In a western blot procedure, proteins are first separated on an SDS-PAGE gel and then transferred to a membrane. This membrane replica is treated with antibodies that specifically recognize a protein or epitope of interest. Additional processing steps generate a signal at the position of the bound antibody. Between the steps, various washes are done to increase the signal-to-noise ratio on the final, developed blot. The major steps in a typical western blot are diagrammed on the following page and discussed in greater detail in sections that follow:
- Electrophoretic transfer of proteins from an SDS-PAGE gel to a membrane
- Blocking of nonspecific protein binding sites on transfer membranes
- Incubation of the membrane with a primary antibody specific for the epitope of interest
- Incubation with a secondary antibody that recognizes primary antibodies
- Visualization of bound antibodies
Electrophoretic transfer of proteins from an SDS-PAGE gel to a membrane
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. In our experiments, we will use membranes made of polyvinylidine fluoride (PVDF), a kind of plastic. PVDF membranes are hydrophobic and the dry membranes do not wet properly with water. Therefore, PVDF membranes are first wet with methanol, then rinsed with deionized water, and finally rinsed with transfer buffer. They must not be allowed to dry out during the transfer and immunoblot procedures. If they do dry out, they must be re-wet with methanol and rinsed with water before proceeding.
During 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). During the electrophoretic transfer, current should flow evenly across the entire surface area of the gel. It is important, therefore, that air bubbles are not trapped between the gel and membrane. 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.
Blocking of non-specific protein binding sites on membranes
The transfer membranes used in western blots bind proteins nonspecifically. 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. Think of this step as analogous to an artist priming a canvas with a lower quality paint before the more expensive media is applied. 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
target proteins. In our experiments, we will use casein proteins from milk as blocking reagents. Because our experiments do not require high sensitivity, rehydrated non-fat dry milk (direct from the grocery store!) is an adequate source of caseins.
Primary antibody binding
Either polyclonal or monoclonal antibodies can be used as the primary antibody on western blots. Antibodies can be directed toward a naturally-occurring protein or toward an epitope attached to an overexpressed protein (as we are doing). Increasingly, researchers are using epitope-tagged proteins in their experiments, because antibodies against naturally- occurring proteins are expensive and time-consuming to prepare. In addition, an antibody directed against an epitope can be used to detect many different proteins carrying that same epitope. 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.
Secondary antibody binding
The secondary antibodies used in western blots are designed to bind the FC fragments
of primary antibodies, taking advantage of cross-species differences in antibody sequences. Secondary antisera are generally prepared by injecting an animal with FC fragments of IgGs from a second species. The first animal recognizes the FC fragments as foreign antigens and produces antibodies that bind the FC fragments. The secondary antibody in our experiment is a rabbit polyclonal antibody prepared against the FC domains of mouse IgGs. The antibody will bind the FC domains of the mouse anti-V5 antibodies bound to the pYES2.1-encoded proteins.
Visualization of bound antibody
In this final step, the western blot is incubated with substrates for the enzyme that has been conjugated to the secondary antibody. Our secondary antibody has been conjugated to HRP, a hardy enzyme with a high turnover number. (The turnover number is the number of product molecules produced at an enzyme’s active site per second.) HRP catalyzes the reaction of hydrogen peroxide and 3,3’,5,5’ - tetramethylbenzidine (TMB), which generates a dark blue- grey reaction product that precipitates at the reaction site on the western blot. Colored reaction product accumulates with time until the reaction is stopped by washing away unreacted substrate. The reaction should be terminated before nonspecific antibody binding becomes problematic, as evidenced by the appearance of many weakly staining bands.