Concentration from GuHCl ?
flo at unixg.ubc.ca
Sat Feb 12 19:10:15 EST 1994
Summary of Replies :
1. From: "Bob Lauder" <bsa016 at cent1.lancs.ac.uk>
Try centrifugal filtration / concentration step. A number of
manufacturers make these - put the sample in the top - place in a
centrifuge - spin. There is a membrane between the upper chamber (where
your sample is) and the lower. The fluid and small salt molecules can pass
through leaving the protein in the top. Adding some NaCl ensures all of the
GuHCl is lost ( it exchanges for Gu which has bound tightly to the protein)
2. From: phi at nrcbsa.bio.nrc.ca (Dr. Barry Phipps)
Protein samples (column fractions, etc.) can be concentrated with either
acetone precipitation (5 vols -20C acetone, sit 20 min at -20C, sediment
15000 x g at 4C, wash pellet with ether at 0C) or TCA precipitation (equal
vol 0C 20% trichloroacetic acid, sit 15 min at 0C, sediment 15000 x g at
wash pellet with cold acetone) or both. Wash pellet well to get rid of
residual guan-HCl which will screw up your gel lanes. Or use Centricon for
rapid concenration of small volumes, diluting a couple of times with buffer
to get the guan-HCl concentration down below 50 mM or so.
3. from Robert Solomon (Bioc) <rgs at mbfs.biology.cambridge.ac.uk>
a) Add a fifth volume of 50 per cent TCA to an aliquot of the protein in
solution. This precipitates protein and GuCl. After spinning (microfuge,
remove s/n carefully (pellet is soft) and wash with 96 per cent EtOH (1X
volume). The GuCl dissolves, protein stays as a pellet.
b) It turns out that the TCA step can be eliminated, and protein ppted
>= five volumes of EtOH. Dry off remaining EtOH under vacuum.
4. from Michael Morales <mmorales at acpub.duke.edu>
Although I've never tried it in 6M Guan-HCl, the method of Wessel & Flugge
(Anal Biochem, vol 138:141-143 (1984)) has worked well for me w/ dilute
proteins in high salt concentrations.
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