Cell Disruption and Subcellular Fractionation
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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 KCl
3 mM NaCl
1 mM ATP(Na)2
3. 5 mM MgCl2
10 mM PIPES pH 7.3
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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.