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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
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.
Latchman DS: (1997) Transcription factors: An overview. Int J Biochem Cell Biol;29:1305–1312.
Fried M, Crothers DM: (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res;9:6505–6525.
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.
VanGuilder HD, Vrana KE, Freeman WM: (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques;44:619–626.
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.
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.
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.
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.
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights 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