Is there any additives in your buffer which can absorbing at 280nm ? Satya
On Tue, Feb 10, 2015 at 1:31 PM, Keller, Jacob <[email protected]> wrote: > 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]> > 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 > > >
