Coomassie Blue: destaining
Arnoud van Vliet
avvliet at knoware.nl
Fri Jul 14 13:26:22 EST 2000
> I am using PhastGel BlueR coomassie blue dye from Pharmacia, I am
> with destaining the gel without losing the signal from the bands (I have
> around 10ng protein per band)
> I am currently destaining in 10% Acetic acid, this takes quite a long time
> and leaves an uneven blue background. I was considering using methanol to
> improve solubility of the CB stain. How much methanol should I use without
> risking loosing signal from the bands?
I wouldn't use methanol or ethanol, as the Coomassie Blue often becomes
light blue. I get much better (but slower!) destaining using an
approximately 12.5% isopropanol, 10% acetic acid solution. The proteins
remain bright, dark blue, while the gel destains completely. The speed can
be increased by brief heating in the microwave (<15 sec at 700W), followed
by 5-15 min shaking at room temp. Then refresh destain and repeat, etc.
Staining can be quicker by microwave heating as well.
hope this helps
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