Hi...is there any best and easy way to concentrate the protein
without modifying its original structure
Dr Engelbert Buxbaum
(by engelbert_buxbaum from hotmail.com)
Sat Dec 17 13:45:48 EST 2011
In article <mailman.312.1323202165.1853.methods from net.bio.net>,
sudheer.pbm07 from gmail.com says...
> Hi Guys
> My recombinant protein is going to inclusion bodies so I am denaturing it
> Gn-HCl and renaturing with GSH and GSSG then finally purified the protein.
> After purification I am getting lower concentration of the protein of
> course it is in larger volume of the buffer. Now I wanted to concentrate
> the protein....Could anyone please tell me, the best and easy way to
> concentrate the protein with out modifying its original structure.
Personally I love Amicon pressure-concentrators (now sold by Millipore).
These use pressure from a nitrogen bottle to press the solution through
a ultra-filtration membrane. The cutoff of the membrane needs to be
selected just small enough to retain the protein, water and small
solutes (like GdnHCl in your case) should pass freely. Because you are
using only the gas pressure, you can turn off the valve once you started
the process, and you can use "empty" bottles with only a few bar left.
The poor man's alternative is to pour the solution into a dialysis bag,
close it, place it into a plastic container and pour solid PEG over it
(MW larger than the pores of the membrane). The stuff is so hygroscopic
that it will pull water and small molecules out of the bag. You just
need to look after it from time to time, otherwise you concentrate your
sample into non-existence!
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