Dear all, Thank you for the prompt and valuable suggestions. I had made a typo in my earlier mail. I use 50mM NaCl, not 5mM NaCl. I am facing this problem only while using DDM in buffers. My other runs, even with 8M urea run perfectly fine.
I will try increasing the salt concentartion and degas by methods suggested. All the detergents I use are from Anatrace and store them in a vacuum box at -20. But just befre weighing, I don't bring the detergent to room temperature before weighing. Would this be causing the problem, as suggested by Bert? If yes, how do you handle the detergents? Thanks again! -Nazia On Sat, Jul 27, 2013 at 6:09 AM, Ho Leung Ng <[email protected]> wrote: > If your buffer can't go through a 0.2 micron filter easily, you > shouldn't run it through your FPLC. Detergent purity may be an issue. > I also experienced problems filtering a buffer containing CHAPS from > vendor X. When I switched to Anatrace CHAPS, no more clogging. > > > Ho > > Ho Leung Ng > University of Hawaii at Manoa > Assistant Professor, Department of Chemistry > [email protected] > > > -------------------------------------------------------------------------------- > We are trying to purify a membrane protein using different detergents (DDM, > OG etc.). We have tried using 1mM DDM in 20mM Tris, 5mM NaCl and 5% > glycerol buffer to purify the protein. however, we are facing problems in > running the buffer in 16/60 Superdex 200 pg gel filytration coloumn using > AKTA Explorer. The entire machine is in a cold cabinet. The buffer was also > kept at constant slow stirring, thinking that it might be getting > precipitated, which we are not able to see. Still the back pressure is very > high and the in-line filter keeps clogging. We have filtered the buffer > through a 0.2 micron filter, which too was very difficult. the > > Has anyone faced a similar proble? Or is there a way that buffers with > detergents are supposed to be made? Or are there any particular coloumns > meant for such runs. > -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi
