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HeLa细胞裂解+细胞核提取+分离纯化线粒体

来源:未知 作者:admin 人气: 发布时间:2009-04-18
摘要:一、细胞的裂解 1. HeLa cells were pelleted using a Sorvall centrifuge at 800 g for 10 min. 2. Cells were resuspended in lysis buffer [ 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 5 mM EDTA, 50 mM NaF, 1 mM PMSF and a protease inhibito

一、细胞的裂解

1. HeLa cells were pelleted using a Sorvall centrifuge at 800 g for 10 min.
 
2. Cells were resuspended in lysis buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% NP-40, 5 mM EDTA, 50 mM NaF, 1 mM PMSF and a protease inhibitor(PI) cocktail: 25 μg/ml pepstain A, 50 μg/ml leupeptin and 0.2% aprotinin] and allowed to swell on ice for 30 min.

3. Cells were then frozen in a dry ice bath and thawed on ice.

4. Cells were spun at 10 000 r.p.m. for 10 min and thesupernatant was frozen at -80℃.

5. Bradford reagent was used to quantitate the protein concentration per sample.

二、提取细胞核

1. Cells were sedimented at 800 g for 10 min and then washed with 50 vol of phosphate-buffered saline(PBS).

2. The pellet was resuspended in a 5× packed cell volume of a hypotonic buffer (10 mM HEPES, pH 7.9, 0.75 mM spermidine, 0.15 mM spermine, 0.1 mM EDTA and 0.1 mM EGTA).

3. Cells were allowed to swell at 4℃ for 10 min then sedimented at 300 g for 10 min.

4. The supernatant was removed and replaced with 2 vol of fresh hypotonic buffer plus PI cocktail.

5. Cells were homogenized by 3-5 strokes in a Dounce homogenizer and sucrose restoration buffer was added (1 vol of 500 mM HEPES, pH 7.9, 7.5 mM spermidine, 1.5 mM spermine, 2 mM EDTA, 2 mM EGTA and 10 mM DTT in 9 vol of 7.5% sucrose).

6. Nuclei were sedimented in a Sorvall centrifuge for 30 s at 14 000 r.p.m.

7. The pellet was resuspended in lysis buffer and stored in small aliquots at -80℃.

8. The supernatant was saved and used for mitochondrial purification.

三、分离纯化线粒体

1. Mitochondria were sedimented using a Sorvall centrifuge at 12 500 r.p.m. for 30 min.

2. The supernatant was discarded and pellet was resuspended in 25 mM sucrose (in TE, pH 7.4, with PI cocktail).

3. Mitochondria were purified on a step gradient consisting of 1.5 M sucrose (in TE, pH 7.4) and 1.0 M sucrose (in TE, pH 7.4) spun in a SW28 rotor at 25 000 r.p.m. for 1 h.

4. Mitochondria were collected in the 1 M sucrose layer and dialyzed against 10 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA and 2 mM DTT.

5. Mitochondrial suspensions were sedimented at 14 000 r.p.m. for 20 min in a Sorvall centrifuge and the pellet was resuspended in lysis buffer.

6. Centifugation at 14 000 r.p.m. for 20 min clarified the mitochondrial lysate and the supernatant was used for western blot analysis.

7. Protein concentrations were quantified with Bradford reagent and lysates were stored at -80℃ in small aliquots.

作者:admin

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