Assay protein, Western blotting

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). 

Proteins can be detected and quantitated by highly specific antibodies monoclonal antibodies are especially useful because they are homogeneous. Enzyme-linked immunosorbent assays and Western blots of SDS-polyacrylamide gels are used extensively. Proteins can also be localized within cells by immunofluorescence microscopy and immunoelectron microscopy. 

Western Blotting A powerful method for detecting a particular protein in a complex mixture combines the superior resolving power of gel electrophoresis, the specificity of antibodies, and the sensitivity of enzyme assays. Called Western blotting, or immunoblotting, this multistep procedure is commonly used to separate proteins and then identify a specific protein of interest. As shown in Figure 3-35, two different antibodies are used in this method, one specific for the desired protein and the other linked to a reporter enzyme. 

Samples for assay by Western blotting are separated by electrophoresis in SDS before blotting and antibody staining, so the reacting proteins are denatured. The use of an SDS-denatured antigen to generate the monoclonal antibody response to the "viral coat protein could assure that similar specificities are achieved in the test. 

Upon lipopolysaccharide stimulation (1 tg/ml), Prabhu et al. (2002) found significantly higher iNOS transcript and protein expression levels with an increase in NO production in selenium-deficient RAW 264.7 cells than in the Se-complemented cells. Electrophoretic mobility-shift assays, nuclear factor-xB-luciferase reporter assays and Western blot analyses indicated that the increased expression of iNOS in Se deficiency could be due to an increased activation and consequent nuclear localization of the redox-sensitive transcription factor NF-xB. 

The stability of a genetic modification may be analysed at the phenotypic and/or the genotypic level. The stability of phenotypic expression may be determined by trait characterisation or by analysis of sufficient samples, where appropriate, of RNA or protein expression. Some phenotypic traits e.g. resistances) may be quantified under testing conditions with the intact plant. As with other plant genes, expression of inserted DNA will be influenced by the environment. This should be taken into account during a phenotypic consideration of stability. Changes in patterns of expression or expression levels can be quantified in a biochemical reaction mediated by an expressed enzyme or by detection of the expressed protein with specific antibodies e.g. enzyme-Unked immunosorbent assay [ELISA], western blot analysis). 

The use of independent methods, other than IFIC, for quantitative demonstration of proteins is particularly important. Both enzyme-linked immunosorbent assay (ELISA) and Western blot may be employed to confirm the amount of protein in a cell/tissue model, and in the protein-embedding bar code model under both comparable fresh and FFPE samples for accurate... 

Ripe tomato fruits accumulate significant amounts of lycopene, but only trace amounts of xanthophylls. Dharmapuri and others (2002) overexpressed the lycopene (3-cyclase (b-Lcy) and (3-carotene hydroxylase (b-Chy) genes under the control of the fruit-specific Pds promoter, and transgene and protein expression was followed through semiquantitative reverse- transcription PCR, Western blotting, and enzyme assays. Fruits of the transformants showed a significant increase of (3-carotene, (3-cryptoxanthin, and zeaxanthin the carotenoid composition of leaves remained unaltered, and the transgenes and the phenotype were inherited in a dominant Mendelian fashion. 

There exists a wide variety in the setup of ELISA assays (direct binding or competition setups) and the enzymatic reaction utilized [148]. A similar principle to enhance sensitivity by enzymatic coupling is realized after gel electrophoretic separation of proteins. Here proteins are transferred to nitrocellulose ( western blot ) and detected by antibody-coupled enzymes. 

PAMAM dendrimers or modified PAMAM dendrimers (with oxiamine or sulfhydryl surface functionalities) exclusive of one another. These antisera recognize den-drimers in ELISA (enzyme-linked immunosorbent assays), dot blots and Western blots. The immunogenicity of dendrimer-protein conjugates has implications for therapeutic use of dendrimers as vaccines and we anticipate that antidendrimer antibodies will have applications in patterning and assembling nanostructures containing dendrimers. 

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. 

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%,... 

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