丁香实验_LOGO
登录
提问
我要登录
|免费注册
点赞
收藏
wx-share
分享

Cell Disruption and Subcellular Fractionation

互联网

817

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

 

<center> <hr /> </center>

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.

 

提问
扫一扫
丁香实验小程序二维码
实验小助手
丁香实验公众号二维码
扫码领资料
反馈
TOP
打开小程序