coomassie - (May/25/2005 )
I tried to destain my gel previously stained with Coomassie Blue in a solution of 40% methanol/10% acetic acid or in 25% methanol; I need it to do a silver staining.
But gel doesn't destain! It destains just a little and stop! Why? What is the problem?
You don't mention how long you've been destaining for...
I use 5% Acetic acid/50% Methanol.
Give it 30secs in the microwave... leave it three-four hours, and then change the destain.. again in the micowave... and leave it at least 24hours... Another trick is to put a sponge in the destain. That will "suck" the stain out of the destain and the gel...
I had the same problem till yesterday when i made fresh desatining solution and it solved the problem
Two factors affects the destaining rate .
1. temperature: it is faster at about 50C than at room temp. i have heard of people who literally boil in a microwave, but that is hazardous; methanol is higly inflammable.
2. concetration of methanol: 10% methanol is generally recommended in destaing. but better results are obtained with 50%. the idea is that usage of a higher concetration reduces the number of changes required as it can leach out more of the commassie stain. reused destain solutions reduce the rate because of being saturated with the stain. it resuse is necessary, then pass it through activated charcoal.