What is the protocol for Western transfer after native electrophoresis? Is it possible to transfer a native protein that has a higher pI than the pH of the transfer buffer?

Product FAQ


Western transfer of native proteins is difficult due to the relatively low charge density of most native protein molecules. Therefore, the conditions most suitable for transfer of individual proteins vary. In a neutral-pH transfer buffer, proteins with low pI are expected to transfer toward the anode, as in denaturing electrophoresis, while proteins with high pI are expected to transfer toward the cathode.

First, consider whether native transfer is really required. Just because the gel electrophoresis was run under native conditions does not mean that the transfer needs to be native as well. Following a native gel run, proteins will be separated based on their native mobilities in the gel. Transferring with an SDS-containing buffer will not affect their positions on the gel - it will simply denature the proteins and make transfer much more efficient in most cases.

Whether you choose to transfer proteins under native conditions or under SDS (denaturing) conditions depends on the planned downstream analysis and whether your detection method requires proteins to be in their native state. If you do decide that you should transfer your proteins in native mode, and you are using Tris-Glycine gels, here are a few things to try:

1) Use the normal transfer buffer and place membranes on BOTH sides of the gel. Any proteins that are more basic than the pH of the transfer buffer will be captured on the membrane placed on the cathodic side. Both membranes can then be developed in the same manner.

2) Increase the pH of the transfer buffer to 9.2, which will allow all proteins with pI below 9.2 to transfer toward the anode.

3) Prior to blotting, incubate the gel for 15 min in Tris-Glycine Transfer Buffer with 0.1% SDS. During the subsequent transfer, use transfer buffer without SDS. The small amount of SDS in the initial incubation may give the proteins enough charge to move unidirectionally towards the anode and, in most cases, should not denature the proteins.

Blue Native gel electrophoresis is another method that can simplify the blotting of native proteins. G-250 dye is added to the samples, which imparts a slight negative charge to the proteins without denaturing them. Blotting after Blue Native electrophoresis will only require one PVDF membrane on the anode side, NuPAGE® Transfer Buffer, and no SDS. For more details on the Novex® Blue Native gel technology, see NativePAGE™ Novex® Bis-Tris Gels in our online catalog.

Answer Id: E3271

Was this answer helpful?