Protein Isolation using Trizol
Protein Isolation using Trizol				3/3/97
									Lab of KVM

1.  In a sterile14ml tube add 100-500mg of tissue.
2.  Add 1ml Trizol per 100 mg of tissue.
3.  Clean homogenizer 3X with 5ml DEPC-treated H2O.
4.  Homogenize samples 2X/10 seconds/setting=5.
5.  Incubate for 5 minutes at RT to let debris settle.
6.  Aliquot 1ml of supernatant into a 2.2 ml tube.
7.  Add 0.2ml chloroform/ml trizol. Cap then vortex for 15 seconds.
8.  Incubate up to 5 minutes at RT to allow phase separation.
9.  Spin at 12,000.rpm/15 minutes/4'C.
10. Remove the entire upper, aqueous layer (~700ul).
11. Precipitate the DNA wth 0.3 ml of 100% ETOH/ml Trizol.
12. Mix by inversion then store for 2-3 minutes at RT.
13. Spin at 2,000.rpm/5 min./4'C to pellet DNA. Repeat in new tube.
14. Remove the phenol-ETOH supernatant (600ul) containing the 	
	protein from DNA pellet.
15. Place supernatant into a new 2.2 ml tube. Add 1.5 ml RT/IPA.
16. Store for 10 min./RT. Then spin at 12,000rpm/10min./4'C.
17. Wash pellet 3X with 2.0 ml of 0.3M guanidine HCl in 95% ETOH. 	
	Store for 20 min./RT between washes. 
18. Spin at 7,500rpm/5 min./4'C.
19. Rinse in 2 ml of ETOH, sit 20 min., vortex, then spin as above.
20. Aspirate supernatant then dry in speed vac, dissolve in 1% SDS.
21. After dissolved, spin at 10,000rpm/10min./4'C to pellet any 	
unwanter junk. Remove and add to a new 1.5 ml tube. 

Quantitate protein by the Bradford method using Bio-Rad kit.