I have experienced problems applying the protocol of the SilverQuest Silver staining kit. I had to increase the volume of the solutions being used and also the incubation time with the silver solution. In spite of the changes that I made to the basic protocol the spots in the upper side of the gel are unfilled.
Since I read that those spots unfilled, are caused by protein overload I loaded less protein. The first gel I'm showing has 50 ug of protein and the 2nd one has 30 ug and the results are far from being better. Besides the problem of the staining, the 2nd gel has enlarged spots in the bottom side, I don't know why.
Hi Nancy,
Have you came across the 2D gel troubleshooting guide from vendors (E.g. http://www.bio-rad.com/en-cn/applications-technologies/vertical-streaking#gel2).
They should contain suggestions to overcome the observed problems.
http://www.bio-rad.com/en-cn/applications-technologies/vertical-streaking#gel2
Dear Nancy,
negative stainig is due to overload, the mechanism of silverstain is still unknown, most likely a complexformation, try our prtocoll in Methods of malaria research 2008
All the best
Ernst
How old is the kit? Are the DTT and Iodoacetamide fresh? Reagent oxidation from age is a frequently encountered problem, especially with DTT solutions. The SilverQuest™ Silver Staining Kit says it has sub-nanogram sensitivity. That means you are loading more than 30,000 times the detection limit. I understand it is separating across the entire gel, but you may still be overloading.
I did a lot of 2D gels and you silver staining looks fine, I think that you should improve your 1st and 2nd dimension runs. Second gel indicates that you have streaking problems in 2nd dimension separation - so try to use fresh DTT and iodoacetamide as Christopher suggested, and carefully measure time of both treatments (it is crucial that DTT and IAA steps are not too long neither too short).
What I can see is that polymerisation of your 2nd dimension gel isn't highly reproducible between runs, have you tried pre-cast gels?
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