小中大
这是我用过的protocol,提取的是intact的mt,可以直接拿来做酶活性实验,如果做western需要裂解pellet.
All solution and equipments should be precooled to 0 to 4 degree and keep on ice.
1. Wash cells (1*107) with PBS @ RT
2. Scrape cells into PBS and pellet the cells at 1000g, @ RT* 15’
3. Aspirate all of the supernant and resuspend in ice-cold 500ul of CHM buffer.
4. Leave on ice for 2’
5. Homogenize cells with syringe (20-30 times, no bubble). Confirm >90% cells breakage has occurred.
6. Add 200ul of ice-cold CHM containing 1M sucrose (final 0.25M) and mix gently by repeated inversion.
7. Pellet nuclei by cfg 10’@ 1000g, 4 degree
8. Aspirate supernant and centrifuge 10 min @ 10000g, 4 degree.
9. Resuspend the pellet in 1 mL ice-cold sucrose/Mg2+ mediu.
10. Recentrifuge at 10000g, 10’, 4 degree. Resuspend the pellet in ice-cold mt suspension medium I.
CHM
To 100 mL DDW add:
30ul of 1M MgCl2 (150mM final)
0.15g of KCl (10mM final)
5mL of 1M Tris.Cl (25 mM final)
Adjust to PH 6.7
0.4 ml of 0.5M EDTA (1mM final)
Add water to 200 mL
Store up to 1 to 2 days at 4 degree or 2 to 3 month at –20.
cold CHM containing 1M sucrose
Add 68.4g sucrose to 200ml CHM
mt suspension medium I.
To 50mL DDW add:
8.5g sucrose (0.25M final)
2.5mL of 1M Tris base (25 mM final)
Adjust PH to 7.0 with acetic acid
Add water to 100 mL
Store up to 1 to 2 days at 4 degree
