Western blot transfer step

In the transfer step of Western blotting, the proteins are transferred from the gel to a membrane. This membrane is typically made of nitrocellulose or polyvinylidene difluoride (PVDF). Compared to PVDF, nitrocellulose is more suitable for low molecular weight proteins, less likely to have background signal (but is also less sensitive), and less suitable to be stripped of antibodies and reprobed with different antibodies.

As in protein gel electrophoresis, the proteins move from the cathode to the anode. In the classic Western blot procedure the gel and membrane are stacked together and placed into a tank filled with buffer. The gel and membrane are protected by filter paper and sponges and held tightly together by a cassette. Since the ions in the buffer conduct the electricity, all the components of this stack should be thoroughly soaked in buffer. The gel and membrane should also be in close contact with one another without air bubbles. To prevent overheating, an ice pack is added to the tank. The cassette is typically color-coded so that the user can ensure the gel is facing the cathode and the membrane facing the anode. For more information on potential issues, check out Western blot troubleshooting.

Other systems exist for Western blot that look slightly different from the one in this simulation, but they are all based on the same principles.