pyrophoshatasing cell extract for nucleotide depletion?

Kyle Legate legatek at mcmail.cis.mcmaster.ca
Thu Aug 8 18:58:40 EST 2002


On 6 Aug 2002, Johannes Graumann wrote:

> Why should the column step be necessary - someone else proposed a
> depletion from nucleotides by size exclusion (see other post in thread),
> but I don'tlike that idea. Looking for interactions I want to be quick
> about the depletion without messing up the protein stoechiometry by
> dilution even more than I do with the extraction buffer anyway ...
>
The gel filtration horse isn't dead yet so I'll beat it some more. The way
I avoid dilution of the protein sample is by making spin columns from the
equilibrated G25 resin. I equilibrate, then make a column in a syringe
then insert it through a hole in a falcon tube lid and screw it into a
falcon tube to collect the filtrate. I then spin the contraption in an old
clinical centrifuge to spin out the liquid and dry out the column. I find
that when I add my protein mixture and spin it through, the nucleotides
are left behind and most* of the protein is found in the filtrate in the
same volume it started in.

*if I add a small amount of BSA to the equilibration buffer, I lose very
little protein on the column (<20%), if I omit the BSA loss can approach
50%, if the protein solution is dilute.

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

   Tower of Tongues:Thursday PM:10:30-11:30 EDT:http://cfmu.mcmaster.ca
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