Help about TCA
Frank O. Fackelmayer
fof1 at chclu.chemie.uni-konstanz.de
Thu Jun 5 02:31:01 EST 1997
In article <339659D6.53A1 at cica.es>, alvarez at cica.es wrote:
> We have some problems with TCA precipitation of proteins. We use TCA 8%,
> and after whashing the pellet with ethanol:diethylic ether 1:1, we leave
> to dry at room temp. But, when we resuspend the pellet in loading
> buffer, bromophenol blue turns yellow (pH below 5?), then we neutralize
> with Tris base 1 M, pH 10.7. Before loading the sample in SDS-PAGE
> minigel, we dilute it just to salt concentration 250 mM aprox. When the
> gel is silver stained, the front is very dark, the lanes are full of
> smearing. Control lanes (without TCA ect) run well.
> Has someone any solution? Dialization is not a possibility.
Turning yellow of bromophenol blue is quite usual after TCA precipitation.
Although most people think its better to neutralize before SDS PAGE, I
prefer NOT to do it. Buffering capacity of the gel buffer is sufficient to
"neutralize" your sample on the fly (you´ll see it turns greenish-blue as
soon as migrated into the gel). So, usually the gels run nicer without
neutralization because of its lower salt concentration.
Your point of high background is, most likely, not related to your TCA
precipitation/neutralization. We´ve had this problem too, until we
realized that the background smear is DNA (or RNA) that is co-precipitated
by TCA and also stained with silver.
The best thing we found to avoid this problem is to separate DNA and
protein before SDS-PAGE (essentially the method of Wessel & Flügge (1984),
Anal. Biochem. 138, 141-143). Also, this method avoids acidification of
your sample, and is much faster than TCA precipitation.
This is how we do it:
* adjust the sample to 1% SDS, vortex, let stand for 2min at RT
* add 4vol of methanol to your sample, vortex, spin down briefly (10sec)
* add 1vol of chloroform, vortex, spin briefly
* add 3vol of water to separate phases, vortex vigorously, spin for 1min
* remove as much of the upper phase (water with DNA or RNA -> you can
analyse this fraction in an agarose gel after ETOH precipitation)
* BUT DO NOT TOUCH OR REMOVE the interphase and lower phase (protein)
* add 3vol of methanol, vortex vigorously, spin down full speed, 5min
* remove supernatant
* air-dry pellet (15min, methanol has to evaporate completely!!!), then
redissolve in sample buffer and run gel
The original method was described for 100ul samples, but we´ve scaled it
up to 10ml sometimes, and it works perfect.
Hope this helps,
Hope this helps
Dr. Frank O. Fackelmayer
Division of Biology
University of Konstanz
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