General Western Blotting and Detection Procedures

In brief, the transfer consists of a gel and a membrane sandwich, with the gel placed closer to the cathode and the membrane positioned closer to the anode. When current is applied, the negative charge of the proteins (conferred by the SDS applied during electrophoresis) causes them to migrate from the gel onto the membrane. The transfer is conducted under constant voltage, with the time and current settings inversely related (i.e., higher current decreases transfer time). It is important to note that higher currents may overheat the system, resulting in melting of the gel or poor transfer.

Setting Up a Wet or Semi-Wet Transfer

Table 1 shows the recommended settings for transferring proteins in Life Technologies gels using the XCell II™ semi-wet blot module. Note that the expected current listed in the table is for transferring one gel. If you are transferring two gels in the blot module, the expected current will double. Figure on left shows the correct setup of a blot for a wet/semi-wet transfer.

Figure 1. A wet/semi-wet transfer setup for western blotting.

Table 2 contains the recommended settings for transferring proteins using the Novex® Semi-Dry Blotter. The figure below shows the correct setup of a blot for a semi-dry transfer. It is important that the pressure on the gel stack(s) be even without being too firm.

Table 2. Recommended settings for transferring proteins using the Novex® Semi-Dry Blotter.

Table 3 contains the recommended settings for transferring proteins in gels using the iBlot® Gel Transfer Device. Figure 4 shows the correct setup of a blot for a dry transfer using the iBlot® Gel Transfer Device.

Table 3. Recommended settings for transferring proteins in gels using the iBlot® Gel Transfer Device

Once proteins have been transferred onto a membrane, they can be probed with antibodies. The proteins on the membrane are exposed to antibodies either through passive diffusion or through electrically driven binding. Passive diffusion involves submerging the membrane in a solution that contains the diluted antibody, washing of the membrane, and then incubation with a diluted secondary antibody. The extent of dilution that works best will depend on the antibodies used. Electrically driven probing of a membrane is performed using the iBlot® Gel Transfer Device and the iBlot® Western Detection Kits, which have anode and cathode stacks designed to move antibodies toward the transfer membrane for western detection.

After the membrane has been probed with labeled antibodies, the target proteins on the membrane can be detected by the method appropriate to the label. Enzymatic detection involves the addition of an enzyme substrate to the membrane followed by washing unbound reaction products and unreacted substrate away from the membrane. Chromogenic reaction products can be visualized by examining the membrane directly or by analyzing a visible-light image of the membrane. Chemiluminescent reaction products can be detected by exposing film to the membrane or by using a chemiluminescence scanner. Fluorescently labeled antibodies can be visualized directly by placing the membrane in an appropriate scanning instrument that can excite
and detect the bound fluorophore.

When multiple proteins need to be detected on a single membrane, the traditional approach is to strip the primary and secondary antibodies from the blot and reprobe with different primary and secondary antibodies. One common method of antibody removal is the use of a heated, low-pH solution containing glycine. The combination of heat and low pH causes dissociation of the antibody–antigen complex. After the membrane has been stripped, it can be reprobed with different antibodies. When planning to strip and reprobe a membrane, it is recommended that you use PVDF for the transfer. Because PVDF is a more durable material, it is less prone to damage during the stripping process than is nitrocellulose.