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. Thanks -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi
