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. Incubation with a secondary antibody that recognizes primary antibodiesĮlectrophoretic transfer of proteins from an SDS-PAGE gel to a membrane.Incubation of the membrane with a primary antibody specific for the epitope of interest.

Blocking of nonspecific protein binding sites on transfer membranes.Electrophoretic transfer of proteins from an SDS-PAGE gel to a membrane.The major steps in a typical western blot are diagrammed on the following page and discussed in greater detail in sections that follow: Between the steps, various washes are done to increase the signal-to-noise ratio on the final, developed blot. Additional processing steps generate a signal at the position of the bound antibody. This membrane replica is treated with antibodies that specifically recognize a protein or epitope of interest. In a western blot procedure, proteins are first separated on an SDS-PAGE gel and then transferred to a membrane.

Sds page vs western blot vs elisa