TCA Precipitation of Proteins in Guanidine

TCA Precipitation of Proteins in Guanidine

Dominik Haudenschild August 11, 1993
Boston Notebook 3 pg. 93 at CBRC

Used for precipitating fractions run on S-400 or Octyl-Sepharose columns in Guanidine so that they can be run on a SDS-PAGE gel.

1) Take 100µl aliquots from column fractions

2) Add 100µl ice cold 20% TCA and vortex well
-should get white precipitate in most sample

3) Allow to precipitate on ice or 4° for at least 30 minutes

4) Spin 15 minutes top speed in eppendorf centrifuge
-should get a large white pellet
-discard supernatant

5) Resuspend pellet in 500µl 100% ethanol and vortex until completely clear

6) Spin 10 minutes top speed in eppendorf centrifuge
-should get smaller pellet
-discard supernatant by carefully inverting tube on paper towel

7) Wash pellet again with 500µl 100% ethanol

8) Spin 10 minutes top speed in eppendorf centrifuge
-discard supernatant by carefully inverting tube on paper towel

9) After pellet is completely dry, resuspend in 1x SDS-PAGE sample buffer
-if pellet still has ethanol, then sample will "float" out of wells!!