Wittrup: TCA Protein Precipitation

1.7	TCA Precipitation of Intracellular Lysate

Adapted from Anne Robinson’s protocol

1.	Induce protein expression through growth in SG-CAA or YPG. Collect 4 OD600 of cells. Wash 1x in 1 mL of PBS.

2.	Spin down. (Cells can be stored at –20°C at this point).

3.	Resuspend cells in 100 μl TCA buffer plus protease inhibitors (1x Halt Cocktail, or Complete protease cocktail.) Transfer to 1.5 mL screwcap vial containing 600 μl pre-washed glass beads and 100 μl 20% TCA. (You can wash beads in Alconox, but be sure to try them thoroughly.)

4.	Pulse beads and cells in bead beater for 2 50 second bursts resting for 1 minute on ice in between.

5.	Collect fluid in eppendorf with gel loading tip. Wash 2x with 500 μl of 1:1 mixture of TCA/TCA buffer collecting the wash in the eppendorf.

6.	Centrifuge at top speed for five minutes. Discard the supernatant and be sure the pellet is dry.

7.	Resuspend in pellet in 200 μl resuspension buffer and boil five minutes. You can aliquot the protein and freeze what you don’t use at –70°C.

Solutions 20% TCA

20% TCA Buffer 20 mM TrisCl, pH 7.9 50 mM ammonium acetate 2 mM EDTA

TCA Resuspension Buffer 3% SDS 100 mM Tris base, pH 11 3 mM DTT (store at –20°C in 1 mL aliquots)

Andy Rakestraw May 5, 2006