pyrophoshatasing cell extract for nucleotide depletion?
dk at no.email.thankstospam.net
Thu Aug 8 21:47:17 EST 2002
Kyle Legate <legatek at mcmail.cis.mcmaster.ca> wrote:
>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.
I consider the horse alive and well :-)
I find that in most cases acrylamide-based P-6 from Bio-Rad performs
better than G-25 in terms of recovery _and_ purity. Recovery heavily
depends on 1) concentration of the total protein in the sample (thus,
when I can afford it, I add BSA to 1 mg/ml from a stock of 100 mg/ml),
2) volume of the sample; I like 1/4 column volume best; 1/5th when
purity is more important, 3) geometry of the column; the best yeild/purity
compromise appears to be at lenght/diameter ratio of 4-5,
4) pre-spin; I find it very helpful to "spin-pack" -> "mock spin" first
with buffer or buffer/BSA -> only then load sample.
In practical terms, I was not able to make good spin columns larger than
5 ml (thus limiting sample volume to 1.25 ml). With 1 ml columns
(Bio-Rad sells great geometry empties for this purpose), I was able
to routinely buffer-exchange >25 chromatography fractions for functional
analysis. Does not really make any difference whether it's 5 or 30 samples.
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