Submitochondrial fraction protocol 1.docx

1、Submitochondrial fraction protocol 1Protocol for SubMitochondrial Fractionation1,000g10minsupernatantpellet1,000g10minsupernatant1,000g10minsupernatantpelletAbout 21/22 livershomogenatehomogenizedAbout 50ml homogenization buffer (10ml homogenization buffer per 22.5g tissue)Washed with homogenization

2、 bufferMinced with scissorweighliverssacrificed11 C57 mice were starved for 18 to 24 hoursAbout 1/22 liver Crude nuclearhomogenizeResuspended in about 50ml homogenization bufferpellet15,000g15minsupernatantpelletsupernatant50ml homogenization buffer144,000g1hrhomogenizecytosolmicrosomehomogenate15,0

3、00g15minsupernatant5ml homogenate45ml homogenate15,000g15minCrude mitochondriasupernatant15,000g15minCrude mitochondria（applied to purification）supernatantCrude mitochondria（applied to purification）Resuspended in 24ml 25% Nycodenz bufferhomogenize12ml homogenate12ml homogenate52,000g 90min5ml 34% Ny

4、codenz8ml 30% Nycodenz12ml 25% Nycodenz8ml 23% Nycodenz3ml 20% NycodenzApplied to Nycodenz density gradient centrifugationBand (30% / 25%)Combine togetherBand (30% / 25%)Diluted with homogenization buffer to about 10ml1ml homogenate9ml homogenate15,000g 15min15,000g 15minPurified mitochondriaPurifie

5、d mitochondria( for submitochondrial fraction)Purified mitochondria( applied to submitochondrial fraction)Suspension 0 20 min, gentle stirring50ml swollen buffer0.5M ATP（500）; 1.0M MgCl2 (1000)Suspension35,000g20min0 5 min, gentle stirringpelletintermembrane50ml swollen buffer2 strokes of a tight-fi

6、tting pestle1,900g15minhomogenate supernatantmitoplast35,000g20minCrude out membraneintermembraneAlkali buffer (0.5mg/ml)Hand-driven homogenize0 20 min 100,000g30minmatrixCrude inner membraneCrude outer membraneContact sites(37.7% and 51.3%)Outer membrane (25.2% and 37.7%)121,000g1hr1ml homogenate1.

7、2ml 25.2% Sucrose1.2ml 37.7% Sucrose1.2ml 51.3% SucroseApplied to sucrose density gradient centrifugationhomogenateHand-driven Potter homogenizer1ml Sucrose dilution bufferMitoplast(below 51.3% )Alkali buffer (0.5mg/ml)Hand-driven homogenize0 20 min 10V Sucrose dilution buffer184,000g1hr100,000g30mi

8、nOuter membraneContact sitesInner membranematrixInner membrane(below 51.3% )Crude inner membraneContact sites(37.7% and 51.3%)Outer membrane (25.2% and 37.7%)121,000g1hr1ml homogenate1.2ml 25.2% Sucrose1.2ml 37.7% Sucrose1.2ml 51.3% SucroseApplied to sucrose density gradient centrifugationhomogenate

9、Hand-driven Potter homogenizer1ml Sucrose dilution buffer10V Sucrose dilution buffer184,000g1hrContact sitesOuter membraneInner membrane11 C57 mice are starved for 1824 hours and sacrificed. The livers were excised immediately and rinsed with ice-cold homogenization buffer to remove the remnant bloo

10、d. Weigh the livers and separate about 1/22 as total liver. The remnant 21/22 were minced with scissors and homogenized, using about 50ml homogenization buffer (about 10ml homogenization buffer per 22.5g tissue). The homogenate was then filtered through a wire gauze to remove some bigger tissue.The

11、flow-through homogenate was centrifugated in 1,000g for 10 minutes. The pellet was crude nuclear and the supernatant was centrifugated again in 1,000g for 10 minutes. This step was repeated for 2 times.The final supernatant was centrifugated in 15,000g for 15 minutes. The pellet contained primarily

12、mitochondria, and the supernatant was again centrifugated in 144,000g for 90 minutes to get microsome pellet and cytosol supernatant. The mitochondria-contained pellet was rinsed with homogenization buffer and centrifugated in 15,000g for 15 minutes. This step was repeated for 23 times and the final

13、 pellet was resuspended in about 50ml homogenization buffer. About 5ml homogenate was separated and centrifugated in 15,000g for 15minutes,and the pellet was used as crude mitochondria. The other 45ml homogenate was centrifugated in 15,000g for 15minutes too and the pellet was applied to further pur

14、ification.The crude mitochondrial pellet was purified from Nycodenz density gradient centrifugation. The pellet was resuspended in 24ml 25% Nycodenz buffer. And divided into two equal part, 12ml for each, and applied to two separated density gradient centrifugation. The gradient was consist of 5ml 3

15、4% Nycodenz, 8ml 30% Nycodenz 12ml 25% Nycodenz (which contained the sample), 8ml 23% Nycodenz and 3ml 20% Nycodenz. After centrifugation in 52,000g for 90 minutes, the band in the interface of 30% and 25% Nycodenz of the two tubes was collected, combined and diluted 15,000g for 15minutes, with the pell