Immunoprecipitation and Blotting

Immunoprecipitation and blotting both use antibodies (normally, but not exclusively monoclonal antibodies) to detect and quantitate specific protein antigens in complex cellular or protein mixtures. Immunoprecipitation has an advantage in that the antigens are allowed to react with the antibodies in their native conformation prior to their subsequent separation and quantification. A further advantage is that a protein at a very low concentration can be concentrated from the relatively large volume of 1–2 mL. The major disadvantage is that the proteins normally have to be radiolabeled to facilitate their detection. In Western blotting the proteins do not have to be labeled, but they have to be separated by electrophoresis in polyacrylamide gels prior to their transfer to either nitrocellulose, polyvinyldifluoride (PVDF), or nylon membranes. This seriously restricts the size of the sample, and hence the protein antigen has to be present at higher concentrations. A further disadvantage is that the antigen is not normally in its native conformation when it reacts with the antibody, because the electrophoresis usually being carried out in the presence of sodium dodecyl sulfate (SDS) to maximize the resolution of the separated proteins. If an antibody has a lower affinity for an antigen, it may well immunoprecipitate an antigen but not react with it on a Western blot. This is the reason why some workers slot-blot their protein mixtures rather than separate them by electrophoresis. This maintains their native conformation. The main problem encountered here is that crossreactions of the primary antibody with all the other proteins in the mixture can outweigh the antigen-specific binding. An unlabeled antigen is often therefore prepurified using an immobilized lectin or even immunoprecipitation itself prior to its quantitation on a slot-blot. Such a combination of lectin and antibody techniques has tremendous potential both in more precise analysis of cellular antigens and in the characterization of disease progression. These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.