I have done multiple SDS-PAGE but since few weeks I am getting a blue smear that does not allow to see bands lower than 30 - 25 kDa.
My gels are 15% Polyacrylamide.
I have change all solutions, add a sample control ( to make sure they were not my samples the issus), rinse well after using alcool (between resolving and stacking gel to get a perfect resolving up-line gel).
It's not super easy to see what's going on, but it seems like you have a pretty low concentration of proteins (looks like a cell extract) loaded on the gel. And de-staining is not complete. So the bands could be still there, but you would need to de-stain it better.. Your marker looks fine, so it's not an issue with the gel itself.
If even with complete de-staining you cannot see the bands, then I would suggest silver staining, which will give you higher sensitivity. Or just load more material.
Good luck!
As suggested by Ana, It seems that you have loaded a low concentration of proteins, and the de-staining is not complete. If you de-stain a bit more, you may able to see the band. Alternatively, you can increase the protein load and try.
Thank you all for your reply. Even if i do more destain I am not able to see bands because of the smear and blue spot (gel 2). About increasing protein, I will try! Thank you :D
Instead of increasing the protein concentration, you can use a more sensitive stain like RuBPS (see for example doi:10.1007/978-1-59745-542-8\_14 for a step-by-step guide to this simple and cheap method).
In your second gel, the bands in the middle lanes look funny. This isn't a problem with the gel, as other lanes look normal. It is a problem with the sample, I can think of several reasons:
- Too much salt in the sample or a very inappropriate pH. Precipitate the protein with MeOH/CHCl3 according to doi:10.1016/0003-2697(84)90782-6 before addition of sample buffer.
- Heavily glycosylated protein. Use CTAB-PAGE instead of SDS-PAGE (doi:10.1016/S0003-2697(02)00639-5)
- Protein degradation. Take measures to control proteases.
- Protein not properly unfolded. Ensure that you have SDS and either ßME or (better) DTT present in the sample buffer. Heat the sample 15' at 60 °C, do not boil. There are (very, very few) proteins that do not unfold under these conditions, check the literature about your protein.
Thank you Engelbert Buxbaum for all the references. I will check :D
Consider tricine-based SDS-PAGE, and if you're only interested in the smaller proteins, consider maybe one of those dialysis tubing based protein separators with a MW cutoff a little above the mass you're interested in, and if necessary some concentrating maybe by desiccation.
- Badges
- Science method