Western blotting technique

Third Step The Western blotting technique, applied to cell extracts, was used to confirm the pRB immunostaining results in two bladder cancer cell lines of T24 and J82, giving quantitative results for pRB in the two cell lines, comparable with that demonstrated by IHC (Fig. 1.3). 

Diamandis, E.P. (1993) Time-resolved fluorometry in nucleic acid hybridization and Western blotting techniques (Review). Electrophoresis 14, 866-875. 

West Basin Reclamation Plant, 26 84 Western blotting technique, 9 756... 

Patients and blood donors are routinely screened for exposure to HIV by means of ElISA and Western blot assays of blood samples (F uie 1-7-15). The assays are designed to detect antibodies to HIV in the blood of the test subject The ELISA is used as the primary screening assay because it is very sensitive. Because the reference interval for the test is set to include everyone with antibodies to HIV, it also gives false positives and thus has a rather low positive predictive value, especially in low-risk populations. The Western blot (or immunoblot) is used as the confirmatory test for HIV exposure. In the Western blot technique, specific HIV proteins are separated by gel electrophoresis and blotted to a filter. The filter is incubated with the test sample. If the sample contains antibodies to HIV, they will bind to the proteins on the filter. The filter is next washed and incubated with a labeled goat anti-human IgG to visualize any bound human antibodies. The Western blot is highly specific. The combination of an ELISA and Western blot has a positive predictive value of greater than 99%,... 

Liu, R. H. Jacob, J. Tennant, B. Chemiluminescent detection of protein molecular weight markers in western blot techniques. Biotechniques 1997, 22(4), 594-595. 

Wu, M., Stockleym P. G., and Martinm W. J. 2nd (2002) An improved western blotting technique effectively reduces background. Electrophoresis 23,2373-2376... 

Alterations of the mitochondrial membrane may precede those that occur in the nucleus. These changes can involve both the inner and the outer membrane, leading to a dissipation of the transmembrane potential and/or to the release of intermembrane proteins through the outer membrane. The main group of proteins responsible for mitochondrial alterations consist of the proteins known as Bax, which form pores in the outer membrane, causing the release of cytochrome c to the cytoplasm (Loeffler and Kroemer, 2000 Arden and Betenbaugh, 2004). Western blot techniques can be used to specifically detect the presence of cytochrome c in the cytoplasm of apoptotic cells. However, complex purification protocols are required, and there is the possibility of incomplete separation of mitochondria from the cytoplasm therefore, this technique is not very popular. 

Western blots have shown that the antibody raised to aphid myrosinase (Wye Q) was highly specific to a single band in crude extracts of B. brassicae by SDS PAGE analysis. Wye Q did not cross react with proteins (also using Western blotting techniques) from Sinapis alba and did not show a reaction to proteins from other Brassica pests tested (data not shown). Anti-plant myrosinase antibodies did not cross-react with B. brassicae proteins, and anti-aphid myrosinase does not cross react with plant myrosinase. The results of the Western blots are summarized in Table 6.1. 

The three best-characterized monoclonal antibodies to desmin are designated D33, DER-11, and DEB-5. By the Western blot technique, they have been shown to recognize desmin epitopes between residues 324 and 415 and to have no cross-reactivity with other lEPs. These reagents show tissue-specificity but species-non-specificity. 

Smith, H. V., Mayambo, T. M., Dunlop, E. M., and Holmes, P. H., Analysis of drug binding to serum macromolecules using a biotin-strepatvidin enhanced chemiluminescent western blot technique. In Bioiuminescence and Chemiluminescence Current Status (P. E. Stanley and... 

In the Eastern-Western procedure, lipids are first applied to a solid support such as nitrocellulose. Next, proteins subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis are transferred by standard Western blotting techniques to the solid support in such a manner that the protein of interest is transferred to the lipid patch. During electrotransfer of protein to the solid support, protein, lipid, and sodium dodecyl sulfate mix and as transfer continues the sodium dodecyl sulfate is removed leaving behind the protein to refold in the presence of lipid. Attachment of the refolded protein to a solid support allows one to probe protein structure using conformation-sensitive antibodies or protein function... 

Contrast the Southern, Northern, and Western blotting techniques. 

Figure 3.4 Western blotting technique. (A) Size separation of proteins by SDS-PAGE. (B) Transfer of the separated proteins onto nitrocellulose (NC) or poly-vinylidine fluoride (PVDF) membrane by electro blotting. (C) Visualization of proteins of interest by specific enzyme-conjugated antibody using immuno-staining. (D) Visualization of whole-protein extract by Coomassie Blue staining (left side), and immunostaining (right side), respectively.

Antibodies against R. rubrum Fj-ATPase are used for quantification of the protein in the chromatophore membrane by the Western blotting technique. As seen in Fig. 1, there is a linear relationship between the amount of Fi added and the area of the peaks for the a- and 3-subunits obtained in the recordings of laser scans of the autoradiographs. From four samples of chromatophores the mean value of Fi present in the chromatophores could be calculated to 3.5 pg Fi (nmol BChl) l. If 383 kDa (2) is used as the Mw of Fi, the chromatophores contain 9.1 nmol Fi (ymol BChl)" and 13.8% of the protein content is Fi-ATPase. Only a few Fo-moieties seem to be avoid of Fi since the chromatophores are well coupled. ATP synthesis exhibited high rates, 9 Vimol ATP (pmol BChl)"l and the ATPase activity was stimulated 3-4 fold by uncouplers. Thus it is assumed that the Fj-content is equal to the FqFi content. 

Fig. 1. Quantification of Fi in chromatophores from R. rubrum by the Western blotting technique. SDS-polyacrylamide gel electrophoresis (11) was performed with isolated Fj-ATPase from R. rubrum (o) and with chromatophores (0.95 nmol BChl, 1.9 nmol, ). Western blotting was performed as described in ref. 9. A laser scanner (LkB) was used to determine the optical density of the dots for the a- and 3-subunits on the radioautograph.

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