Troubleshooting with silver staining - (Nov/28/2013 )
I follow the quick silver staining protocol-
I am facing lot of problems in this-
- It takes lot of time in development, I start to see some bands only after 15 min.
- Background is too high.
- I add 10% acetic acid to stop the development, as soon as I add this, the faint bands just disappear.
If anyone is using this protocol, please help me in troubleshooting.
P.S. I am always using fresh reagents, including DTT
our silver stain procedure is a little different than yours (we use a method modified from that of merrill, et al).
however, if you add a crystal (small to medium) of sodium thiosulfate to your developer then your background should remain translucent. also, we wash 2x with 1/3 of the developer before developing with the final 1/3. we stop development with 1% acetic acid (actually, we usually use our coomassie destaining solution (30% methanol, 7% acetic acid) so that the gel shrinks back to normal) after removing the developer and washing with water a couple of times (to remove thiosulfate, it doesn't like acid).
careful with the water washes after silver incorporation. too much washing will remove too much silver (it is not bound tightly to the protein).
i almost forgot, make sure sds removal is as complete as possible, it binds silver like crazy and creates very strong background.
Like you said that your method is different from our method, earlier we were using vorum's method, where we used to use sodium thiosulfate for sensitization. But in this method, we are not using sodium thiosulfate, instead we are using DTT. I was wondering that principle of both the methods could be different in terms of components ... therefore I don't know if sodium thiosulfate can be used with this method?
And what do you mean by SDS removal? It should be removed by washes?...DO I need to increase methanol washes?
the method we perform uses dichromate to sensitize. we add the thiosulfate to the developer as a clearing agent.
we use 10% ethanol/5% acetic acid to wash out sds.