Cell Disruption and Subcellular Fractionation

Cell Disruption and Subcellular FractionationContributor: Suprya Jayadev Date: July 31, 1991 Nitrogen Bomb 1) Harvest cells and wash 2 times with PBS. 2) Resuspend final pellet in relaxation buffer. (20 ml/1X109 cells) 3) Pressurize with N2 for 20 minutes at 350 psi, 40C with constant stirring in a nitrogen bomb. 4) Collect cavitate dropwise into EGTA, pH 7.4. --> The final EGTA concentration should be 1.25 mM. Fractionation 5) Pellet the nuclei and unbroken cells by centrifuging the cavitate at 500 g, 40C for 10 min. --> This pellet is known as the P1 fraction. 6) The supernatant should be decanted and layered onto a precooled (to 40C)Percoll gradient.

Reagents Relaxation buffer (without EGTA):100 mM KCl3 mM NaCl1 mM ATP(Na)23. 5 mM MgCl210 mM PIPES pH 7.3

NOTE Relaxation buffer , a high-potassium, low-sodium, calcium-free buffer containing MgATP, was designed to mimic cytoplasmic conditions in the neutrophil, based in part upon conditions shown to promote cytoplasmioc relaxation in nonmuscle contractile systems.