Artifacts only in Reduced samples!!! - (Sep/09/2010 )
Since the basis of silver staining is that the silver ions in solution are reduced to silver metal in the presence of proteins, I wonder if the reducing agent in your samples is causing metallic sliver deposits to appear as background. If this is the case, it's not due to the purity of your agents, but perhaps due to the quantity used?
Prep! on Wed Sep 22 03:28:35 2010 said:
hi md... the thing is the only difference between the reduced and non-reduced sample is the addition of DTT in the sample buffer... apart from that everything is same right from the stock solutions of gels till the running buffer and even the staining solutions!!!! that leaves my scratching my head!!!!
that's right, the artifact is released by the reducing agent. we eliminate the artifact by omitting the reducing agent from our samples (where we can) or by using freshly purchased reducing agent to prepare the sample.
i can't locate the paper but we found a procedure that uses sodium metabisulfite to eliminate the artifact (it didn't appear to work in our hands, we may have applied it incorrectly).
ultimately, we explained the artifact (with references) and ignored it in our publications.
mdfenko on Wed Sep 22 14:36:49 2010 said:
i can't locate the paper but we found a procedure that uses sodium metabisulfite to eliminate the artifact (it didn't appear to work in our hands, we may have applied it incorrectly).
Maybe it was this one (see at the bottom, just above "Anticipated Results")?
hey guys what do ya suggest... what shud be the final quantity of DTT in the sample... right now its 50 mM in my case... and the loading amount is 5 micrograms. I will decrease the concentration of DTT and see its effect... I dont wanna lose the imputiries too if they are present!!!
HomeBrew on Wed Sep 22 15:37:03 2010 said:
not that one, it's too new and is showing a destaining solution for silver stained gels (in the section that you cite), not something that will prevent the artifact.
if i get the opportunity i'll search for the paper(s) and give the reference (i have xerox copies, not electronic).
tat would be cool... and yesterday i did different amounts of DTT and i observed sumthing strange... with decreasing DTT concentration say from 100 mM to 10 mM in my final 1X sample buffer... the lower mol wt impurities increased in signal strength!!! can this be explained??? is it tat 10 mM is reducing everything but in excess the reaction is getting reverced.. which is not satusfactory explanation to convince myself!! haha 
another explanation can be that with increasing the DTT concentration the product is getting degraded more ( fragmentation or sumthing) and so the intensity is recucing with increasing DTT... this means that DTT is not only cleaving my disulp binds but also playing around with my protein.. is this normal??!!!
P.S. i have attached the picture.... the loading order is marker, 10 mcg protein with 10 mM, 50 mM, 75 mM and 100 mM DTT
also the artifacts have reduced.... i just ran the gel at lower voltage...
Just to be sure -- you did the experiment in such a way that not only the total amount of protein per sample was the same (10 ug), but also that the protein concentration per lane was the same for each sample (ug/ul)?
yeah hb... right from the sample concentratiopn to the loading volume.. everything is same in the gel except for the molarity of DTT!!!
ok guys... i found out the reason for the artifacts.... i know its a bit too late.. but dint get time for a loon time.... my protein being highly glycosylated is not a very efficient silver stainer... so it takes more time to completely stain the gel in which the artifacts start developing.. so the artifacts with DTT as u people pointed out is a very common phenomenon... i used the same DTT stock for another protein which efficiently takes up the silver stain and NO artifacts!!!! 
moreover i also tried TCEP... its a better reducing agent than DTT and NO artifacts!!! 