I have found that ion exchange is a good way to concentrate protein.
If there is no reason that you can't pass the whole 450 mls through
the column then you should do that. Another way to do this would be to
add ion exchange resin straight into the 450 mls, stir for a bit, let
it settle, pour off the supernatant and load the remainder into an
empty column for washing and elution. I'm assuming from the wording of
your email that your refolding buffer is a suitable binding buffer for
the subsequent ion exchange step.
Other things you might like to try are to engineer a hexahis tag onto
your protein. After resolubilisation you can bind the protein to metal
affinity resin in a centrifuge tube in the presence of urea, pellet
the resin, remove the urea and resuspend the resin in refolding
buffer. Wash a few times and elute the protein in as small a volume as
you like.
Another way to keep the initial volume low would be to dialyse your
urea away instead of refolding by dilution. I realise that the slower
pace of dialysis and the higher protein concentration might reduce
your refolding efficiency but if you haven't tried it then the
advantages of smaller volumes might make it worth while.
In short, if there was a way that I could avoid routinely having to
use cross-flow to reduce 450 mls of buffer then I'd be pursuing that.
I hope that was helpful.
Nathan
Begin forwarded message:
From: Exec <[EMAIL PROTECTED]>
Date: 27 June 2008 1:28:59 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Concentrating protein
Reply-To: Exec <[EMAIL PROTECTED]>
Dear All,
we have GCSF protein produced in inclusion bodies. we solubilise it
refold
it and then concentrate it using proflux system. still the
concentration
of the protein we get is less and volume is more for us to load in Ion
exchange chromatography. is there any simple technique that can be
performed in lab without using any hi-fi instrument to concentrate the
protein in small volume of buffer. the protein we obtain is about 0.7
mg/ml and we get 450 ml solution. our column is 110ml lab scale and we
have to work in that only. i have heard of NH4SO4 precipitation. but
it
requires protein conc more than 1 mg/ml.
kindly help me to progress in my experiment.
