The most common reason that we TCA precipitate proteins is to concentrate
them prior to SDS-PAGE. After SDS-PAGE the proteins are visualized either
by staining the gel or by western blotting. This protocol is for this
application of TCA precipitation. There are slight differences in the
protocol if when used for other purposes. These are noted at the end. Trichloroacetic
acid (TCA) is very nasty! Remember the point here is to denature
and precipitate proteins and YOU are made of
proteins. It is very important to wear proper
protective outerwear when using TCA. Minimally you need a lab coat and
safety glasses and latex gloves. Remember - A
small puncture in your gloves can lead to a nasty burn. You should prepare some 100 mM sodium bicarbonate buffer in case you have
a spill or get some TCA on an unprotected area of skin by accident. If
you get any TCA on you or your clothes douse with 100 mM sodium bicarbonate
buffer and then wash with lots of water.
TCA precipitate the chilled products by adding 2 volumes of ice cold
20% TCA, vortex, and incubate from 10 mins to several hours on ice.
Alternatively you can use 1/2 volume of 50% TCA (w/v) but obviously 50% TCA is
more hazardous than 20%. The idea is to end up with ~15% TCA as the
final concentration.
Spin out precipitate 15 to 20 seconds in microfuge, aspirate sup., add 400
ul ice
cold ethanol:ether (1:1 v/v) spin again and aspirate sup. Ether is also
hazardous - carefully check any bottle of Ether for crystals. If there is any crystalline precipitate in a bottle of ether do not use it.
Carefully return it to the flammables cupboard and arrange for disposal. Let the pellets dry in a fumehood for 5-10 minutes.
Add 1X SDS-PAGE loading Buffer. If you use loading buffer
containing bromphenol blue and it turns yellow then there is still some acid
left in your precipitate. You can neutralize this with 1M Tris of
whatever pH you use for the stacking gel. If you use loading
buffer with red dye then dissolve your pellets in 0.1 M Tris pH 8.9 (add a volume equal to
1/2 the amount you want to load in a lane on the gel). Vortex, and if the
precipitate does not go into solution immediately incubate at 37ºC for half an
hour vortexing every 5-10 minutes.
NOTES:
If you want to immunoprecipitate the protein (for example when using in
vitro translation products and an antibody that recognizes the denatured
protein or if you want to immunoprecipitate your protein without any
putative binding partners) dissolve your pellets in 100 ul of 0.1 M Tris pH 8.9,
1% SDS. Vortex, and if the precipitate does not go into solution
immediately incubate at 37ºC for half an
hour vortexing every 5-10 minutes. If that doesn't work heat the dissolved samples in boiling bath for 2-4 minutes.
Dilute samples with 20 volumes of 1 X TX-SWB to take up all excess SDS into
Triton micelles (final Triton conc is approx. 1%, final SDS conc is approximately .05%.)
Add antiserum and proceed as for previous immunoprecipitation protocol starting
with step 3.
If you are TCA precipitating reticulocyte lysate make sure you don't try
to precipitate more than 1 ul or you will get concrete. Ammonium
sulphate precipitation is probably better for retic.
