Anisha Patel COSMOS UC Davis Cluster 1: Biotechnology

6. Grind approximately five grams of frozen cell tissue in a mortar.i. Add liquid nitrogen, grind slowly as it boils off. Repeat grinding step three times.7. Add a small amount of liquid N2, and swirl to break loose the ground tissue.8. Transfer ground tissue to a 4 degrees C mortar and add 8 mL of Tris extraction bufferi. Grind until tissue and buffer are thoroughly mixed.ii. Prepare Tris Extraction Buffer: 40 mM Tris-HCl (pH 9.5), 50 mM MgCl2, 2%PVPP, 125 U endonuclease/mL, 1 mM PMSF9. Pour mixture into 13 mL centrifuge tube, vortex, and sonicate.i. Vortex for 1 minute, set on ice for 1 minute.ii. Sonicate for 10 pulses (Each Pulse = 2 sec). Set on ice for 3 minutes.iii. Repeat (a) and (b) for a total of two times.10. Centrifuge at 5,000 g for five minutes (6,500 rpm) to pellet debris and recover the supernatant.11. Precipitate protein by adding 4.33 mL of 37.5% TCA plus 1.0% β-mercaptoethanol,bring to a final volume of 13 mL using 37.5% TCA, incubate on ice for 45 minutes,and centrifuge at 14,000 g for 15 min.12. Wash protein pellet 3 times in 80% acetone containing0.05% β-mercaptoethanol.i. Break up protein pellet using small metal spatula.ii. Sonicating in a water bath for five minutes will help disrupt the pellet*.13. Air dry protein pellet in laminar flow hood until all the liquid is gone.