I run 15% SDS-PAGE gels, at about 100 mA for 2 hours. When I do only 1D my samples show up fine, lots of protein bands. However, when I use IPG strips (3-10), do IEF overnight and run the gels, no spots appear... The colored MW markers run normally in the gel, I can see them fine, but no spots appear. I initially thought that maybe my agarose sealant was the problem, since it didn't solidify very well, but I made a fresh batch and now it's fine.
Common problems I have read about no spots in silver staining include:
- temperature (too low, or too high), which is not the case,
- wrong silver nitrate dilution (also not the case, and it's a new reagent from Sigma)...
- Using bicarbonate instead of carbonate for developing solution (also not the case...)
- the only problem I could think of was the thiosulphate, although I have used the same batch before and it worked FINE... and when I used NEW GE healthcare thiosulphate no spots appeared either...
What bugs me the most is that I KNOW the MW markers are there, and not getting stained!!!
Anyone help? Suggestion?
Thanks a lot!
Edited by rachelhauser, 13 August 2010 - 05:10 AM.