i am trying to get rid of unexpected protein band by centricon - (Aug/24/2014 )
my target protein at 34kDa, but when i analysis by SDS-PAGE i got another band at 26kDa so i want to use 30kDa Milipore Centricon to remove that band. but i do not know how to use centricon.
Would anyone help me any information about that such as washing, loading, and centrifuge speed etc...
Any help would be appreciated
Thanks in advance
The instructions usually come with the package (well, they used to, a number of years ago). Basically you should spin at about 1000 RCF until you reach the volume you require.
This is, unfortunately, unlikely to work. The cutoff molecular weight on these columns is not at all sharp, and you will have great difficulty separating a 26 from a 34 kDa protein. A mono-q column, or perhaps even just salting out with ammonium sulfate would probably be a better strategy.
Agree with phage...a filter membrane is not meant to separate proteins from other proteins. I've had monodisperse monomeric GFP (28kDa) be largely retained by a 100kDa filter.
Cation or anion exchange is your best bet. Or use a different affinity tag. Or it's possible your 26kDa protein is a proteolytic breakdown product of your target protein, in which case throw in some protease inhibitors (and use clean, fresh filtered buffers, work at 4C or on ice, etc).
Lots of people who start working with proteins are told all proteins can be purified with the same protocol, i.e. with just an affinity column followed by gel filtration. This is very commonly not the case. Expect to put in a little effort to purify quality protein if it's anything other than a well behaved, very well expressed, super stable and soluble protein (which most aren't ). Don't get caught up being protocol oriented when working with proteins. I see too many people doing it these days and their work suffers because of it.
EDIT: in grad school I had a protein with an intraprotein (internal) crosslink. The crosslinked protein traveled about 5kDa faster on a gel than the non-crosslinked. In other words, if you can convince yourself both bands on your gel are your target protein (in-gel digest + MS/MS will do the trick), consider in addition to proteolytic cleavage other post-translational modifications that could alter electrophoretic mobility such as internal crosslinks.