Once the proteins have been separated, they need to be made visible to the eye. A slippery and fragile gel is not the best thing to keep experimenting with, nor is it very compatible with the antibodies of development chemicals in the following steps of the Western blot. Therefore, the proteins need to be transferred onto something more practical Ė namely a thin membrane.
The membrane is usually made of either PVDF (polyvinylidene
fluoride) or nitrocellulose. It
is positively charged, and
when it is placed upon the gel, and an electrical current is applied to
the system, the negatively charged proteins are attracted to the
membrane. As a result, they are transferred to the membrane in the exact
same pattern as they had on the gel.
Tell me more about the different kinds of membrane! How do I know which one to use?
A nitrocellulose and a PVDF membrane vary in porosity, thickness, hydrophobicity, solvent compatibility, prize etc. However, it seems to be quite impossible to recommend one over the other in general terms of Western blotting application. Rather, which one of the two membranes that work best seems to be a totally individual preference, and it is probably a good idea to devote one Western blot run to determine which membrane that works best for your specific sample.
Whichever membrane you choose,
it is important not to forget that you need to treat the two membranes
in different ways prior to the transfer.
While a nitrocellulose membrane can be treated with
transfer buffer directly, a PVDF membrane first needs to be treated with
methanol and H2O in order to eliminate air bubbles which may
interfere with the transfer. On the other hand, a nitrocellulose
membrane is instantly ruined (it sort of dissolves) by methanol
How do I know that the transfer has been successful if I canít see the proteins yet?
Staining of the gel once the transfer is completed is an excellent way of investigating how effective the transfer has been. A Coomassie blue solution is very commonly used for this. After the staining, all proteins left on the gel are visible. Logically, the more thorough the transfer process has been, the smaller amount of proteins are left on the gel. Silver stain or ethidium bromide are two other common ways of staining.