Problem with protein concentration - (Jan/08/2004 )
i am working in structural biology, thus i try to produce enough protein to obtain crystals ... as you know you have to reach a very high concentration to produce crystals ... the only problem is that i loose more than 90% of the protein when i concentrate it with spining concentration unit such as centricon. I can't concentrate the protein with ion exchange column because i need to obtain the protein in a very precise buffer
Is it possible to use a speedy vac to concentrate them? The principle relies on centrifugation under vacum. The only problem may be that it will concentrate salts in your buffer
That's the problem, for crystallography you want to be sure that youre prot is in the right buffer, you can't monitor the salt concentration with the speedvac
It could be crashing due to interaction with the membrane of the column probably
Either that or the membrane cut off maybe too high
maybe just lyophilize the protein, and then resolve it with water, or using water before lyophilize and then resolve it in your buffer.
Concentrate vs PEG or with dry Sephadex(for example) or precipitate protein with ammonium sulphate and change the buffer solution. Concentrate by solution freezing, then collect unfreezed fraction(use little pipe) and change the buffer.
Long ago I have encountered with same problem and now I know that good decision in such case is incubate centricons(after concentration step, and any systems that contain membranes) overnight.
do you mean incubate the centrifuged centricons in room temperature?
or could I use a prolonged inverted centrifugation step to get back my concentrated protein?
I mean incubation at 4`C. Inverting centrifugation resulted in protein drying and denaturation ( i mean protein that adsorbed on membrane), so it`s no good. Just leave yours centricons (do not remove concentrated solution) overnight at 4`C and possibly you will win (depend on protein nature).
thanks your sharing
so the best way to recover the concentrated sample is incubation followed by pipette out the retentate?
also, precipitation method is common to concentrate protein sample. mm...any people compared the centricon to precipitation method?
1. In such case (see topic), possibly yes
2. In such case (see topic), yes