Immunoprecipitation antibody specificity determination

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. 

The basic principle behind the ChIP method is relatively simple It is based on the selective enrichment of a chromatin fraction containing a specific antigen (e.g. transcription factors, DNA binding proteins, modified histones, etc.) by an immunoprecipitation step. Specific (important, see below ) antibodies that recognize a protein of interest or the modified form of a protein can be used to determine the relative abundance of it within DNA regions. 

N]-, [ C]-, pHjleucine or p Sjmethionine in the case of proteins) for various periods, after which the cells are lysed and the protein of interest is purified (often by immunoprecipitation with specific antibodies). The time course of isotope incorporation gives information about the rate (slope of curve) and extent (amplitude of curve) of the proteins synthesis. To measure degradation, cells are first pulse-labeled (i.e., exposed to radiolabeled precursor for a fixed period, after which sufficient nonla-beled precursor is added to reduce the radiospecific activity of the precursor). Then, the cells are further incubated, and the radiospecific activity of a particular protein of interest is determined (again usually after immunoprecipitation or some other means for achieving its isolation from other cellular proteins). The key point is that the chase allows one to stop radiolabel uptake almost instantaneously, thereby permitting the kinetic... 

Lysates of frozen tissue containing 900 pg of protein were immunoprecipitated with specific monoclonal antibody. The resultant immunocomplexes were resolved in 12.5% polyacrylamide slab gels containing SDS Western blotting was performed. After electrophoretic transfer of protein from the gel onto the nitrocellulose filter, protein was detected by the use of polyclonal antibody and 125I-labeled protein A. Quantitative differences in the levels of protein are determined by densitometric analysis. 

EMSA assays can also be exploited to measure STAT nuclear localization, which is, similar to NFkB localization, a measure of STAT activity. Determination of JAK phosphorylation is carried out by immunoprecipitation of the JAK proteins from cell lysates, followed by SDS-PAGE electrophoresis, immunoblotting with antiphosphotyrosine antibody and JAK-specific antibody re-probing [99]. 

MAPK is active only when phosphorylated on two specific amino acids, one threonine and one tyrosine. Because of this, MAPK activity can be measured by determining the content of phosphotyrosine in the enzyme using highly specific antiphosphotyrosine antibodies. Known amounts of protein are separated by polyacrylamide gel electrophoresis, transferred to sheets of nitrocellulose (or polyvinylidine difluoride), and probed with antiphosphotyrosine antibodies. To increase the signal produced by the detection system, it may be necessary to immunoprecipitate MAPK from known quantities of tissue, or to partially purify MAPK, biochemically, before immunoblot analysis. An increase in specific antiphosphotyrosine antibody binding implies that MAPK activity increases. 

Immunoprecipitation analysis can be used to determine if a specific protein, for which an antibody is available, becomes associated with a microinjected RNA. Moreover, antibodies against particular RNA modifications (e.g., 5 cap structures or modified bases) can be employed to determine if an injected RNA undergoes a particular posttranscriptional modification following oocyte injection. 

Immunoprecipitates prepared from metabolically radiolabelled cells can be used not only to assess the molecular size of a given protein but also to determine if it is phosphoiylated or is part of a multiple structure. Antibody-containing culture supernatants from cloned hybridomas can be used for immunoprecipitation by first binding the mAbs to a bead support coated with anti-Fab antibodies, then incubating the specific beads with extracts of the cells made with non-ionic detergents such as Triton X-100 or Nonidet 40. 

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