Or use copper staining—just add 300 mM copper chloride to your gels right after running, see your results in ~5 min, when non-protein parts of the gel become foggy white. Should be indifferent to the nature of the protein, and this method is actually super-sensitive.
JPK From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Glenn Masson Sent: Tuesday, February 10, 2015 1:26 PM To: [email protected] Subject: Re: [ccp4bb] Vanishing protein in coomasie stained SDS-PAGE gel 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 <[email protected]<mailto:[email protected]>> 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
