The first thing is that you'll have to increase the salt concentration in your buffer. 5 mM is way too low and may cause non-specific binding of the protein to the resin. 100 mM is the minimum you should use. There is nothing in your buffer that will precipitate, so you should't have to worry about that. Running the columns at 4C and using 5% glycerol will result in a relatively high back pressure even for a new column, especially if you have the flow restrictor in place. But its hard to see how anything in your buffer could precipitate and clog things up. Are you storing the DDM at -20 and let it get to roomtemp before you weigh it out? If not I could see it getting hydrolysed over time and form alcohols which are poorly soluble.
Bert ________________________________ From: CCP4 bulletin board [[email protected]] on behalf of Nazia Nasir Phd2009,ProteinCrystall.Lab [[email protected]] Sent: Friday, July 26, 2013 1:45 PM To: [email protected] Subject: [ccp4bb] Off topic: Gel filtration of membrane protein 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
