Isolation of Protein using Trizol
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.