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 >> > > >
