Advertisement

Western Blotting Against Tagged Virulence Determinants to Study Bacterial Pathogenicity

  • Gili Aviv
  • Ohad Gal-MorEmail author
Protocol
  • 1.6k Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 1734)

Abstract

Western blotting is a common approach to detect the presence of a target protein in biological samples or proteins mixture using specific antibodies. This method is also useful to study regulation of virulence determinants by analyzing changes in protein expression between different genetic backgrounds or under varying environmental conditions. To avoid the need to raise specific antibodies for each studied protein, commercial antibody against commonly used peptidic epitopes can be utilized if the right target tagged version is constructed. Here we describe a C-terminal fusion between a protein of interest and the two hemagglutinin A (2HA) tag. The tagged protein is cloned into a low-copy number vector and expressed under its native promoter in Salmonella enterica. Then, the expression of the tagged protein can be analyzed by Western blotting and commercially available anti-2HA antibodies.

Key words

Western blotting Gel electrophoresis Protein tagging Immunoblotting Hemagglutinin Antibodies 

Notes

Funding Information

The research in Gal-Mor lab is supported by a grant number 1096.39.11/2010 from the German-Israeli Foundation for Scientific Research and Development (GIF); by a grant number 999/14 from the Israel Science Foundation (ISF) and by grant number 3-0000-12435 from Infect-ERA and the Chief Scientist’s Bureau in the Israeli Ministry of Health.

References

  1. 1.
    Kurien BT, Scofield RH (2015) Western blotting: an introduction. Methods Mol Biol 1312:17–30CrossRefPubMedGoogle Scholar
  2. 2.
    Elhadad D, Desai P, Rahav G et al (2015) Flagellin is required for host cell invasion and normal Salmonella pathogenicity island 1 expression by Salmonella enterica serovar Paratyphi a. Infect Immun 83:3355–3368CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Uzzau S, Figueroa-Bossi N, Rubino S, Bossi L (2001) Epitope tagging of chromosomal genes in Salmonella. Proc Natl Acad Sci U S A 98:15264–15269CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Wang RF, Kushner SR (1991) Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene 100:195–199CrossRefPubMedGoogle Scholar
  5. 5.
    Rose RE (1988) The nucleotide sequence of pACYC184. Nucleic Acids Res 16:355–356Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.The Infectious Diseases Research LaboratorySheba Medical CenterTel-Hashomer, Ramat GanIsrael
  2. 2.Department of Clinical Microbiology and Immunology, Sackler Faculty of MedicineTel Aviv UniversityTel AvivIsrael

Personalised recommendations