Protein precipitation - acetone?
(by stanleycheungkk from yahoo.com)
Sun Mar 14 21:53:03 EST 2010
Hi Nick and everyone,
I tried the 0.02% deoxycholate/10-20% TCA method. It works. However, I faced problem in samples with high volume, the resultant precipitate formed a large and unbreakable lump. The lump retained TCA which turned the color of sample buffer to yellow, however, boiling in sample buffer also can't break down the lump.
Is there any limitation on using this method, like the amount of total proteins?
Would the lump formed by some insoluble components (salts?) rather than proteins?
I read some articles mentioned this problem when preparing samples for 2D-PAGE and sonication can improve it. Any other methods can break down the lump since I don't have this apparatus in my lab so that the TCA can be washed away and the proteins are fully resolubilized in sample buffer?
From: Nick Theodorakis <nick.theodorakis from gmail.com>
To: methods from magpie.bio.indiana.edu
Sent: Fri, March 12, 2010 12:50:28 AM
Subject: Re: Protein precipitation - acetone?
On Mar 11, 10:56 am, "Iraz Toprak Aydin" <iraz.ay... from epfl.ch> wrote:
> Dear all,
> I have to do a western blot, but my protein concentration is very low, and I
> have to run a mini gel. So I was thinking of precipitation the proteins. I
> have never done this before. Does acetone have a bad effect on the blotting?
> Are there any points that I should be careful about?
Acetone shouldn't affect blotting or detection, but depending on the
protein, you may need to add several volumes of cold acetone, which
may be more volume than will easily fit in one centrifuge tube. Many
people have good luck with TCA/deoxycholate precipitation. Add sodium
deoxycholate to 0.02% and TCA to 10%. Ice down for about half an hour,
then spin hard (e.g., full speed in a cold microfuge for 15 min). Wash
the pellet once or twice with cold acetone (or cold ethanol) to remove
the TCA, air dry, and resuspend in SDS/gel loading buffer. If the
color goes yellow, you didn't wash all the TCA out, but you can add a
microliter or two of saturated Tris to bring it back to blue, and be
more careful next time. Heat and serve.
nick_theodorakis from hotmail.com
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