HELP: dialysis of protein

[Richard Mandel]

NAME RAVI PRAKASH .D (sl16s at cc.usu.edu) wrote:
: Dear Netters,
: I have attempted to dialyse a recombinant protein (in 0.5M NaCl, 300mM
: imidazole, 6M urea) into buffer with 150mM NaCl, 20mM tris, 2.5mM CaCl2. Within
: few minutes of dialysis there is a precipitate formation in my dialysis bag.
: Can anyone have any suggestions as to how I can rescue my protein from the
: precipitate? Or any better way to dialyse the protein in future?
: Thankyou in advance for any suggestions or comments.

You could try using a linear gradient for the dialysis to more slowly remove
the urea from the buffer.  Use two beakers, the first with the final buffer
the second with the starting buffer with a filled tube connecting them.  Place
the second buffer on a stirrer but have the levels of the two beakers equal.
Finally hook up a tube from the second beaker to a small flask containing
the dialysis bag and a small volume of the starting buffer.  Seal the
top of the flask with a two hole stopper, having an input and outlet and
control the rate of throughput to give a gradual change in the solution
that bathes the dialysis bag.

I hope that is comprehensible.
--

Ciao,
Rich 

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Richard Mandel			|	E-mail address: rmandel at acs.bu.edu
Boston Univ. School of Medicine	|	Phone No. 617-638-4512	
Boston, MA 02118		|	Fax No. 617-638-4085
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