I am working on the brain tissue of mice (10% SDS-PAGE gel). The protein sample was extracted with lysis buffer (Tris-HCL, Triton, and B-mercaptoethanol). I think this leakage is due to the interaction between the protein sample and Triton. To be sure of this idea, I tried to do a control gel in which only the lysis buffer and the marker were loaded, where a well-separated marker refers to good electrophoresis, and after the staining step, the background was clear. what should I do?
1.centrifuge the sample and take supernatant for gel and dilute the sample to 1:10
2. for leakage there must be an issue with gel
Hi Basima
Try out these things:
1. Ensure your gel is properly solidified and not shrinked.
2. I would highly encourage to go for different concentrations of acrylamide for stacking and resolving part.
3. Sometimes, higher concentration of sample could be a trouble.
4. If sample is loaded for long without any voltage, the sample gets dissolved and spreads out of the wells.
*In the provided image, if second lane is the marker.... then it appears like the gel did not ran properly.
**In this case, I would recommend re run of a freshly made or preordered but intact gel .. if possible, a gradient gel... with a marker loaded in one of the lanes (to have a better idea)
Hope this helps :)
- Badges
- Science topic