Buffer Exchange - (Jul/26/2007 )
I need to do buffer exchange for my protein. Can anyone tell me the amount of buffer I need to buffer exchange a 10 mL sample?
I've read somewhere that the ratio was like 1:800? so I need like 8L of buffer? or maybe I can concentrate it down first?
Are you doing this buffer exchange by dialysis? If so, you can do the 10 ml sample against 1 or 2 liters of buffer, dialyze for an hour or two then change the buffer. It is more efficient to dialyze against smaller volumes and do several buffer changes than it is to do dialysis against a large volume with no changes.
i tried to concentrate my protein but i think it precipitated! what would i do in this case? can i still use it? ( the reason i was concentrating it down was so i could do a second step purification)
How did you concentrate your protein? It could be that you've just aggregated the protein, which may be reversed by re-diluting (I know it sounds like one step forward, one step back, but if you dilute in the new buffer, your job might be half done). I think dialysis may be the easiest way forward. You will require several hundredfold excess of the new buffer, so you get close to complete exchange.
If you have to go and start again, you could also try using a fast desalting column, like a PD-10 from GE Healthcare (or whatever company name they're operating under this month). Your volume goes up by about 50%, as I recall.
If the protein is precipitated, you could denature with urea, then try to refold by slowly diluting it into fresh buffer.
What were you planning to use for the second stage purification?