When performing a Western blot, proteins within a sample are separated by size, via gel electrophoresis, and then transferred from the gel to a membrane for detection by specific antibodies. There are wet, dry, and semi-dry transfer methods, though traditional wet transfer in a tank remains the most common method. During protein transfer in a tank, heat is generated, and the buffer can become hot. As the buffer temperature increases, the resistance lowers, and protein transfer becomes less efficient. The buffer also develops hot spots, resulting in inconsistent transfer efficiency across the membrane surface area. If the buffer temperature rises too much, the gel can melt and adhere to the membrane.
To avoid these issues, there are a variety of options for cooling the buffer. Many tanks are equipped with refillable ice packs or ice chambers, or the entire tank can be placed in an ice bucket or cold room. Still, these options lead to variations in temperature across the tank. To address this, many Western blotting protocols utilize magnetic stirrers to circulate the buffer during protein transfer to maintain a consistent temperature and avoid hot spots that can inhibit transfer.
Another crucial step to prevent overheating and ensure a high-quality transfer is to wash the gel before the transfer. This step is essential to remove salts from the gel electrophoresis running buffer, which can potentially contaminate the transfer buffer. By effectively removing these salts, the quality of the transfer can be significantly improved. To do this, soak the gel in a small amount of transfer buffer for 5-10 minutes prior to setting up the protein transfer. This incubation helps remove salts that have permeated into the gel. Discard the transfer buffer used to equilibrate the gel; don’t transfer it to the tank for protein transfer. If your instrumentation uses the same tank for gel electrophoresis as protein transfer, make sure to thoroughly rinse all components prior to transfer.
Disclaimer: Some protocols utilize a lower-wattage transfer in a cold room overnight, but for safety concerns, it is never recommended to leave the transfer running unattended. Additionally, it is not advisable to use a hot plate stirrer, as the heating component could be accidentally turned on, ruining your experiment and equipment.
