Which type of blot could be used to facilitate proteomic analyses?
Added by Allison S.
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Proteomics is the large-scale study of proteins. Show more…
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Western Blotting is a protein detection technique that combines the separation power of agarose and 2D gel electrophoresis, DNA isolation and ethidium bromide staining, chromatography and centrifugation, SDS-PAGE together with high recognition specificity of antibodies, Southern and Northern blotting.
Sri K.
The method of western blotting, also known as immunoblotting, is the most widely used technique for determining the approximate molecular weight of a protein. It involves gel electrophoresis, which is a method in which macromolecules are separated based on their size, charge, and other physical properties. SDS-PAGE is a form of electrophoresis in the presence of SDS (sodium dodecyl sulfate), an anionic detergent. SDS not only unfolds proteins but also gives them a negative charge. Calibration of the gel with marker proteins allows us to estimate the molecular weight of unknown proteins. The sieving properties of the gel are determined by the concentration of the gel. The range of separation of proteins is usually given in kilodaltons (kDa). Immunoblotting, also called blotting, begins by transferring the protein bands from the gel onto a nitrocellulose sheet. Once the blotting is complete, the next step is to treat it with primary and secondary antibodies.
A good way to increase total proteome coverage without using 2D PAGE is to: "Use two orthogonal types of chromatography." Enrich for phosphopeptides only. Analyze whole proteins instead of proteolytic peptides.
Adi S.
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