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The quantitative immunoblot is a similar approach to ELISA. It uses the same principles as the ELISA, a reliance on the highly specific antibody antigen interaction to measure the amount of a given protein in a complex mixture. The major distinguishing feature of this method is the inclusion of a step to electrophoretically separate the proteins in the sample according to their molecular weights. Thus, all samples can be solubilized using a standardized procedure and analyzed concurrently for a number of interesting proteins.

The separation step allows the researcher to be more confident that the signal generated actually results from the target protein in question because it is spatially resolved from cross-reactions which would confound an ELISA system. When quantitated standards are included on the blot, the samples can be quantitated using the available software. A tool to define the contour is used to select the area for quantitation and the values are background subtracted to give an adjusted volume in counts for each standard and sample.


Source: www.mosbio.eu