I have tried running my samples on a 10% separating gel after heating them for 5 minutes at 98C and placing them on ice. They were heated in standard laemmli buffer. The ladder separates fine, but all my samples consistently stay at the top of the gel, with only a couple managing to produce an additional band of around 70kDa (in the attached photo the white horizontal line corresponds to the junction of the stacking and resolving gels). I run the gel at 150V for 1 hour and 45 minutes until the dye front completely leaves the bottom of the gel. The samples are proteins purified by sucrose gradient and resuspended in 0.01M sodium phosphate buffer.
It seems that your protein is highly aggregated. One thing you might try is to skip the heating step. This step sometimes causes problems. You may need to add fresh reducing agent to the sample buffer if it has become oxidized.
Hi Elizabeth,
Since this looks like a solubility issue, I would use a stronger, eg., 5x sample buffer to solubilize the proteins.
As already suggested by Adam B. Shapiro above, the sample buffer needs to have freshly prepared reducing agent added to it, just before electrophoresis. I assume that your 0.01M phosphate buffer is above pH 7?
Good luck,
Hediye.
Hello! Adam ended up being correct. I tried rerunning my samples without heating them yesterday, and I was able to get beautiful separation of bands.
Wonderful!
That is the value of this site. Thank you Adam.
Best,
Hediye
- Badges
- Science topic
- Similar topics
- Linguistics
- Composition Studies
- Publication