Antibodies immunoprecipitation

Peltz, G., et al. (1987). Monoclonal Antibody Immunoprecipitation of Cell Membrane Glycoproteins, Anal. Biochem. 167 239-244. 

Figure 4. Phosphorylation of cytokeratins in intact hcpatocytes. Hepatocytes were loaded with Pi and further incubated for 15 min in the absence or in the presence of 3() M KN-62. Incubations were continued for an additional 15 min period with or without O.S fiM OA. Cells were subsequently disrupted and cytokeratins were immunoprecipilatcd with an anti-pan cytokeratin monoclonal antibody. Immunoprecipitates were subjected to SDS-PAGE and autoradiography. The experiment was repeated three times and similar results were obtained.

Langer PR, Waldrop AA, Ward DC (1981) Enzymatic synthesis of biotin-labeled polynucleotides novel nucleic acid affinity probes (nucleotide analog/DNA and RNA polymerase/avidin-sepharose/antibiotin antibody/immunoprecipitation). Proc Nat Acad Sci USA 78 6633-6637... 

Fig. 6. Early (1962) glycoclusters used in quantititative immunoprecipitation of anticarbohydrate antibodies.89...

Heat at 37°C for lOminutes to fully solubilize and maintain crosslinked proteins, and then enrich specific complexes by immunoprecipitation using an immobilized antibody specific for the bait protein that was used. Alternatively, heat at 96°C for 20 minutes to solubilize and break all crosslinks (this may be used as a control). 

The advantage of such co-purification protocols is that the fully processed protein serving as the bait can allow interactions in a native environment and cellular location to allow isolation of multicomponent complexes. One limitation with this approach is the necessity for an antibody with specific immunoreactivity and immunoprecipitative capability for the bait protein. This drawback can be addressed by expression of the protein with an epitope tag. Excellent antibodies to a variety of epitope tags are available and can be utilized for immunoaffinity purification. Tags such as 6-histidine and GST allow purification using affinity characteristics to nickel and GSH beads, respectively. 

Immunohistochemistry is a powerful method for identification of proteins in cells and tissues, but control procedures are necessary. The specificity of the results depends on two independent criteria the specificity of the antibody and of the method used (Burry 2000). The antibody specificity is best determined by immu-noblot and/or immunoprecipitation. The specificity of the method is best determined by both a negative control, replacing the primary antibody with the corresponding IgG (must be the same species as primary antibody at the same concentration), and a positive control, testing the primary antibody with tissues known to contain the antigen. 

Alternatively, GFP can be visualized using rabbit polyclonal antibody raised against GFP purified directly from A. victoria. This anti-GFP antibody facilitates the detection of native GFP, recombinant GFP, and GFP-fusion proteins both by immunofluorescence and brightfield microscopy, as well as by western blot analysis and immunoprecipitation. Direct anti-GFP conjugates made from a complete serum or from an IgG fraction are available from Invitrogen (http //www.invitrogen.com/ site/us/en/home.html). Additional options for your research offered by Invitrogen include two mouse monoclonal antibodies and a chicken IgY fraction. 

Major progress in the analytical use of antibodies occurred with the development of several fundamental techniques the ability to detect cell-bound antibody (Coombs Test, 1945) immunoprecipitation in gels (Ouchterlony, 1953) radioimmunoassay (Yalow and Berson, 1960) and monoclonal antibodies (Kohler and Milstein, 1975). These, together with a con-... 

Gels are used in immunoprecipitation techniques to stabilize the precipitate, enabling both the position and the area of the precipitate to be measured. The point has already been made that maximum precipitation occurs when the equivalent proportions of both antigen and antibody are available. Hence, if a high concentration of antigen is permitted to diffuse into a gel that contains a uniform concentration of antibody, at some point in the concentration gradient of antigen that is... 

Amongst other techniques are those involving the immunoprecipitation of the bound fraction using a second antibody, which reacts with the proteins of the first antibody. This second antibody may be produced by immunization ... 

This assay method (RIPA) is used primarily in research. It is too technically demanding for routine use in clinical laboratories. HIV is cultured in radio-labeled cells, or viral proteins are directly labeled with a radioisotope. The virus is disrupted and then exposed to the test specimen. Specific antigen-antibody complexes are concentrated and isolated by immunoprecipitation. After exten-... 

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