![]() Secondary antisera are generally prepared by injecting an animal with FC fragments of IgGs from a second species. ![]() Of primary antibodies, taking advantage of cross-species differences in antibody sequences. The secondary antibodies used in western blots are designed to bind the FC fragments In our western blots, we will use a mouse monoclonal antibody that binds the V5 epitope on Met and LacZ proteins expressed from the pYES2.1 plasmid. In addition, an antibody directed against an epitope can be used to detect many different proteins carrying that same epitope. Increasingly, researchers are using epitope-tagged proteins in their experiments, because antibodies against naturally- occurring proteins are expensive and time-consuming to prepare. Antibodies can be directed toward a naturally-occurring protein or toward an epitope attached to an overexpressed protein (as we are doing). Because our experiments do not require high sensitivity, rehydrated non-fat dry milk (direct from the grocery store!) is an adequate source of caseins.Įither polyclonal or monoclonal antibodies can be used as the primary antibody on western blots. In our experiments, we will use casein proteins from milk as blocking reagents. If the transfer membranes are not adequately blocked before the antibody is applied, the nonspecific sites on the membranes will absorb some of the antibodies, reducing the amount of antibody available to bind the ![]() Think of this step as analogous to an artist priming a canvas with a lower quality paint before the more expensive media is applied. 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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