![]() Nitrocellulose (NC) and polyvinylidene difluoride (PVDF) membranes with a pore size of 0.2 μm are the most common types of membranes used for the transfer of proteins. Plastic- and cellulose-based solid surfaces, in the form of thin, microporous sheets of about 100-μm thickness, are used for the transfer of biomolecules, such as nucleic acids and proteins. This study was conducted with a set of three randomly chosen membrane proteins of high, medium and low molecular weight. Here, our focus is on three of the variables, including type of membrane, type of blocking agents and concentration of methanol in Towbin's transfer buffer (TTB), that we believe play a critical role in enhancing the detection sensitivity of proteins. In addition to the ratio between protein applied to the gel and the concentration of primary and secondary antibodies used, other variables also influence protein detection sensitivity in western blotting. The optimal combination of primary and secondary antibodies is then used to obtain the maximal signal for the protein of interest. Primary and enzyme-conjugated secondary antibodies are titrated at various dilutions against a specific amount of protein sample that is then electrophoresed. To establish the optimal detection conditions for specific protein in western blotting, immunotitration is generally performed. However, obtaining maximal sensitivity for the detection of a specific protein remains a fundamental issue, leading for new ways of enhancing detection sensitivity by making changes in the protocol for specific proteins. ![]() The techniques used in western blotting have evolved greatly since its inception in 1979 by Towbin et al. The term ‘western’, for protein transfer from gel to membrane, has been used to maintain a geographical naming tradition after ‘Southern’ blotting, first described by EM Southern, for the transfer of DNA to membrane and later ‘northern’ blotting for the transfer of RNA to membranes. In this procedure, crude lysates are first separated based on their molecular weight by SDS-PAGE, transferred to a solid membrane surface and detected with the help of protein-specific antibodies. Western blotting is a commonly used procedure for the detection and characterization of proteins.
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