Methods Digest, Vol 58, Issue 10 - TCA precpitation

Priscila Peña Diaz via methods%40net.bio.net (by ofiura from gmail.com)
Mon Mar 15 12:27:18 EST 2010


> 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?
>
>
> This might happen when precipitating with TCA/deoxycholate.  You would have
to homogenize the sample with a potter homogenizer.  If you do routine
protein work, there should be one (or several) in your lab.  It usually
works nicely, but be careful with samples already in Laemmli buffer, because
the bubble formation can be a pain.

good luck


P.


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