Immunoprecipitation method

Immunoprecipitation method for elF2B assay (Immunoprecipitation of endogenous elF2B complexes)... 

The use of an immunoprecipitation method has been reported for detecting Hu, Ri, and Yo antibodies [165, 166], This method is highly specific and sensitive, as it can detect low levels of antibodies not demonstrated by immunohistochemistry or immunoblotting, and is useful in prevalence studies. However, low levels of antibodies are often not correlated with clinical disease and must be regarded with caution in patients without neurological symptoms. For instance, 25% of patients with SCLC have low levels of Hu antibodies [167]. There are also case reports of individuals with cancer and high antibody levels without PNS, even after prolonged observation [166, 168],... 

Weinmann AS, Farnham PJ. Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. Methods 2002 26(1) 37 17. 

Jialal I, Hirany SV, Devaraj S, Sherwood TA. Comparison of an immunoprecipitation method for direct measurement of LDL-cholesterol with beta-quantification (ultracentrifugation). Am J Clin Pathol 1995 104 76-81. 

The relative amounts of polypeptides in the cotyledons were determined by a Western blotting analysis. The relative synthesis rates of proteins were determined by in vivo labeling with (135s] Met and the immunoprecipitation method. The relative amounts of hybridizable mRNA were determined by a Northern hybridization method. 

Immunoprecipitation methods may be employed in conjunction with electrophoretic separations in at least two ways In the first, specific antibodies or mixtures of them are used to form precipitation arcs with the individual electrophoretically separated proteins. As the method is ordinarily employed in immunoelectrophoresis, small differences in electrophoretic mobility are difficult to detect. However, modifications have been developed which make the method more precise. For example, chimpanzee transferrin, haptoglobin, and a2-niacroglobulin have mobilities different from their human homologues. When human and chimpanzee sera are run in parallel, and antiserum is diffused in from both sides and allowed to form complete ovals around the antigens, these mobility differences are readily observed (Fig. 3), as shown by Williams and Wemyss (1961). Methods for increasing the resolution of this system are now under development in the MAN Program laboratories at Oak Ridge and may solve the problem. 

Immunodiffusion and immunoprecipitation, developed ia the 1940s as a means to identify and semiquantitate specific proteias, were the direct precursors to the development ia 1953 of Immunoelectrophoresis, a method used ia many clinical laboratories (5). Single- and double-gel immunodiffusion and immunoelectrophoresis were, ia effect, the first standardized and routinely used immunoassay methods (see Electroseparations, electrophoresis). 

Niranjanakumari, S., Lasda, E., Brazas, R., and Garcia-Bianco, M. A. (2002). Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo. Methods 26, 182—190. 

The concentration of protein in the lysate should be determined as a guide to even loading of gels or the amount of material to be subjected, for example, to immunoprecipitation. A simple and reliable method for this is that of Bradford (Bradford, 1976). 

However, FLIM measurements by both frequency and time domain using commercially available software suffers from variation in the measured lifetime from region to region in cells [36], which can be confusing. In the light of the potential pitfalls associated with each of the above-mentioned FRET techniques, potential protein associations should ideally be tested independently by a combination of two or more FRET-based methods in addition to biochemical techniques such as co-immunoprecipitations. 

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. 

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-... 

For immunoprecipitations from native tissues, one requires antibodies directed against both the fish and the bait proteins. Further, these antibodies should not bind to epitopes within the putative protein-protein BDs. It is technically difficult to determine low affinity or transient association among proteins by immunoprecipitation because low-affinity interactions may be lost by washing immune pellets to remove nonspecifically bound proteins. Also, one cannot manipulate protein concentrations to favor protein association as one can in a pull-down assay. Under these circumstances, probably the best method to use would be FRFT. 

Although the standard MSP is an appropriate approach for promoter methylation status screening, our results clearly showed that subsequent evaluation of the differential promoter methylation level should be conducted by quantitative MSP (qMSP) to quantify the degree of these alterations to make clear correlations to the severity of the psychiatric disorders (H.M. Abdolmaleky and S. Thiagalingam, unpublished). In addition, it could be further validated using a complementary quantitative method such as immunoprecipitation (IP) of the methylated DNA to confirm the results of the qMSP (34). 

The specificity of fluorescence localization is dependent on the specificity of the primary antibody, a property that must be tested and controlled by other methods, such as immunoprecipitation or immunoblotting. Controls for the labeling procedure described include deletion of the primary antibody step, which controls for the second-step reagent, or inclnsion of a similar, but nonreactive antibody as a first step. In the case of the availability of purified primary antigen, competition controls can be used, but they only control for the reactivity of the antibody with one antigen, and do not mle ont the possibility of a crossreactive, but unrelated antigen. 

Chromatin Immunoprecipitation as a Powerful Method to Study Chromatin... 

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. 

The combination of chromatin immunoprecipitation with DNA microarrays allows the genome-wide analysis of the distribution of an antigen. The immunoprecipitated DNA is quantitatively amplified, labeled and used to probe DNA microarrays. In principle ChIP-on-chip methods can be divided into two basic groups, depending on the content of the microarrays which are used (i) microarrays/promoter tiling arrays and (ii) genome tiling arrays. 

---