urea dialysis question

Kyle Legate legatek at mcmail.cis.mcmaster.ca
Sat May 18 10:01:19 EST 2002


Hello, all. I'm trying to remove nucleotide from a GTPase by a urea
dialysis method that had worked for mammalian ARF1 in the past. Thusfar I
have been able to remove the nucleotide, but I have been unable to refold
the protein in such a way as to restore GTP-binding activity.
	I understand that simply dissolving solid urea into the buffer is
not appropriate since there are charged forms of urea in the mix that can
harm the protein. It is recommended that urea be passed over a column of
some sort to remove the charged species prior to adding the protein.
	My question is this: can anyone provide me with a protocol for
cleaning up the urea prior to adding it to the dialysis buffer (I recall
something about pouring the urea through an Amberlite column?) Thanks for
your help.

... . . .  .  .  .    .    .    .     .     .     .      .      .      .
legatek at mcmaster.ca		Kyle Legate            legatek at hotmail.com

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