Ammonium sulfate precipitation from solutions with high sugar
Dr Engelbert Buxbaum
(by engelbert_buxbaum from hotmail.com)
Sat May 19 09:53:38 EST 2012
In article <mailman.239.1335827494.3456.methods from net.bio.net>,
novalidaddress from nurfuerspam.de says...
> Hi Pow, long time no see :)
> My big problem is that there is almost no protein in the sample ->
Iwon't see any bands. I expect about 1mg from 50 ml. I have started with
50ml of fruit juice concentrate, diluted with 50ml water and then added
ammonium sulfate until saturation: No visible turbidity, No (visible)
pellet after 2hrs @ 4000g (except some ammonium sulfate crystals.
Unfortunately, I do not have access to a high speed centrifuge for such
volumes. Spinning 24 eppis with 2ml each @ 15.000g in my small benchtop
centrifuge might be an option, however.
> Could it make sense to add some unrelated protein, eg BSA or OVA
> (that's what I have in my fridge), as co-precipitant (like one does
> with glycogen or poly-acrylamide for DNA)?.
what Wo presumably was referring to was a self-forming gradient (similar
to CsCl). This can be done, but requires an ultracentrifuge.
In your situation, I would try either dialysis against high MW PEG or an
Amicon pressure cell to remove most of the sugar and fluid. You could
even dilute the product of such an operation with buffer and repeat.
More information about the Methods