I have experienced this myself. Is your protein especially acidic or basic? When purifying small proteins/peptides/single domains (less than 150 aa) that are either one or the other, coomasie blue may fail to stain the protein.
The way I got around this, was to first use a stain called Stains-All, and then silver-staining the gel. A protocol can be found here: http://sites.bio.indiana.edu/~ybelab/procedures/stainsallsilver.pdf It is time-consuming protocol, but in subsequent preps you know your protein is very pure when you see nothing on a coomaise stained gel :) Glenn On 10 February 2015 at 18:14, xaravich ivan <xaravich.i...@gmail.com> wrote: > Hello everyone, > > I was wondering whether anyone has had such an experience!!! > > I loaded a 35 mg/ml concentrated protein (in a buffer containing 50 mM Arg > and 50 mM Glu) for SEC in Superdex 200. I get a nice peak ~ 300mAu and have > a volume 12 ml consisting of a few fractions included in the peak. However > I cannot see anything in the coomasie gel when I load 20 uL of the > fractions from the peaks. I concentrated the protein to ~ 20-25 mgs/ml and > ran the gel and still nothing. > > The absorbance at 280 is high with a nice peak, but I can't see any band > in the gel. > > Has it happened to anyone? Any suggestions or comments will be invaluable > as always!!! Would silver staining help? > > Thanks in advance, > Ivan >
