I am currently trying to run a western blot on BRCA2, a nuclear protein that is ~ 400kDa. I have done the SDS PAGE and membrane transfer a few times and I am getting no signals except for faint bands just underneath the wells. The transfer seems to be okay (coomassie stain of the gel after the transfer shows a clean gel). I have run the sample in standard Laemmli buffer with a reducing agent in both a 5% gel and a 4-15% gradient gel at 125V for more than 2 hours (until I run out all the visual markers except for the 220kDa marker) and cannot seem to get the band any further into the gel. The coomassie stain suggests that some other proteins >220kDA migrate into the gel which is very confusing.

Any suggestions or tips on running immunoblots on very large proteins it would be very much appreciated.

Joseph Ahn

Joseph Ahn,

You are in a tough range to visualize by Western blot.

In "Molecular Cloning, A Laboratory Manual " (Sambrook, Fritsch, Maniatis) The effective range of separation of SDS-Gels is

15% = 12-43 kD
10% = 16-68 kD
7.5% = 36-94 kD
5% = 57-212 kD

Which explains why your protein is basically in the wells since it is barely entering the gradient gel.

That being said I have been able to detect apoB100 (550kD) by Western blot using a 4-20 % gradient SDS-PAGE gel run until the markers stop, then running the gel for 2 min after switching the leads (positive to negative), then transferring onto 0.45 um Nitrocellulose for 1hr on a Biorad semi-dry apparatus in Bjerrum buffer. Ive also heard of people transferring at low voltage overnight at 4C on wet blotters.

IF those fail can you get away with a dot-blotting your protein?

Rb