Ultrafiltration Membrane Cloggged - (Nov/13/2007 )
Hi,
I am trying to concentrate and perform buffer exchange from a crude tissue extract of rat cartilage tissue, which was extracted overnight in 4M Guanidine with 10mM EDTA, 50mM Na-acetate (pH 5.8) and 1x Roche Protease inhibitor. To the best of my reading, Urea or detergents will not work for the ECM proteoglycans I'm trying to recover, and I would prefer not to perform precipitations because my sample is really small and I'm afraid of losing it. Nonetheless, I need to get rid of the Guanidine, and concentrate the sample about 10-fold for down stream applications. Therefore, I'm trying ultrafiltration of 500 ul of extract using a 3.5kD MWCO spin column with PES (polyethersulfone) as the filter material. So far, I've gotten only about 20ul to flow-through after an hour of centrifugation, at 21,000 x g @ 4`C, so I'm left with ~480ul that I'd like to be closer to 50ul.
So I'm wondering....
- I am clogging up the column (too much protein, nucleic acid, carbohydrate)?
- Is my buffer not appropriate for this filter media (Pall says this buffer is not tested for this system)?
- Should I pre-wash my filter media with water or some other buffer prior to adding my sample?
Any suggestions would be greatly appreciated!-JAH
I am trying to concentrate and perform buffer exchange from a crude tissue extract of rat cartilage tissue, which was extracted overnight in 4M Guanidine with 10mM EDTA, 50mM Na-acetate (pH 5.8) and 1x Roche Protease inhibitor. To the best of my reading, Urea or detergents will not work for the ECM proteoglycans I'm trying to recover, and I would prefer not to perform precipitations because my sample is really small and I'm afraid of losing it. Nonetheless, I need to get rid of the Guanidine, and concentrate the sample about 10-fold for down stream applications. Therefore, I'm trying ultrafiltration of 500 ul of extract using a 3.5kD MWCO spin column with PES (polyethersulfone) as the filter material. So far, I've gotten only about 20ul to flow-through after an hour of centrifugation, at 21,000 x g @ 4`C, so I'm left with ~480ul that I'd like to be closer to 50ul.
So I'm wondering....
- I am clogging up the column (too much protein, nucleic acid, carbohydrate)?
- Is my buffer not appropriate for this filter media (Pall says this buffer is not tested for this system)?
- Should I pre-wash my filter media with water or some other buffer prior to adding my sample?
Any suggestions would be greatly appreciated!-JAH
1. Try adding some DNase to your crude preparation, prior to the urea. That should fix up the DNA problem, and DNase will be easy to remove later (not that there is much to remove!).
2. If urea or detergents won't help you get the proteoglycans, why are you adding them?
3. Have you tried doing the buffer exchange first, using a fast desalting column? Once the sample is in the required buffer, then try concentrating.
4. good luck, and please tell us how things go.
Have you thought about a size-exclusion column? GE/Amersham sephacryl would probably clean up the sample, which then could be concentrated with a microcon filter, or even by spinvac.
I would suggest you to first do a dialysis against the buffer you wish to exchange, then put the dialysis bag in a shallow dish, like a 100 mm culture dish, and cover it with 1/2 cm thickness of PEG 8,000 powder, check it every 5 min to monitor the concentration step till your satisfactory. Simple and fast.