Losing Low Molecular Weight Proteins in SDS-PAGE - (Jul/11/2006 )
I have been running protein samples on 15% gels at 15mA, and over the course of the day, my 15 and 10kD markers are no longer visible. I then used a wet transfer at 5v for 1 hour onto 0.2u PVDF membrane. Ponceau staining showed no proteins under 20kD at all. I also checked with a histone H2a (13kD) antibody and saw nothing. My loading control, b-tubulin (~50kD), looked fine. I've done this experiment before on mini, ready-made gels, but I wanted to improve my separation using a larger gel, which I poured myself. I followed the protocol for pouring the gel exactly and it was pretty straight-forward. Is it possible that my proteins might be diffusing off the gel while they are running for some reason? I know it's possible to transfer them through the membrane, but it looked like the markers were gone before I transferred it. Also, my transfer protocol regularly works for small proteins on the ready-gels. One other oddity was that it looked like some of my samples precipitated when I loaded the wells. I've used these samples before with no problem. I would appreciate any ideas that anyone might have. Thanks!
you should stop when dye reaches the front. It sounds like you have run it for too long. There are pre-stained marker (Bio-Rad) to allow you to see where the bands are in gel as well as on the blot.
Histon is cationic protein and can form precipitates with SDS (which is anionic). you may want to use acid-urea buffer system.
Histon is cationic protein and can form precipitates with SDS (which is anionic). you may want to use acid-urea buffer system.
well, if you lose only the low molecules it COULD be possibel that you ran the gel too long:
do you have a prestained marker on the gel? then you would be sure not to run the electrophoresis too long so the small molecules have already run through.
do you check the gel after transfer by coomassie staining?
Cause the question is: didn't the low molecules transfer to the membrane or have they already passed through? to check the second possibility you could put a second membrane on top of the first for transfer...
running the gel should be no prob as with prestained marker you can see the progress - the transfer is another thing - I normally run it for 1h at 100V, 350mA and get good results as I am looking for a protein with about 20kD molekular weight...