《线粒体生物学与医学》课程教学课件（Mitochondria Biology and Medicine）8 线粒体生物医学 Research Protocol for MITOCHONDRIAL STUDY

ResearchProtocolforMITOCHONDRIALSTUDYJiangangLong, PH.DInstituteofMitochondrial Biologyand Medicine

1 Research Protocol for MITOCHONDRIAL STUDY Jiangang Long, PH.D. Institute of Mitochondrial Biology and Medicine

Basic Methods. Isolation and subfractionation of Mt·Determination of OCR and ECAR: Biochemical assays of Mt enzymes activity: Reactive oxygen species (ROS) andMitochondrial Membrane Potential (MMP)MtDNAcopynumbers: Assays for Mt morphology2

2 Basic Methods • Isolation and subfractionation of Mt • Determination of OCR and ECAR • Biochemical assays of Mt enzymes activity • Reactive oxygen species (ROS) and Mitochondrial Membrane Potential (MMP) • MtDNA copy numbers • Assays for Mt morphology

Mitochondrial Isolation3

3 Mitochondrial Isolation

Mt Isolation表5-1-1不同亚细胞组分的大小以及离心性质（肝脏）（选译至Dealtry,GB编撰的《Cellbiologylabfax》一书）亚细胞颗粒大小（μm)离心力（gav）时间(min）细胞核4~125~10500~1000线粒体10~150.4~2.51000~10000溶酶体10~200.4~0.86000~15000过氧化物酶体0.4~0.86000~1500010~20高尔基体1.0~2.020~3010000~20000内质网0.05~0.3530000~10000030~604

4 Mt Isolation

Mt Isolation表5-1-2不同离心力下的细胞组分（肝脏）（选译至Dealtry,GB编撰的（Cellbiologylabfar》一书）沉淀组分离心力（ga）*时间（min）P11000*10未破碎的细胞，细胞核，细胞膜复合物，重线粒体P23000*10细胞膜复合物片段，重线粒体P36000*10线粒体，溶酶体，过氧化物酶体，完整的高尔基体P410000*10线粒体，溶酶体，过氧化物酶体，高尔基体膜片段P520000*10溶酶体，过氧化物酶体，高尔基体膜片段，粗面内质网P6100000*50内质网，质膜，高尔基体等的膜片段小泡5

5 Mt Isolation

Mt Isolation53[6]MAMMALIANBRAINMITOCHONDRIA10%Brainhomogenate2000gx3minpelletsupernotant12,500gx8minCrudemitochondrialpellet(CM)Cresuspendin3%Ficol3%Ficoll6%Ficollresuspendinisolationmedium11,500gx30min11,500gx10minfreemitochondria(CandNM);rn

6 Mt Isolation

General Points for Mt preparations Performance at 4oC on ice. Cell rupturing by mechanical and /orchemical means and resuspension inisolation bufferDifferential centrifugation（差速离心）Storage. Criteria of purity and intactness?

7 General Points for Mt preparations • Performance at 4oC on ice • Cell rupturing by mechanical and /or chemical means and resuspension in isolation buffer • Differential centrifugation（差速离心） • Storage • Criteria of purity and intactness

Equipments for Mt Isolation· Tissue aliquot8

8 • Tissue aliquot Equipments for Mt Isolation

Homogenator 均质机MPOLYTRON9

9 Homogenator 均质机

Centrifuge离心机10

10 Centrifuge 离心机