Hi Nazia, The DDM concentration you mention seems fine to me. I routinely run the FLPC at 4C with buffers containing 0.05% (1mM) DDM and 2% glycerol without issues. I have also used buffers with 5% glycerol in the past but I usually adjust the flow rate, as needed and when I know all else is good with the column and the machine.
In addition to what Bert's comments and suggestions, here are a few more common culprits, which have to do with general FPLC and column maintenance and wear and tear: (1) Air bubbles in the system, which need to be manually purged out. (2) A Superdex200 column clogged with years of protein buildup (3) A clogged in-line filter (ideally should be replaced every couple of months (i) If your buffer is the problem, then flowing filtered water through the system without any columns hooked up should drop the pressure. (ii) If the inline filter is the problem, then the pressure will be higher than usual even in water or buffers without glycerol/urea/GuHCl and without any column attached. In that case, swap out the old one for a new inline filter. (ii) And if the column is the problem, the appropriate column-specific cleaning procedures are outlined in the manufacturer's manual. Also, in case you are not aware, suggestions and procedures for all the above can be found in the FPLC manual. Hope this helps. Raji On Fri, Jul 26, 2013 at 8:57 AM, Bert Van-Den-Berg < [email protected]> wrote: > 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 > -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
