Cell Disruption and Subcellular Fractionation
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 KCl
3 mM NaCl
1 mM ATP(Na)2
3. 5 mM MgCl2
10 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.
Reference
Borregaard, N. et al. (1983) J. Cell Biol. 97, 52-61.
