![]() Before the membranes are incubated with specific (and expensive) antibodies, they must be pretreated with blocking solutions that contain high concentrations of abundant (and cheap) proteins to saturate non-specific binding sites. The transfer membranes used in western blots bind proteins nonspecifically. After the electrophoretic transfer, which can be done in a few hours or overnight with reduced voltage, the membrane replica with the transferred proteins can be allowed to dry out and stored for later visualization with antibodies.īlocking of non-specific protein binding sites on membranes It is important, therefore, that air bubbles are not trapped between the gel and membrane. ![]() During the electrophoretic transfer, current should flow evenly across the entire surface area of the gel. If they do dry out, they must be re-wet with methanol and rinsed with water before proceeding.ĭuring the transfer process, the gel and membrane are placed directly against each other within a “sandwich” of pre-wet filter papers and foam pads (see the figure below). They must not be allowed to dry out during the transfer and immunoblot procedures. Therefore, PVDF membranes are first wet with methanol, then rinsed with deionized water, and finally rinsed with transfer buffer. PVDF membranes are hydrophobic and the dry membranes do not wet properly with water. ![]() In our experiments, we will use membranes made of polyvinylidine fluoride (PVDF), a kind of plastic. The first step in a western blot is to generate a replica of the SDS-PAGE gel by transferring proteins electrophoretically to a synthetic membrane with a high protein binding capacity.
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