Protein-blot analysis

Fig. 1.2 Protein blot analysis of human therapeutic protease inhibitor (HTPI) produced in alfalfa cell cultures using different promoters and subcellular targeting peptides as shown. Equal amounts of total soluble proteins from cell cultures were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinyldifluoride (PVDF) membrane. Monoclonal anti-HTPI IgGs were used for detection.

Fig. 1.4 Protein blot analysis of C5-1 assembly in agroinfiltrated alfalfa leaves. Total leaf soluble proteins, extracted 4 days after infiltration were separated by SDS-PAGE under non-reducing conditions and blotted onto a PVDF membrane. Polyclonal antimouse IgGs were used for detection. Purified C5-1 was mixed with total soluble proteins from control infiltrated alfalfa leaves and loaded as a standard.

Protein-Blot Analysis of Glycoproteins and Lectin Overlays... 

Gershoni,]. M., Bayer, E. A., and Wilchek, M. (1985) Protein blot analysis of glycoconjugates Enzyme-hydrazide—A novel reagent for the detection of aldehydes. Anal. Biochent. 146, 59-63. 

J.M. Wilson, P.E. Daddona, H.A. Simmonds, K.J. Van Acker, and W.N. Kelley, Human adenine phosphoribosyltransferase immunochemical quantitation and protein blot analysis of mutant forms of the enzyme, Biol. Chem. 257 1508-1515 (1982). 

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). 

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. 

Acridinium ester—labeled chemiluminescent probes have been utilized to detect the specific protein-coding transcripts and to distinguish between transcripts that code for the 190-kDa protein and the two closely related 210-kDa proteins. The assay is called the hybridization protection assay (D3). In this assay, RNA isolated from the patient s white blood cells is first amplified by PCR. The amplified product is incubated with the chemiluminescent probe. The unhybridized probe is removed by selective hydrolysis in sodium tetraborate buffer, containing surfactant Triton X-100 at pH 8.5, in an incubation step at 60°C for 6 min. After the sample is cooled to room temperature, the chemiluminescence of the hybridized probe is measured in a luminometer. The procedure is reported to detect one leukemic cell in a population of a million or more normal cells. It is also rapid, requiring less than 30 min. Its reliability has been attested to by correlation with results obtained on karyotypic and Southern blot analysis (D3). 

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. 

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