TCA/Acetone Protein Extraction Protocol

1. Grind tissue very finely in LN2.

2. Add to polypropylene tube containing 10% TCA in acetone plus 0.07% 2ME (we've been using 20-25 mls for 2 g tissue. For young leaf, 0.5 g is plenty of tissue, but you still need to use 20-25 ml TCA/acetone so the centrifuge tubes have enough volume for a hard spin). Vortex.

3. Precipitate at -20^oC for at least 45 min.

4. Centrifuge at 35,0000xg (15,600 rpm in SA-600 Sorvall rotor), 15', 4^oC.

5. Decant and wash the pellet with 20-25 mls acetone containing 0.07% 2ME, 1mM PMSF and 2mM EDTA. Make sure you break up the pellet by vortexing or with a pipet tip or spatula. Precipitate @ -20^oC (I don't usually precipitate at this step and my yields have been fine).

6. Spin, 35000xg, 10-15', 4^oC.

7. Decant and repeat. After last wash, make sure you decant off as much acetone as possible; it's not good for the speed vac.

8. Dry in the speed vac (One 2 g sample takes around 25-30 min to dry) to 10 mTor. Make sure your sample is dry or the residual TCA will cause problems. For 2 g starting tissue, the dry powder usually weighs around 0.2 g.

9. At this step, you can freeze the pellet and resolubilize the protein later, or continue with the protocol. Freezing may help remove residual TCA.

10. Resuspend the powder in 8M urea, 4% CHAPS, 20mM DTT, 0.2-0.5% biolytes or other appropriate resolubilization buffer. Right now I'm usually using 1.0 ml buffer/g starting material. Vortex strongly for 5' and sonicate in an ultrasonic bath for 5'. (I've also seen a version that lets it sit at RT for 1 hr. I think a sonicating probe would also be good, and fast.)

11. Spin 10', 12000xg, 10^oC. Recover the supernatant.

12. Repeat the resolubilization.

13. Check protein concentration and rehydrate an IPG strip.