Western blot assay

Positive enzyme-linked immunosorbent assays are repeated in duplicate and if one or both tests are reactive, a confirmatory test is performed for final diagnosis. Western blot assay is the most commonly used confirmatory test, although an indirect immunofluorescence assay is available. 

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%,... 

Constantine, N. T. Bansal, J. Zhang, X. Hyams, . C. Hayes, C. Enhanced chemiluminescence as a means of increasing the sensitivity of western blot assays for HIV antibody. J. Virol. Methods 1994, 47(1-2), 153-164. 

Leong, M. M L., Fox, G. R., and Hayward, J. S. (1988) A photodetection devise for luminol-based immunodot and western blotting assays Anal Biochem 168, 107-114. 

To determine the extent of biotinylation of the BCCP fusion protein, cany out a supershift Western blot assay (again with an anti-His primary antibody) in which equivalent crude lysate samples are pre-incubated with or without streptavidin (0.1 g/mL) (Fig. 3 see Note 6). 

Ida, N Hartmann, T., Pantel, J et al. (1996) Analysis of heterogeneous A4 peptides in human cerebrospinal fluid and blood by a newly developed sensitive Western blot assay. J. Biol. Chem. 271,22908-22914. 

DNA, RNA, or protein, which can then be probed with a labeled sequence or antibody (Western blot assays), microscopy embedding, electrotechniques, skin protectants, microfilters, and others. Nitrocellulose continues to be used in photography, the manufacture of lacquers, patent and natural leathers, artificial pearls, process engraving, and cements. 

A western blot assay on treated cancer-derived cell lines (Fig. 8.9a, b) was performed to determine the in-cell specificity of WBZ 4. The immunoblots revealed specificity toward C-Kit consistent with the selective anticancer activity on the GIST882 cell line that expresses C-Kit. Thus, the activating phosphorylation of C-Kit at sites Tyr703 and Tyr721 in ST882 cells is inhibited by WBZ 4 in a dose-sensitive manner similar to imatinib (Fig. 8.9a). By contrast, phosphorylation of Bcr-Abl at Tyr 412 (22) in K562 cells was not significantly inhibited (<15%) by WBZ 4, while densitometry revealed an imatinib-induced inhibition of 85% (Fig. 8.9b). 

Fig. 8.9 (a) Western blot of C-Kit inhibition. WBZ 4 inhibits phosphorylation of C-Kit kinase in ST-882 GIST cells. Gel bands from the western blot assays of C-Kit and its phosphorylated (P) form in GIST cells treated with WBZ 4 and imatinib. The fi-actin assay was adopted as control, (b) Western blot of Bcr-Abl inhibition. Phosphorylation of Bcr-Abl kinase is not significantly inhibited by WBZ 4 in K562 CML cells. Electrophoretic gel bands for western blots for Bcr-Abl kinase and its phosphorylated (P) form in CML cells treated with WBZ 4 and imatinib. Reprinted from the [14], copyright 2007 with permission from the American Society for Clinical Investigation... 

L8. Leong, M. M. L., and Fox, G. R., Enhancement of luminol-based immunodot and western blot assays by iodophenol. Anal. Biochem. 172, 145-150 (1988). 

Fig. 3. Essential fatty acid deficiency-induced epidermal hyperproliferation and protein kinase C (PKC)-P activity are suppressed by dietary linoleic acid. Membrane-associated epidermal PKC isozyme (a and p) activities were determined in the epidermal extracts from control, essential fatty acid-deficient (EFAD) and reversed guinea pigs (EFAD guinea pigs restored to normal). Specifically, epidermal high-speed particulate membrane fractions were prepared and PKC isozyme activities from each dietary group were assayed as described previously (16). The upper portion of the figure represents PKC-p and PKC-a activities of the three dietary groups values are means SD (n = 12) from three separate experiments. The lower portion of the figure represents the expression of the 2 PKC isozymes. To determine PKC-P and PKC-a expression, 30 mg protein of solubilized epidermal membrane preparation from each dietary group was subjected to gel electrophoresis (SDS-PACE, 10 % gel) followed by Western blot assay with specific PKC-a and PKC-p. The gel electrophoresis data were reproducible in three separate experiments.

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