Lab Manual
biological reagents
Cell Free Systems
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misc methods

TCA precipitation of proteins

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.

  1. 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.
  2. 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. 


bullet 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.
bullet 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%.)
bullet Add antiserum and proceed as for previous immunoprecipitation protocol starting with step 3.
bullet 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.