Western blot antibodies specificity determination

Many ancillary techniques have been developed in recent years, but several stand out in their ability to extend the usefulness of 2D6EL. This final section will discuss four of these Western blotting, antibody production from gel spots, and metabolic labelling to determine amino acid composition and tyrosine-specific phosphorylation. 

Chemokine receptor expression on the VP can be determined by Western blot using specific antibodies, by ELISA on VP-coated plates, or by flow cytometry. In this last case, small particle size limits measurement of the surface proteins by conventional techniques, and particles are coupled to latex beads to allow detection. 

The Western blot method is often used in the analysis of host cell impurities. It can be used to identify a recurring impurity. O Keefe et al. used a Western blot to identify an E. coli protein impurity in the preparation of the recombinant fibroblast growth factor (aFGF).29 By using specific antisera to the E. coli host cell proteins, they were able to isolate the impurity and determine its N-terminus amino acid sequence to confirm its identity. Antibodies could be used to determine the concentration of this impurity in sample preparations. 

P-gp is constitutively expressed in nearly all barrier tissues. Techniques involving Northern blots (37) or Western blots with monoclonal antibodies such as C219 (38) and MRK 16 (39) have been used extensively to determine the tissue distribution of P-gp. It is expressed in adrenal cortex, kidney, liver, intestine, and pancreas endothelial cells at blood-tissue barriers, namely, the CNS, the testis, and in the papillary dermis (3,4,38,40,41). P-gp displays specific subcellular localization in cells with a polarized excretion or absorption function. More specifically, P-gp is found at the apical (AP) canalicular surface of hepatocytes, in the AP membrane of the columnar epithelial cells of colon and jejunum, and the AP brush border of the renal proximal tubule epithelium (3,4,40 1-2). In endothelial cells, P-gp is located in the luminal membrane (4,43). 

The main advantage of the ELISA over Western blotting is its ability to analyze many samples at one time. In addition, modifications to the general ELISA protocol can be used to quantify the exact concentration of antigen in an unknown solution, the concentration of a specific antibody in an unknown solution, the dissociation constant for a particular antigen-antibody interaction, or the relative affinity of two different antibodies for the same antigen. An example of an indirect ELISA, and its use in the determination of the concentration of an antigen in an unknown solution, is the subject of Experiment 17. 

GST-Raf-RBD protein complexes is resolved by Western blotting. High-quality, isoform-specific anti-Ras antibodies, which allow determination of activation of a specific Ras isoform, are widely available (41). Only GTP-bound Ras can bind to GST-Raf-RBD, so this affinity reagent provides a quick and easy way to determine the relative levels of cellular GTP-Ras when compared with the total Ras protein. For example, resting cells contain only 5% Ras-GTP compared with total Ras, whereas growth factor-stimulated cells may show 50% to 70% Ras-GTP. 

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. 

Specificity of the active caspase-7 ELISA was determined by analyzing captured polypeptides. Capture antibody is the caspase-7-specific antibody coated in the ELISA 96-well plates and is different from the anti-caspase-7 antibodies used for western blotting. Polypeptides that are bound by the capture antibody are referred to as captured. Jurkat cells were incubated with STS for 0-4 h and then with bzVKD-fmk for an additional 1 h. Cell extracts were incubated in 6-well plates coated with caspase-7 capture antibody. After washing, polypeptides captured on the plate were solubilized in SDS sample buffer and Western-blotted. Captured polypeptides were blotted with anti-caspase-7 LSU and anti-caspase-7 SSU to detect polypeptides derived from caspase-7. Captured polypeptides were blotted with HRP-streptavidin to detect the polypeptides covalently modified with the bzVKD-fmk inhibitor. Captured polypeptides covalently modified with bzVKD-fmk is the material that the ELISA quantifies. 

To determine if differential glycosylation of fibronectin (Fn) in inflammatory synovial fluid (SF) included expression of an oncofetal epitope (Onf Fn) previously detected only on Fn derived from embryonal or neoplastic tissue [61], Fn was purified from plasma, SF and synoviocyte conditioned medium by affinity chromatography and analyzed by sodium dodeeyl sulfate-polyacrylamide gel electrophoresis and Western blotting using a monoclonal antibody (FDC-6) specific for the Onf Fn. The Onf Fn was not expressed on... 

Closely linked to bioactivation is the formation of hapten-carrier complexes. Generally the binding capacity of reactive compounds to model carrier peptides or nucleophilic amino acids is determined. For this also a number of assays are available for instance using HPLC or LC, in combination with MS or NMR (Singh et al., 2004 Ahlfors et al., 2005). If the new chemical can be radiolabeled, binding to peptides can be easily detected. In cases where specific antibodies to the compound or to structurally related compounds are available, immunochemistry techniques such as Western blotting or even immuno-histology can be applied. 

Specificity - A description of how the producer determined that the antibody binds only the listed antigen. Most of the time this is a western blot (with a blot shown), but it can be immunocytochemistry (with an image shown). Sometimes data are included about binding to other related proteins or to posttranslationally modified (e.g., phosphorylated) proteins. Some vendors who use a peptide for making an antibody will also sell the peptide for an absorption control. More information about specificity is discussed in the Chapter 9, Controls. Frequently, there are no data given for the specificity. 

Detection of the symporter protein can also take place through various means. In western blotting, proteins harvested from cells or tissues can be separated on a denaturing polyacylamide gel, transferred to membranes, and then probed with specific antibodies directed toward the symporter protein that can indicate whether the symporter was present in that specific tissue. These specific antibodies may also be utilized in a process called immunohisto-chemistry. In this method, the tissue remains whole and is sectioned into thin slices for easy study under a microscope. Treatment of this tissue with the antibody directed against the symporter then allows for the detection of the protein within the tissue slice. A final method of determining symporter expression and iodine uptake makes use of the fact that some iodine isotopes are radioactive. The location of these radioactive isotopes can be determined by using cameras that detect iodines radioactive emissions. This allows... 

Proteins are usually detected by immunohistochemical procedures. Some proteins can be detected directly (e.g. by their errzymatic activity). However, the number of detected proteins depends on the availability of specific antibodies and often determined by the quality and the quantity of the surgical specimen. Immunohistochemical procedures discussed in the prior section have the advantage that the morphology of the tumor is still conserved and can be studied simultaneously with additional information regarding cellular antigens and their localization. In addition to immunohistochemistry, modern proteomics offers new approaches to detect proteins by western blotting, 2-dimensional gel electrophoresis or by protein arrays. 

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