Help with protein precipitation

Theresa Gallagher via methods%40net.bio.net (by gallaght from umbi.umd.edu)
Wed Nov 5 10:49:10 EST 2008


Hi,

I am purifying 6-His tagged proteins from inclusion bodies using Talon
resin.

All goes well until I try and dialyze out the 8 M urea.

I have tried two methods:

1.       Step wise dialysis from 8M urea to 0M (in hepes buffer) when the
dialysis buffer reached 2M the whole lot precipitates out into the cassette

2.       Using decreasing concentrations of urea to wash the column - when
it came to elution time I had no protein eluted.

 

If anyone could give me some advice on additives for the urea or a method
that would be more suitable I would appreciate it!

 

Thanks in advance!

 

	
		

 

 



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