Kyle Legate <legatek at mcmail.cis.mcmaster.ca> wrote:
>>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.
Most GTPases readily lose nucleotide when there is no Mg around.
Try dialysis against EDTA first
> 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.
Much bigger problem is that many proteins are very difficult if not
impossible to refold. And that has nothing to do with urea decomposition.
> 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.
Purely unnecessary IMHO, but if you insist, indeed, run solution through
ion-exchanger. Discard first 2-3 column volumes flow-through, collect the
rest. Make note of resin's capacity, generously overestimate ion concentration
of ions in your solution and use no more than 1/2 of column capacity.
DK
