Concentration of large volumes is a common bane of many biochemistry/protein production labs. Diaflow can be quite expensive and regular concentrators tend to take forever. Thus, my preferred solution is not to use concentrators at all.
1. AS cuts do not really *require* high protein concentrations. Hit it with high enough AS, and most proteins will precipitate. However, high AS cuts present a different problem - namely the density of the 'floaties' can be close enough to (or even less than) the density of the AS solution so that settling via centrifugation becomes difficult. Therefore: 2. Why concentrate at all when you can instead dilute the protein solution down to the ionic strength where the Q column is binding, then load the whole thing. As a rule of thumb, if you dilute to below 5-8 mS/cm most proteins (with the exception of seriously basic ones) will bind to Q resins. You can load in batch (by stirring the resin for an hour or two with your diluted sample), then spin the resin (gently), resuspend in small volume of buffer and pack a column. This will help you avoid flow issues, if your column cannot take enough pressure to allow fast flow loading. When you elute the protein from the column it will act as a concentrator since the elution profile may be controlled by means of gradient adjustments. If you have e.g. His-tag on the protein, you can use it to concentrate as well, via the same general principle. Do the math: at 20 ml/min (which is not even remotely close to what a properly packed XK 50 column would allow) you will only need 50 minutes to pass 1 liter of solution through your ion exchange column. In comparison, concentrating 1 liter down to e.g. 20 ml can take 30-60 minutes on a diaflow device, and several hours using a conventional concentrator. If you use batch, then the volume becomes nearly irrelevant and the time scheme looks like this: Stir (or nutate) protein solution with resin - 2 hrs. Spin the whole thing - 30 minutes at 5000 rpm. Resuspend, pack a column - 20 minutes. Run column - 30 minutes. This way you can process arbitrary amounts of stuff (up to the limit of your centrifugation volume, typically 6 liters) in about 3 hours. Artem P.S. It's kind of sad that people forget the 'poor student' techniques of protein biochemistry. -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Exec Sent: Thursday, June 26, 2008 11:29 PM To: CCP4BB@JISCMAIL.AC.UK 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.
