TCA precipitating small amounts of protein (5/19/00)
The following method is based on observations I made while developing a
way to do in vitro protein-DNA crosslinking. Some of these crosslinking
experiments involved minute amounts of protein, and a TCA precipitation
was used to remove free radioactive nucleotides that were present after
crosslinking and nuclease digestion.
Adjust the solution containing protein so it contains 0.5% NP40 (or Triton
X 100) and 0.1% Sarkosyl.
Add 1/5 volume of 100% TCA (w/v) so the final TCA concentration is 20%.
The stock solution of TCA is stored at 4oC.
Place tubes on ice for 15 minutes - the solution should become cloudy.
Spin samples in a cold microfuge for 15 minutes.
At the end of the spin, immediately place the samples on ice. As
you remove samples from the microfuge, you should see a separate phase
at the bottom of the tube. This may be clear or cloudy, but it will
become cloudy when the samples are place on ice.
Carefully remove the clear solution located above the cloudy layer at the
Add 100 ul of acetone to each sample and place the samples back on ice
(the acetone added to the samples should be cold and stored at -20oC).
You may need to add more acetone if you've precipitated
the protein from a large volume.
After adding acetone to each sample, gently flick the side of each
tube 10 times so the material at the bottom of the tube begins to dissolve
in the acetone. This is a critical step. Most of the material
at the bottom of the tube begins to dissolve in the acetone but the protein
will form a precipitate which can easily be lost (the protein precipitate
often appears as a thin chip). Keep everything in the lower half
of the tube so this precipitate is not inadvertantly lost somewhere on
the lid of the tube. Ten flicks is not enough to dissolve the material
at the bottom of the tube so keep repeating this procedure with each sample,
moving from sample to sample so that there is a period when each sample
sits on ice between periods of flicking.
The material at the bottom of the tube looks to me like an oily layer.
Once this has dissolved completely, leave the samples on ice for 5 minutes.
Centrifuge in the cold for 10 minutes and then transfer the sample immediately
Carefully remove the supernatant. The protein precipitate frequently
dislodges from the side of the tube so one must be careful not to inadvertently
suck it up into the pipette and discard it (removing the supernatants with
a drawn out glass pipette is advised). Set the tubes on their sides
at room temperature for about 10 minutes so the acetone evaporates.
Dissolve the protein precipitates in "Alkaline SDS-PAGE sample buffer".
Because of the TCA, the samples are usually acidic. To neutralize
the samples, the sample buffer contains Tris at pH 8.
1X Alkaline SDS-PAGE sample buffer.
50 mM Tris pH 8.0
100 mM DTT
Boil samples for 5 minutes before loading them on SDS-PAGE.
In this procedure, I think the detergent is acting as a carrier.
Although I don't know the details, I recall seeing another procedure where
deoxycholate was used as a carrier.