Protein precip. from Guanidine

Klaus Lehnert k.lehnert at auckland.ac.nz
Wed Oct 13 23:02:21 EST 1999


Kresten,

I am routinely using the protocol supplied in the Quiagen handbook. It
starts from 6M Gua-HCl (which is easy to adjust to), always works, but I
don't know whether it is quantitative or whether it ppts all of the 40µg in
your sample. Anyway, here it is:


Add equal volume of 10% TCA, mix;
Incubate on ice for 20min;
Spin 15 min full speed in microfuge (I do it at room temp).
Wash pellet with 100µl ice-cold EtOH;
Dry (I do it in a SpeedVac)
Resuspend in sample buffer (haven't tried in anything else...)

Hope it helps


Klaus



Kresten <kresten at my-deja.com> wrote in message
news:7tvkd5$taj$1 at nnrp1.deja.com...
> Could anybody please suggest a method for precipitation of protein for
> subsequent SDS-PAGE.
>
> My sample volume is 200 microliters and contains 40 micrograms of (pure
> protein). Furthermore the sample is 6.8M in Guanidine Hydrochloride.
>
> I have tried to precipitate by diluting with water to 900 ul and adding
> 100 ul 100% TCA w. deoxycholate. I then get a big fluffy pellet, which
> is much larger than in a sample without guanidine. I tried washing the
> pellet with acetone but this completely removed pellet (including
> protein).
>
> I also tried to add (to another sample)
> 300 ul water
> 500 ul methanol
> 125 ul chloroform
>
> then spin 1 min., remove the top-phase (right?) and add 500 ul Methanol.
>
> Spinning for 2 min. (15000 g) showed no pellet so instead I speed-vac'ed
> the whole mixture, redisolved in loading buffer and loaded on the gel.
> Most of the protein had been lost.
>
> I could of course desalt on a small column but would much prefer simply
> to precipitate, so any suggestions are highly appreciated.
>
> Thank you
> Kresten
>
> --
> The address kresten at my-dejanews.com is for
> spambots only. Please mail me at LysLeuLeu at crc.dk
> transforming the pre at -part into my initials.
> Kresten Lindorff Larsen
>
>
> Sent via Deja.com http://www.deja.com/
> Before you buy.





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