I have been running gel zymography (7.5% polyacrylamide co-polymerized with 1% gelatin) loaded with 250 µg of protein / well from colonic lysate with the hopes of visualizing MMP-2 and -9. I had a few good gels, but recently I have been getting these white spots on my gels (see attached) post-Coomassie staining and de-staining, following a protocol by Frankowski et al., 2012 (doi: 10.1007/978-1-61779-452-0_15).
Can someone explain where these big spots are coming from? It makes it impossible to quantify my images. I thought it was the Coomassie itself, perhaps due to chunks of undissolved Coomassie; however, I filtered the stain and still have the same problems. My most recent gel looks the exact same as this image, with the same two white circles.
Additionally, is using 1% gelatin appropriate? I have seen researchers use 0.1-0.2% gelatin co-polymerized with the polyacrylamide, but the source I listed above used 1%.
Thank you!
Dear Aydin Herik
It looks like a reflection from light bulbs illuminating the gel from top. Then, these "spots" should not be visible by naked eye. If so, the problem is with the gel image capturing, not the gel itself.
Regards
White spots appearing on gel zymography, post-Commassie staining and destaining could indicate that the protein bands are not completely destained. This can occur if the destaining solution is too dilute or the destaining process is not continued long enough.
Hi Tomasz Fraczyk ,
Thank you for the reply. The spots are visible with the naked eye as areas where the staining is less intense (i.e., lighter blue).
Dear Aydin Herik
Is it possible that in the lanes with white spots there is much more MMPs than in other lanes? Are those spots appearing for the same samples in different gels or are they located in the same place of the gel no matter which samples are in there?
Hello Tomasz Fraczyk
Thank you again for the reply! The left most side contains samples from mice infected with C. rodentium which should (in theory) contain more MMPs. The samples on the right contain control mice proteins. However, the samples in the C. rodentium and control groups also included liver samples, which evidently have a lot more MMP content. I will re-run my gels tomorrow with only loading buffer, and a set of gels with a lower concentration to see if this occurs again. The protein concentration may be too large as 50 mg of protein / well does not result in this weird staining.
Thank you for your help, I will update you soon!
- Badges
- Science topic
- Similar topics
- Computer Science and Engineering
- Bioinformatics