Analysis of Host-Cell Responses by Immunoblotting, ELISA, and Real-Time PCR
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 conv
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1. Introduction 1.1. Immunoblotting
Detection of proteins by immunoblotting is a well-established technique, enabling the detection of specific proteins and confirmation of their molecular weight by use of electrophoresis and antibodies (1, 2). It can be used to discriminate between a precursor protein and the processed form by size, and its usefulness in identifying intracellular signal pathway activation has been further enhanced by the development of antibodies capable of distinguishing a modified protein from its unmodified form—e.g. antibodies that recognise the phosphorylated form of a protein only (phosphospecific antibodies). Whenever immunoblotting is being used to demonstrate changes in the levels of either a protein or its modification, it is important to also establish the levels of a loading control protein—e.g. a-actin or tubulin. This is to ensure that the levels of protein loaded for each sample are equivalent, enabling
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_23, © Springer Science+Business Media, LLC 2012
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direct comparison of observed bands. In this chapter, we give a standard methodology for carrying out immunoblot analysis on protein samples, from generating the samples to developing the blots. 1.2. Immunoprecipitation
Immunoprecipitation is a technique which uses one antibody coupled to an insoluble matrix to selectively isolate one protein or a group of proteins for further analysis by a second antibody. Generally, it is used to determine whether two different proteins are physically associated in a sample. However, when phosphospecific (or other modification-specific) antibodies for a protein are not available, the modification state of a protein can be determined by immunoprecipitation methods using an antibody to either the modification or the protein to isolate it from the sample and then a second antibody to detect the presence of protein/modification in an immunoblot. Whatever the investigation, it is important that either the two antibodies are derived from different species or that the antibody to be used in immunoblotting is biotinylated. The choice of antibody for the immunoprecipitation step is important. Many antibodies that are good for use in immunoblotting are not necessarily good for immunoprecipitation. This is due to the potential difference in the epitopes recognised. Whilst antibodies used in immunoblotting recognise linear epitopes (as the proteins are denatured for running on SDS-PAGE gels), immunoprecipitation requires antibodies that will recognise conformational epitopes in a folded protein. The lysis buffer used to generate samples is also important in immunoprecipitation. If too strong a lysis buffer is used, or a buffer which denatures proteins is used, then either any protein–protein interactions will be degraded or the antibody being used to precipitate the target group will be damaged and unable t
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