Western blot analyses

A. Western blot analysis of membrane fractions from P. vulgaris probed with a PGIP-specific antibody. 1) PGIP purified from P. vulgaris] 2) and 3) membranes 4) supernatant of the membrane preparation. 

Further characterization by SDS-PAGE and Western blot analysis of the culture filtrate of a 6 day-old culture of lindemuthiaimm showed that only one endoPG of the same molecular weight (42 kDa) as the pure endoPG (Hugouvieux et al., 1995) was induced on pectin. 

The entire eluted fraction and the aliquots containing 3% of input and 3% of flow-through are then mixed with 4x sample loading buffer, boiled for 3 min at 95°, and subjected to Western blot analysis, as has been described in Section 2.4. 

Unfortunately, there are no universal methods to detect all types of protein oxidation, because the products formed can be so diverse in nature. However, some forms of protein oxidation can be assayed using chemical modification (Davies et al., 1999 Shacter, 2000). In particular, the formation of carbonyl groups on proteins can be targeted using the reagent 2,4-dinitrophenyl-hydrazine (DNPH). This compound reacts with aldehydes to form 2,4-dinitrophenylhydrazone derivatives, which create chromogenic modifications that can be detected at high sensitivity in microplate assays or Western blot analysis (Buss et al., 1997 Winterbourn et al., 1999). 

Fig. 8.1 Western blot analysis of transgenic lines showing the expression of an assembled monoclonal antibody in transgenic chloroplasts. Lane 1 Extract from a chloroplast transgenic line, Lane 2 Extract from an untransformed plant. Lane 3 Positive control (human IgA). The gel was run under non-reducing conditions. The antibody was detected with an AP-conjugated goat anti-human kappa antibody.

The advent of immunoproteomics made possible the identification of highly immunogenic proteins that can be used for vaccine development. Proteins that have the greatest potential for eliciting a protective immune response are collectively referred to as the pathogen s immunome. Immunoproteomics has been utilized to characterize the immu-nome of B. anthracis for the development of a safer and equally efficacious vaccine. The immunoreactive proteins are first identified by using 2DE Western blot analysis in conjunction with mass spectrometry. In B. anthracis, for example, antisera from humans post-infected with anthrax were used to probe Western blots of its various... 

Alternatively, GFP can be visualized using rabbit polyclonal antibody raised against GFP purified directly from A. victoria. This anti-GFP antibody facilitates the detection of native GFP, recombinant GFP, and GFP-fusion proteins both by immunofluorescence and brightfield microscopy, as well as by western blot analysis and immunoprecipitation. Direct anti-GFP conjugates made from a complete serum or from an IgG fraction are available from Invitrogen (http //www.invitrogen.com/ site/us/en/home.html). Additional options for your research offered by Invitrogen include two mouse monoclonal antibodies and a chicken IgY fraction. 

Fig. 1.8 Western blot analysis (via His6-tag) of the expression of the SH3 domain using the wild-type DNA-sequence (lanes 1 and 2) or the optimized DNA-sequence (lanes 3 and 4). On lanes 1 and 3 0.25 pL DNA were loaded on the gel, while on lanes 2 and 4 0.125pLwere applied.

There are many examples of ELIS As used for detecting host cell impurities in the literature. Pauly et al.12 developed an ELISA to detect impurities in erythropoietin that had a detection limit of around 0.05 ng/ml. SDS polyacrylamide gel and Western blot analysis were used to confirm the spectrum of proteins detected and to demonstrate the specificity of the antibody preparation. Anicetti et al.14 describe an assay for the detection of E. coli proteins in recombinant DNA-derived human growth hormone. Whitmire and Eaton15 report on an immuno-ligand assay for quantitation of process-specific E. coli host cell contaminant proteins in a recombinant bovine somatotropin. 

Covalent protein adducts of quinones are formed through Mchael-type addihon reachon with protein sulfhydryl groups or glutathione. Metabolic activahon of several toxins (e.g., naphthalene, pentachlorophenol, and benzene) into quinones has been shown to result in protein quinone adducts (Lin et al, 1997 Rappaport et al, 1996 Zheng et al., 1997). Conversion of substituted hydroquinones such as p-aminophenol-hydroquinone and 2-bromo-hydroquinone to their respective glutathione S-conjugates must occur to allow bioactivation into nephrotoxic metabolites (Dekant, 1993). Western blot analysis of proteins from the kidneys of rats treated with 2-bromo-hydroquinone has revealed three distinct protein adducts conjugated to quinone-thioethers (Kleiner et al, 1998). 

Figure 3. (a) Western blot analysis of breast cancer derived cell lines showing an induced Cyclin D1 levels that correlates to a decreased SMARl expression, (b) A schematic representation of SMARl recruitment of HDACl on CyclinDl promoter, that switches off the transcription... 

Cerfain proteins were confirmed by Western blot analysis. The 2D PAGE analysis can resolve up to 2000 proteins. In this study, 820 spots were mapped to proteins but only 165 were used in the protein-to-gene analysis. Presumably these represent the highest fold expressed genes from the Ixmg tumors that were also visible on the gels by silver staining. 

Expression level as determined by Western blot analysis... 

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