Submitochondrial fraction protocol 1.docx

Submitochondrial fraction protocol 1.docx

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Submitochondrialfractionprotocol1

Protocolfor

SubMitochondrialFractionation

1,000g×10min

supernatant

pellet

1,000g×10min

supernatant

1,000g×10min

supernatant

pellet

About21/22livers

homogenate

homogenized

About50mlhomogenizationbuffer

（10mlhomogenizationbufferper2~2.5gtissue）

Washedwithhomogenizationbuffer

Mincedwithscissor

weigh

livers

sacrificed

11C57micewerestarvedfor18to24hours

About1/22liver

Crudenuclear

homogenize

Resuspendedinabout50mlhomogenizationbuffer

pellet

15,000g×15min

supernatant

pellet

supernatant

50mlhomogenizationbuffer

144,000g×1hr

homogenize

cytosol

microsome

homogenate

15,000g×15min

supernatant

5mlhomogenate

45mlhomogenate

15,000g×15min

Crudemitochondria

supernatant

15,000g×15min

Crudemitochondria

（appliedtopurification）

supernatant

Crudemitochondria

（appliedtopurification）

Resuspendedin24ml25%Nycodenzbuffer

homogenize

12mlhomogenate

12mlhomogenate

52,000g×90min

5ml34%Nycodenz

8ml30%Nycodenz

12ml25%Nycodenz

8ml23%Nycodenz

3ml20%Nycodenz

AppliedtoNycodenzdensitygradientcentrifugation

Band（30%/25%）

Combinetogether

Band（30%/25%）

Dilutedwithhomogenizationbuffertoabout10ml

1mlhomogenate

9mlhomogenate

15,000g×15min

15,000g×15min

Purifiedmitochondria

Purifiedmitochondria

（forsubmitochondrialfraction）

Purifiedmitochondria

（appliedtosubmitochondrialfraction）

Suspension

0℃20min,gentlestirring

50mlswollenbuffer

0.5MATP（500×）;1.0MMgCl2（1000×）

Suspension

35,000g×20min

0℃5min,gentlestirring

pellet

intermembrane

50mlswollenbuffer

2strokesofatight-fittingpestle

1,900g×15min

homogenate

supernatant

mitoplast

35,000g×20min

Crudeoutmembrane

intermembrane

Alkalibuffer（0.5mg/ml）

Hand-drivenhomogenize

0℃20min

100,000g×30min

matrix

Crudeinnermembrane

Crudeoutermembrane

Contactsites

（37.7%and51.3%）

Outermembrane

（25.2%and37.7%）

121,000g×1hr

1mlhomogenate

1.2ml25.2%Sucrose

1.2ml37.7%Sucrose

1.2ml51.3%Sucrose

Appliedtosucrosedensitygradientcentrifugation

homogenate

Hand-drivenPotterhomogenizer

1mlSucrosedilutionbuffer

Mitoplast

（below51.3%）

Alkalibuffer（0.5mg/ml）

Hand-drivenhomogenize

0℃20min

10VSucrosedilutionbuffer

184,000g×1hr

100,000g×30min

Outermembrane

Contactsites

Innermembrane

matrix

Innermembrane

（below51.3%）

Crudeinnermembrane

Contactsites

（37.7%and51.3%）

Outermembrane

（25.2%and37.7%）

121,000g×1hr

1mlhomogenate

1.2ml25.2%Sucrose

1.2ml37.7%Sucrose

1.2ml51.3%Sucrose

Appliedtosucrosedensitygradientcentrifugation

homogenate

Hand-drivenPotterhomogenizer

1mlSucrosedilutionbuffer

10VSucrosedilutionbuffer

184,000g×1hr

Contactsites

Outermembrane

Innermembrane

11C57micearestarvedfor18~24hoursandsacrificed.Theliverswereexcisedimmediatelyandrinsedwithice-coldhomogenizationbuffertoremovetheremnantblood.Weightheliversandseparateabout1/22astotalliver.Theremnant21/22weremincedwithscissorsandhomogenized,usingabout50mlhomogenizationbuffer（about10mlhomogenizationbufferper2~2.5gtissue）.Thehomogenatewasthenfilteredthroughawiregauzetoremovesomebiggertissue.

Theflow-throughhomogenatewascentrifugatedin1,000gfor10minutes.Thepelletwascrudenuclearandthesupernatantwascentrifugatedagainin1,000gfor10minutes.Thisstepwasrepeatedfor2times.

Thefinalsupernatantwascentrifugatedin15,000gfor15minutes.Thepelletcontainedprimarilymitochondria,andthesupernatantwasagaincentrifugatedin144,000gfor90minutestogetmicrosomepelletandcytosolsupernatant.

Themitochondria-containedpelletwasrinsedwithhomogenizationbufferandcentrifugatedin15,000gfor15minutes.Thisstepwasrepeatedfor2~3timesandthefinalpelletwasresuspendedinabout50mlhomogenizationbuffer.About5mlhomogenatewasseparatedandcentrifugatedin15,000gfor15minutes,andthepelletwasusedascrudemitochondria.Theother45mlhomogenatewascentrifugatedin15,000gfor15minutestooandthepelletwasappliedtofurtherpurification..

ThecrudemitochondrialpelletwaspurifiedfromNycodenzdensitygradientcentrifugation.Thepelletwasresuspendedin24ml25%Nycodenzbuffer.Anddividedintotwoequalpart,12mlforeach,andappliedtotwoseparateddensitygradientcentrifugation.Thegradientwasconsistof5ml34%Nycodenz,8ml30%Nycodenz12ml25%Nycodenz（whichcontainedthesample）,8ml23%Nycodenzand3ml20%Nycodenz.Aftercentrifugationin52,000gfor90minutes,thebandintheinterfaceof30%and25%Nycodenzofthetwotubeswascollected,combinedanddiluted15,000gfor15minutes,withthepell