Thanks, David. I believe the leaked column was probably my case, although I didn't see any visible leakage I re-tightened connections anyway. But the weird thing was that after I equilibrized the column for a while, the dry patches and cracks disappeared. Everything returned to normal even a standard ran fine. That's why I suspected the mixing of reagents in the column has something to do with it in the beginning.
Nian Huang, Ph.D. UT Southwestern Medical Center On Sun, Aug 28, 2011 at 5:14 PM, David Briggs <drdavidcbri...@gmail.com> wrote: > Hi Nian, > > It can be a number of things - but typically, air getting into the > column, or a leaky seal somewhere letting it dry out. > > Switching from 150mM NaCl to 20% EtOH directly shouldn't cause a problem. > > The reason I suggest it is that I have had the same situation as > described in the original post (protein comes off across entire volume > of SEC column elution) and it turned out the column had a leak and had > dried out in places. > > Repacking the column solved the problem. > > Cheers, > > Dave > ============================ > David C. Briggs PhD > Father, Structural Biologist and Sceptic > ============================ > University of Manchester E-mail: > david.c.bri...@manchester.ac.uk > ============================ > http://manchester.academia.edu/DavidBriggs (v.sensible) > http://xtaldave.wordpress.com/ (sensible) > http://xtaldave.posterous.com/ (less sensible) > Twitter: @xtaldave > Skype: DocDCB > ============================ > > > > On 28 August 2011 22:35, Nian Huang <huangn...@gmail.com> wrote: >> Hi, David, >> What is the common cause of knackered SEC column? Will equilibrizing a >> buffer containing 150 mM NaCl directly into a 20% EtOH or vise versa cause >> the problem. There was no problem just after packing the column. >> >> Nian >> >> On Sun, Aug 28, 2011 at 9:24 AM, David Briggs <drdavidcbri...@gmail.com> >> wrote: >>> >>> Following on from Roger's fine suggestions: >>> >>> 8. Your column is knackered. Can you see fine lines or cracks in the >>> column? Good packing is v.important for SEC columns. >>> >>> HTH, >>> >>> Dave >>> >>> >>> ============================ >>> David C. Briggs PhD >>> Father, Structural Biologist and Sceptic >>> ============================ >>> University of Manchester E-mail: >>> david.c.bri...@manchester.ac.uk >>> ============================ >>> http://manchester.academia.edu/DavidBriggs (v.sensible) >>> http://xtaldave.wordpress.com/ (sensible) >>> http://xtaldave.posterous.com/ (less sensible) >>> Twitter: @xtaldave >>> Skype: DocDCB >>> ============================ >>> >>> >>> >>> On 28 August 2011 10:25, Allan Pang <a.p...@qmul.ac.uk> wrote: >>> > Hi there everyone, >>> > >>> > What does it mean when you have proteins eluting in almost the whole >>> > column >>> > volume of S200? >>> > >>> > I ran a gel with fractions from 8ml to 20ml and saw band for my protein >>> > all >>> > throughout. >>> > >>> > Judging peaks on chromatogram is not useful as it doesn't have any >>> > aromatic >>> > rings. >>> > >>> > Cheers, >>> > >>> > Allan >>> > >>> > -- >>> > Allan Pang >>> > >>> > PhD Student >>> > >>> > G35 Joseph Priestley Building >>> > Queen Mary University of London >>> > London >>> > E1 4NS >>> > >>> > Phone number: 02078828480 >>> > >> >> >
