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Contributor: Suprya Jayadev
Cell Disruption and Subcellular Fractionation
Date: July 31, 1991
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.
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
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
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
Borregaard, N. et al. (1983) J. Cell Biol. 97,