Skip to main content

Analysis of Host-Cell Responses by Immunoblotting, ELISA, and Real-Time PCR

  • Protocol
  • First Online:

Part of the Methods in Molecular Biology book series (MIMB,volume 845)

Abstract

Intracellular responses to external pathogens/stimuli are crucial to the host’s response to infection. The methods used to analyse these responses fall into many categories. Activation of proteins as part of a signal cascade can be screened for using conventional immunoblotting techniques or immunoprecipitation to identify the presence of modified proteins or protein complexes. Transcription factor activity can be screened for by EMSA or ELISAs to identify DNA binding of these factors. Finally, expression of activated genes can be quantified using real-time PCR methods. Here, we will show how to perform these assays and discuss the relative merits of each.

Key words

  • Immunoblotting
  • Transcription factor
  • Cell signalling
  • Immunoprecipitation
  • ELISA
  • Real-time PCR
  • Electrophoresis
  • Phosphorylation
  • Protein modification

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-61779-539-8_23
  • Chapter length: 16 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   149.00
Price excludes VAT (USA)
  • ISBN: 978-1-61779-539-8
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   199.00
Price excludes VAT (USA)
Hardcover Book
USD   279.99
Price excludes VAT (USA)
Fig. 1.
Fig. 2.

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Renart J, Reiser J, Stark GR: (1979) Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: A method for studying antibody specificity and antigen structure. Proc Natl Acad Sci USA;76:3116–3120.

    CAS  CrossRef  Google Scholar 

  2. 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 USA;76:4350–4354.

    CAS  CrossRef  Google Scholar 

  3. Latchman DS: (1997) Transcription factors: An overview. Int J Biochem Cell Biol;29:1305–1312.

    CAS  CrossRef  Google Scholar 

  4. Fried M, Crothers DM: (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res;9:6505–6525.

    CAS  CrossRef  Google Scholar 

  5. Garner MM, Revzin A: (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: Application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res;9:3047–3060.

    CAS  CrossRef  Google Scholar 

  6. VanGuilder HD, Vrana KE, Freeman WM: (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques;44:619–626.

    CAS  CrossRef  Google Scholar 

  7. Ngoka LC: (2008) Sample prep for proteomics of breast cancer: Proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers. Proteome Sci;6:30.

    CrossRef  Google Scholar 

  8. 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.

    CAS  CrossRef  Google Scholar 

  9. 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.

    CAS  CrossRef  Google Scholar 

  10. Livak KJ, Schmittgen TD: (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−delta delta c(t)) method. Methods;25:402–408.

    CAS  CrossRef  Google Scholar 

  11. Shapiro AL, Vinuela E, Maizel JV, Jr.: (1967) Molecular weight estimation of polypeptide chains by electrophoresis in sds-polyacrylamide gels. Biochem Biophys Res Commun;28:815–820.

    CAS  CrossRef  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Julian R. Naglik .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Moyes, D.L., Naglik, J.R. (2012). Analysis of Host-Cell Responses by Immunoblotting, ELISA, and Real-Time PCR. In: Brand, A., MacCallum, D. (eds) Host-Fungus Interactions. Methods in Molecular Biology, vol 845. Humana, Totowa, NJ. https://doi.org/10.1007/978-1-61779-539-8_23

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-539-8_23

  • Published:

  • Publisher Name: Humana, Totowa, NJ

  • Print ISBN: 978-1-61779-538-1

  • Online ISBN: 978-1-61779-539-8

  • eBook Packages: Springer Protocols

We’re sorry, something doesn't seem to be working properly.

Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.