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.

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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.
 

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Reference

Borregaard, N. et al. (1983) J. Cell Biol. 97 , 52-61.