Zestern analysis

Zestern analysis is a form of immunoblot analysis developed to address the issues with existing immunoblot methods. This method provides a new solution to the cross-reactivity problem of the antibodies to significantly increase the efficiency of protein analysis.
Western blot analysis and Dot blot analysis are the most commonly used forms of immunoblot analysis. Western blot analysis is no doubt the most popular form of immunoblot analysis. The advantage of Western blot analysis over Dot blot analysis is that Western blot analysis is able to satisfactorily address the cross-reactivity issue of the antibodies. However, Dot blot analysis remains the technical basis of all forms of enzyme-linked immunosorbent assay (ELISA) and protein microarrays.
Western blot analysis is able to address the issue of the cross-reactivity of antibodies by the electrophoresis step. Proteins in the lysates are first separated based on their molecular weights in the electrophoresis step side by side with protein markers (A mixture of protein molecules with known molecular weight). In the detection step, the bands shown on the membrane are further differentiated by their molecular weights in reference to protein markers. Only the band with the expected molecular weight is considered a valid band, while other bands are considered non-specific bands due to the cross-reactivity of the antibodies. Nonetheless, this step also significantly limits the number of protein samples that can be analyzed at one time.
Zestern analysis is able to eliminate the electrophoresis step in the process of immunoblot analysis without sacrificing the specificity of the assay. In an otherwise a typical Dot blot analysis, an elution step is added before the detection step. Immunocomplex formed on the membrane is allowed to access a solution containing excessive amount of competing molecules. The competing molecule can be synthesized antigen, or partial antigen, or multiple repeats of antigen or partial antigen within one molecule. The competition is able to happen due to the reversibility of the antigen-antibody interaction. Antibodies are liberated by competing molecule from membrane into the elution solution. Quantification of the amount of antibodies in elution solution through reporter assay give reliable indication of the amount of antigen in the protein samples.
The advantages over other methods of immunoblot analysis
Compared with existing methods of immunoblot analysis, Zestern analysis has several advantages.
* It eliminates the electrophoresis step to increase the efficiency of protein analysis;
* The result can be directly quantified, rather than being image-based as in the case of Western blot and Dot blot analysis; and
* The process of peptide blocking (Peptide competition assay) is well incorporated in this process to validate the result.

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