Hi...is there any best and easy way to concentrate the protein without modifying its original structure

Dr Engelbert Buxbaum via methods%40net.bio.net (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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