RE: [ccp4bb] Concentrating proteinInstead of using solid NaCl with dialysis tubing use PEGs which have molecular weights larger than the cutoff of the dialysis membrane. This avoids the need to remove the NaCl later. If you can get away with loading the solution directly on the column or in batch that is the way to go as far as time is concerned, as suggested previously. Jeremy Lohman University of Oregon
----- Original Message ----- From: First, Eric To: [email protected] Sent: Friday, June 27, 2008 8:16 AM Subject: Re: [ccp4bb] Concentrating protein Back in the old days, we used to put the protein in dialysis tubing and surround it with solid NaCl (a 2 liter graduated cylinder works well for this). After several hours, rinse off the outside of the dialysis tubing, readjust the dialysis clips and remove excess tubing, and dialyze against your favorite buffer. I suggest wrapping parafilm around the ends of the dialysis clips, as the dialysis tubing will swell when you dialyze out the NaCl. You lose some protein due to adhesion to the dialysis tubing, but it works fairly well. Eric First Dep't of Biochemsitry and Molecular Biology LSU Health Sciences Center in Shreveport -----Original Message----- From: CCP4 bulletin board on behalf of Exec Sent: Thu 6/26/2008 10:28 PM To: [email protected] Subject: [ccp4bb] Concentrating protein Dear All, we have GCSF protein produced in inclusion bodies. we solubilise it refold it and then concentrate it using proflux system. still the concentration of the protein we get is less and volume is more for us to load in Ion exchange chromatography. is there any simple technique that can be performed in lab without using any hi-fi instrument to concentrate the protein in small volume of buffer. the protein we obtain is about 0.7 mg/ml and we get 450 ml solution. our column is 110ml lab scale and we have to work in that only. i have heard of NH4SO4 precipitation. but it requires protein conc more than 1 mg/ml. kindly help me to progress in my experiment.
