Protein blotting is an analytical method that involves the immobilization of proteins on membranes before detection using monoclonal or polyclonal antibodies. There are different blotting protocols (dot blot, 2D blot); one of the most powerful is western blotting.
In western blotting (or immunoblotting), prior to protein immobilization on the PVDF or nitrocellulose membranes, sample proteins are separated using SDS polyacrylamide gel electrophoresis (SDS-PAGE) providing information about molecular weight and the potential existence of different isoforms of the proteins under study.
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1. Many people reuse SDS PAGE running buffer. This may lead to higher background signal as some of the proteins from previous samples may be pulled into the current gel. These extra proteins may be deposited on the membrane by electrophoretic transfer.
2. Air pockets are not compatible with the passage of electric current. Pressing the gel and membrane ‘sandwich’ together by gently using a Pasteur pipette as a rolling pin can reduce the occurrence of air pockets between the layers.