced luminescence procedure for sensitive determination of peroxidase-labelled conjugates in immunoassays. Nature 305, 158–159.

    Article  CAS  Google Scholar 

  23. Lundin, A. and Hallander, L. O. B. (1987) Bioluminescence and Chemiluminescence: New Perspectives, Wiley, New York.

    Google Scholar 

  24. Bronstein, I., Edwards, B., and Voyta, J. C. (1989) 1,2 Dioxetanes: novel chemiluminescent enzyme substrates. Application to immunoassays. J. Biolumines. 4, 99–111.

    Article  CAS  Google Scholar 

  25. Johnson, D. A., Gautsch, J. W., Sportsman, R., and Elder, J. H. (1984) Improved technique utilizing non fat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal. Tech. 1, 3–8.

    Article  CAS  Google Scholar 

  26. Center for Disease Control (1989) Interpretation and use of the Western Blot assay for serodiagnosis of human immunodeficiency virus type 1 infections. MMWR 38(S-7), 1–7; MMWR 51(18), 395-396 and references therein.

    Google Scholar 

  27. McDougal, J. S., Kennedy, M. S., Nicholson, J.K.A., et al. (1987) Antibody response to human immunodeficiency virus in homosexual men: relation of antibody specificity, titer, and isotype to clinical status, severity of immunodeficiency and disease progression. J. Clin. Invest. 80, 316–324.

    Article  PubMed  CAS  Google Scholar 

  28. Lange, J. M. A., Coutinho, R. A., Krone, W.J.A., et al. (1986) Distinct IgG recognition patterns during progression of subclinical and clinical infection with lymphadenopathy associated virus/ human T lymphotropic virus. Br. Med. J. 292, 228–230.

    Article  CAS  Google Scholar 

  29. Esteban, J. I., Shih, J. W., Tai, C.C., et al. (1985) Importance of Western blot analysis in predicting infectivity of anti-HTLV-III/LAV positive blood. Lancet 2, 1083–1086.

    Google Scholar 

  30. Goudsmith, J., Lange, J. M. A., Paul, D.A., et al. (1987) Antigemia and antibody titers to core and envelope antigens in AIDS, AIDS-related complex and subclinical human immunodeficiency virus infection. J. Infect. Dis. 155, 558–560.

    Google Scholar 

  31. Weber, J. N., Clapham, P. R., Weiss, R.A., et al. (1987) Human immunodeficiency virus infection in two cohorts of homosexual men: neutralizing sera and association of anti-gag antibody with prognosis. Lancet 1, 119–121.

    Google Scholar 

  32. Martinez, P. M., Torres, A. R., de Lejarazu, R.O., Montoya, A., Martin, J.F., and Eiros, J. M. (1999) Human Immunodeficiency virus antibody testing by enzyme-linked fluorescent and Western blot assays using serum, gingival-crevicular transudate and urine samples. J. Clin. Microbiol. 3, 1100–1106.

    Google Scholar 

  33. World Health Organization (1990) AIDS: Proposed WHO criteria for interpreting results from Western blot assays for HIV-1, HIV-2, HTLV-I/HTLV-II. Weekly Epidemiol. Rec. 65, 281–283.

    Google Scholar 

  34. Mortimer, P.P. and Parry, J. V. (1994) Detection of antibody to HIV in saliva: a brief review. Clin. Diagn. Virol. 2, 231–243.

    Article  PubMed  CAS  Google Scholar 

  35. Slomka, M. J., Ashley, R. L., Cowan, F. M., Cross, A., and Brown, D. W. (1995) Monoclonal antibody blocking tests for the detection of HSV-1 abd HSV-2 specific humoral responses: comparison with Western blot assay. J. Virol. Methods 1, 27–35.

    Google Scholar 

  36. Rahn, D.W. (1998) Natural History of Lyme disease. in Lyme Disease (ed]Rahn, D. W. and Evans, J., eds.), American College of Physicians, Philadelphia, PA, pp. 35–48.

    Google Scholar 

  37. Dressler, F., Whalen, J. A., Reinhardt, B. N., and Steere, A. C. (1993) Western blotting in the serodiagnosis of Lyme disease. J. Infect. Dis. 167, 392–400.

    PubMed  CAS  Google Scholar 

  38. Craft, J. E., Fischer, D. K., Shimamoto, G.T., and Steere, A. C. (1985) Antigens of Borrelia burgdorferi recognized during Lyme disease: appearance of a new immunoglobulin M response and expansion of the immunoglobulin G response late in the illness. J. Clin. Invest. 78, 934–939.

    Article  Google Scholar 

  39. FDA Medical Bulletin. (1999) http://www.fda.gov/medbull/summer99/Lyme.html.

  40. Hauptl, T., Hahn, G., Rittig, M., et al. (1993) Persistence of Borrelia burgdorferi in ligamentous tissue from a patient with chronic Lyme borreliosis. Arthritis Rheum. 36, 1621–1626.

    Article  Google Scholar 

  41. Preac-Mursic, V., Magret, W., Busch, U., Pleterski Rigler, D., and Hagl, S. (1996) Kill kinetics of Borrelia burgdorferi and bacterial findings in relation to the treatment of Lyme borreliosis. Infection 24, 9–18.

    Article  PubMed  CAS  Google Scholar 

  42. Heikkila, T., Seppala, I., Saxen, H., Panelius, J., Yrjanainen, H., and Lahdenne, P. (2002) Speciesspecific serodiagnosis of Lyme arthritis and neuroborreliosis due to Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii by using decorin binding protein A. J. Clin. Microbiol. 40, 453–460.

    Article  PubMed  CAS  Google Scholar 

  43. Theisen, M., Frederiksen, B., Lebech, A. M., Vuust, J., and Hansen, K. (1993) Polymorphism in ospC gene of Borrelia burgdorferi and immunoreactivity of OspC protein: implications for taxonomy and for use of OspC protein as a diagnostic antigen. J. Clin. Microbiol. 31, 2570–2576.

    PubMed  CAS  Google Scholar 

  44. Magnarell, L. A., Anderson, J.F., and Johnson, R. C. (1987) Cross-reactivity in serological tests for Lyme disease and other sprirochetal infections. J. Infect. Dis. 156, 183–188.

    Google Scholar 

  45. Goossens, H. A., van den Bogaard, A.E., and Nohlmans, M. K. (1999) Evaluation of fifteen commercially available serological tests for diagnosis of Lyme borreliosis. Eur. J. Clin. Microbiol. Infect. Dis. 18, 551–560.

    Article  PubMed  CAS  Google Scholar 

  46. Centre for Disease Control(1995) Recommendations for test performance and interpretation from the second national conference on the serological diagnosis of Lyme disease. MMWR 44, 590–591.

    Google Scholar 

  47. Hauser, U., Lehnert, G., and Wilske, B. (1999) Validity of interpretation criteria for standardized Western blots (immunoblots) for serodiagnosis of Lyme borreliosis based on sera collected throughout Europe. J. Clin. Microbiol. 37, 2241–2247.

    PubMed  CAS  Google Scholar 

  48. Craven, R. B., Quan, T. J., Bailey, R.E., et al. (1996) Improved serodiagnostic testing for Lyme disease: Results of a multicenter serologic evaluation. Emerg. Infect. Dis. 2, 136–140.

    Article  PubMed  CAS  Google Scholar 

  49. Fawcett, P., Rose, C. D., Gibney, K.M., and Doughty, R. A. (1998) Comparison of immunoblot and Western blot assasy for diagnosing Lyme borreliosis. Clin. Diag. Lab. Immunol. 5, 503–506.

    CAS  Google Scholar 

  50. Brenner, C. (2003) Explanation of the Lyme disease Western Blot. Lymenet. www.lymenet.de/labtests/brenner.htm.

  51. Schulte-Spechtel, U., Lehnert, G., Liegl, G., et al. (2003) Significant improvement of the recombinant Borrelia-specific immunoglobulin G immunoblot test by addition of VlsE and DbpA homologue derived from Borrelia garinii for diagnosis of early neuroborreliosis. Clin. Microbiol. 41, 1299–1303.

    Article  CAS  Google Scholar 

  52. Gomes-Solecki, M. J., Dunn, J. J., Luft, B.J., et al. (2000) Recombinant chimeric Borrelia proteins for diagnosis of Lyme disease. J. Clin. Microbiol. 38, 2530–2535.

    PubMed  CAS  Google Scholar 

  53. Wilske, B., Habermann, C., Fingerle, V., et al. (1999) An improved recombinant IgG immunoblot for serodiagnosis of Lyme borreliosis. Med. Microbiol. Immunol. 188, 139–144.

    Article  PubMed  CAS  Google Scholar 

  54. Rikihisa, Y. (1991) The tribe Ehrlichieae and ehrlichial diseases. Clin. Microbiol. Rev. 4, 286–308.

    PubMed  CAS  Google Scholar 

  55. Buller, R. S., Arens, M., Hmiel, S.P., et al. (1999) Ehrlichia ewingii, a newly recongnized agent of human ehlichiosis. N. Engl. J. Med. 341, 148–155.

    Article  PubMed  CAS  Google Scholar 

  56. Chen, S. M, Dumler, J. S., Bakken, J.S., and Walker, D. H. (1994) Identification of a granulocytotrophich Ehrlichia species as the etiologic agent of human disease. J. Clin. Microbiol. 32, 589–595.

    PubMed  CAS  Google Scholar 

  57. Unver, A., Felek, S., Paddock, C.D., et al. (2001) Western blot analysis of sera reactive to human monocytic ehrlichiosis and human granulocytic ehrlichiosis agents. J. Clin. Microbiol. 39, 3982–3986.

    Article  PubMed  CAS  Google Scholar 

  58. Tsang, V. C., Brand, J.A., and Boyer, A. E.(1989) An enzyme-linked immunoelectrotransfer blot assay and glycoprotein antigens for diagnosing human cysticercosis (Taenia solium). J. Infect. Dis. 159, 50–59.

    PubMed  CAS  Google Scholar 

  59. Rodriguez-Canul, R., Allan, J. C., Fletes, C., Sutisna, I. P., Kapti, I.N., and Craig, P. S.(1997) Comparative evaluation of purified Taenia solium glycoproteins and crude metacestode extracts by immunoblotting for the serodiagnosis of human T. solium cysticercosis. Clin. Diagn. Lab Immunol. 4, 579–582.

    PubMed  CAS  Google Scholar 

  60. Greene, R. M., Hancock, K., Wilkins, P.P., and Tsang, V. C.(2000) Taenia solium: molecular cloning and serologic evaluation of 14-and 18-kDa related, diagnostic antigens. J. Parasitol. 86, 1001–1007.

    PubMed  CAS  Google Scholar 

  61. Hancock, K., Khan, A., Williams, F.B, et al. (2003) Characterization of the 8-kilodalton antigens of Taenia solium metacestodes and evaluation of their use in an enzyme-linked immunosorbent assay for serodiagnosis. J. Clin. Microbiol. 41, 2577–2586.

    Article  PubMed  CAS  Google Scholar 

  62. Hubert, K., Andriantsimahavandy, A., Michault, A., Frosch, M., and Muhlschlegel, F. A., et al. (1999) Serological diagnosis of human cysticercosis by use of recombinant antigens from Taenia solium cysticerci. Clin. Diagn. Lab. Immunol. 6(4), 479–482.

    PubMed  CAS  Google Scholar 

  63. Muhlen, C.A. and Tan, E. M. (1995) Autoantibodies in the diagnosis of systemic rheumatic diseases. Semin. Arthritis Rheum. 24, 323–358.

    Article  Google Scholar 

  64. Yago M., Belmonte, M. A., Olmos, M. J., Beltran, J., Teruel, C., and Segarra, M. (1999) Detecting anti-SSA and anti-SSB antibodies in routine analysis: a comparison between double immunodiffusion and immunoblotting. Ann. Clin. Biochem. 36, 365–371.

    PubMed  CAS  Google Scholar 

  65. Vitozzi, S., Lapierre, P., Djilali-Saiah, I., and Alvarez F. (2002) Autoantibody detection in type 2 autoimmune hepatitis using a chimera recombinant protein. J. Immunol. Methods 262, 103–110.

    Article  PubMed  CAS  Google Scholar 

  66. Brichory, F. M., Misek, D. E., Yim, A-M., et al. (2001) An immune response manifested by the common occurrence of annexin I and II autoantibodies and high circulating levels of IL-6 in lung cancer. Proc. Natl. Acad. Sci. USA 98, 9824–9829.

    Article  PubMed  CAS  Google Scholar 

  67. Lieu, T-S., Jiang, M., Steigerwald, J.C., and Tan, E. M. (1984) Identification of the SS-A/Ro intracellular antigen with autoimmune sera. J. Immunol. Methods 71, 217–228.

    Article  PubMed  CAS  Google Scholar 

  68. Hu, G. R., Harrop, P., Warlow, R., Gacis, M. L., and Walls, R. S. (1997) High resolution autoantibody detection by optimized protein-G horseradish peroxidase immunostaining in a Western blot assay. J Immunol Methods. 202, 113–121.

    Article  PubMed  CAS  Google Scholar 

  69. Naour, F. L, Brichory, F., Beretta, L., and Hanash, S. M. (2002) Identification of tumor-associated antigens using proteomics. Technol. Cancer Res. Treat. 1, 257–262.

    PubMed  Google Scholar 

  70. Prabhakar, S. and Bhatia, R. (2001) Diagnosis of Creutzfeld-Jacob disease. Neurol. India 49, 325–328.

    PubMed  CAS  Google Scholar 

  71. Safar, J., Wille, H., Itri, V. et al. (1998) Eight Prion strains have PrSc molecules with different conformations. Nature Med. 4, 1157–1165.

    Article  PubMed  CAS  Google Scholar 

  72. Wadsworth, J. D. F., Joiner, S., Hill, A.F., et al. (2001) Tissue distribution of protease resistant prion protein in variant Creutzfeld-Jacob disease using a highly sensitive immunoblotting assay. Lancet 358, 171–180.

    Article  PubMed  CAS  Google Scholar 

  73. Wiltfang, J., Otto, M., Baxter, H. C., et al. (1999) Isoform pattern of 14-3-3 proteins in the cerebrospinal fluid of patients with Creutzfeld-Jacob disease. J. Neurochem. 73, 2485–2490.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. GE Healthcare Bio-Sciences, Amersham, UK

    Pirkko Soundy & Bronwen Harvey

Authors
  1. Pirkko Soundy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bronwen Harvey
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. University of Hertfordshire, Hatfield, UK

    John M. Walker  & Ralph Rapley  & 

Rights and permissions

Reprints and permissions

Copyright information

© 2005 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Soundy, P., Harvey, B. (2005). Western Blotting as a Diagnostic Method. In: Walker, J.M., Rapley, R. (eds) Medical Biomethods Handbook. Springer Protocols Handbooks. Humana Press. https://doi.org/10.1385/1-59259-870-6:043

Download citation

  • DOI: https://doi.org/10.1385/1-59259-870-6:043

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-288-9

  • Online ISBN: 978-1-59259-870-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation