Western Blotting

Western Blotting

Western Blotting is an application of antibodies that, unlike ELISA, which only allows determination of the presence and concentration of an antigen of interest in a given sample, also reveals data about the nature of the antigen detected, such as its molecular weight, isoelectric point, tertiary structure, and, in some cases, its biological activity. Western Blotting involves fractionation of a protein sample by gel-based methodologies such as SDS polyacrylamide electrophoresis, isoelectric focussing, or two-dimensional electrophoresis, which is used routinely in proteome analysis, followed by transfer of the protein from the gel onto a membrane by force of an electric field. The membrane, which thus displays a replica of the gel pattern, is then probed with specific antibody or other applicable biological probes (ligands, antigens, substrates etc.) and the result is visualized through a marker conjugated to the probe or an appropriate secondary antibody as described under ELISA. In clinical settings, Western Blotting is routinely used to confirm serious diagnosis suggested by ELISA such as HIV seroconversion. In the research lab Western Blotting is used extensively to characterize and quantitate antigens and even probe for biologic activity such as ligand binding or enzymatic activities.

Antibody Solutions has generated many monoclonal and polyclonal antibodies for Western Blotting applications and has developed optimized protocols.

For example, we have made a monoclonal antibody (AS55-P) to SLP-76, a cytoplasmic protein that is expressed in T lymphocytes and myeloid cells and is a substrate for ZAP70 protein tyrosine kinase. As shown in the left image, our antibody detects SLP-76 in total lysate of T-cells but not in B-cell lysate. Activation of the T-cell receptor leads to ZAP70 mediated phosphorylation of SLP-76, which is shown by the right image by Western Blotting with commercial anti-phosphotyrosine antibody on SLP-76 immunoprecipitated from activated T-cells with AS55-P.

Polyclonal peptide-directed antibodies are commonly used to detect the parent protein, which the peptide fragment was derived from, on Western Blots. Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G), is an antiviral factor involved in host defense against HIV which is believed to render many primate species resistant to HIV infection. In human, this defense mechanism is overcome by HIV's viral infectivity factor (Vif) (Sheehy et al., 2002). Using an APOBEC3G-specfic antiserum custom-made by Antibody Solutions in Western Blotting, Stopak et al. (Molecular Cell, 12:591, 2003) showed that Vif alone in the absence of other HIV proteins is sufficient to cause depletion of APOBEC3G in HIV-infected human cells.

Antibody Solutions offers a wealth of expertise in developing antibodies for Western Blotting applications. Please contact us with your specific application needs.

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