Department of microbiology microbial food technology group a diploma in quality assurance in microbiology diploma



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Transfer


In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose orpolyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this "blotting" process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e. binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings.

The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane withCoomassie Brilliant Blue or Ponceau S dyes. Ponceau S is the more common of the two, due to Ponceau S's higher sensitivity and its water solubility makes it easier to subsequently destain and probe the membrane as described below.[6]


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