dialysis of purified protein - (Jun/14/2005 )
I have taken an Abs reading before and after dialysis (3,500MWCO), and after dialysis with 50mM ammonium bicarbonate, it appears that I am loosing nearly half of my purified protein. Does anyone have any suggestions as to what is happening?
1. Did protein ppt. during dialysis? This is common.
2. Did you use the same buffer to take the OD? Different buffers can give different reading.
3. The best thing is to run a few microlitres of your sample before and after dialysis to ensure it
is at the same conc.
Also, why are you using 3500 MW cutoff? I am wondering because Ialso use 3500 MW cutoff tubing for various reasons and I was wondering if you have any particular reason why you use this size as opposed to the 12-14 MW cutoff which seems to be more common?
My protein did not ppt. at all, I had dialyzed in Amm. Bicarbonate O/N, and that is the buffer that I used as a reference. When I compared the Abs reading after dialysis to the Abs reading I had collected before dialysis it was significantly less (and I took it several times in case of a mistake.) The really wierd thing is that I had two dialysis tubes in the Amm. Bicarbonate, one was less concentrated than the other almost by a factor of 3...and I didn't lose very much of it. Although, I lost over half of the concentrated sample. So I am not sure if there was some fluke, or if the concentration comes in to play some how??
I also think that I am using the 3500MW cutoff due to the size of my protein...I am not 100% positive, but I have been instructed to use it over the 12-14.
Hmm. I really don't know if the concentration plays a role. Seems possible that you would randomly lose more of a concentrated sample through a pore.
But if it is a purified protein I would run it on a gel and coomassie stain it just to be sure I am losing it.
I agree, run it on a gel and see if it really did disappear. If it did, I'm willing to bet it stuck to the dialysis bag.
Well, can I run amm. bicarbonate through a polyacrylamide gel?? I was told to exchange the buffer first (that amm. bicarbonate would cause it to streak) which would defeat our purpose of dialysis so we can lyophylize.