TITLE: Mitochondrial Isolation SOP (#10)
AUTHORS: Elena Pumbo, Kimberly Chapman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for mitochondrial isolation used by our group for assays needing intact and mitochondrial lysates.</div></div>

[STEPS]
?. [Tissue Prepration]
Tissue preparation
?. [Tissue Prepration]
For 5 mL of 15-20 mg/mL of liver mitochondria protein we need 10-15 gm of frozen mouse liver tissue. If more of less protein needed scale these numbers up or down.
?. [Tissue Prepration]
Cool down centrifuge and Rotor F-35-6-30 for 6 x 50 mL and 5 x 15 mL tubes. Set temperature +40C.
?. [Tissue Prepration]
·Take livers from -800C freezer put on dry ice.
?. [Tissue Prepration]
·Measure weight of a 10x10 cm square of aluminum foil using Mettler Toledo Analytical Balance and write it down.
?. [Tissue Prepration]
·Put on the foil unwrapped frozen livers up to 15 gm (about 10 livers)
?. [Tissue Prepration]
·Subtract foil weight.
?. [Tissue Prepration]
·Write number.
?. [Tissue Prepration]
·Keep livers on ice.
?. [Tissue Prepration]
·Bring liquid nitrogen in special 1 L liquid nitrogen container.
?. [Tissue Prepration]
·Take ceramic mortar and pestle
?. [Tissue Prepration]
·Fill mortar with liquid nitrogen and put pestle on dry ice cool down.
?. [Tissue Prepration]
·Put metal spatula on dry ice to cool down.
?. [Tissue Prepration]
·After liquid nitrogen evaporate mortar is cold enough.
?. [Tissue Prepration]
·Prepare 50 mL conical tube and put int on dry ice
?. [Tissue Prepration]
·Put 1st frozen liver in the mortar and add ½ liquid nitrogen up to ½ mortar volume.
?. [Tissue Prepration]
·Wrap pestle with paper towel to avoid frost bite and cover mortar with paper towel.
?. [Tissue Prepration]
·Grind liver till there is only powder in the mortar.
?. [Tissue Prepration]
·With cold spatula put grinded tissue in the 50 mL tube.
?. [Tissue Prepration]
·Repeat with next liver until all of them are ground.
?. [Tissue Prepration]
·Put mortar and pestle aside to warm up.
?. [Tissue Prepration]
·Clean the bench with 70% ethanol.
?. [Tissue Prepration]
·Prepare fresh 50 mL conical tube.
?. [Tissue Prepration]
·Measure weight using Mettler Toledo Analytical Balance and wright it down.
?. [Tissue Prepration]
·Transfer in fresh tube 10 gm of grinded liver powder (subtract weight of empty tube from weight of the tube with tissue).
?. [Tissue Prepration]
·Extra grinded liver store in -800 C.
?. [Tissue Prepration]
·To the tube with 10 gm of frozen liver add Tissue lysis Buffer up to 50 mL.
?. [Tissue Prepration]
·Wait till all tissue thawed. Vortex from time to time.
?. [Tissue Prepration]
·Prepare 25 Matrix A 2mL tubes for MP Homogenizer
?. [Tissue Prepration]
·When tissue is fully thawed using transfer pipettes aliquot 50 mL in Martix A tubes.
?. [Tissue Prepration]
·Following instruction for MP Homogenizer choose on touch screen
?. [Tissue Prepration]
-Recommended Settings (Animal: mouse, organ: liver)
?. [Tissue Prepration]
-Run
?. [Tissue Prepration]
·After program finished put tubes with homogenized liver on regular ice.
?. [Tissue Prepration]
·Clean and turn off MP Homogenizer
?. [Tissue Prepration]
·Using transfer pipette transfer homogenate into fresh 50ml tube.
?. [Tissue Prepration]
2. Mitochondria Extraction
?. [Tissue Prepration]
·Prepare 50 mL conical tube with water for balance
?. [Tissue Prepration]
·Using transfer pipette transfer homogenate from Matrix A tubes into fresh 50ml tube.
?. [Tissue Prepration]
·Label tube “Pellet 1”
?. [Tissue Prepration]
·Put 50  mL tube with homogenate and balance into Rotor F-35-6-30
?. [Tissue Prepration]
1st slow centrifugation. Close rotor lid tightlyClose centrifugeSet centrifuge: 2000 rpm, 10 min,
4 °C
?. [Tissue Prepration]
·After centrifugation finished, take tube from centrifuge and put on ice.
?. [Tissue Prepration]
·Transfer supernatant into fresh tube. Label tube “Supernatant 1”
?. [Tissue Prepration]
·Discard pellet.
?. [Tissue Prepration]
·Adjust balance volume to new sample volume.
?. [Tissue Prepration]
·Put 50 mL tube with homogente and balance into Rotor F-35-6-30
?. [Tissue Prepration]
1st fast centrifugation. Close rotor lid, tightlyClose centrifuge, Set centrifuge: 7830 rpm, 20 min,
4 °C
?. [Tissue Prepration]
·After centrifugation finished, take tube from centrifuge and put on ice.
?. [Tissue Prepration]
·With q-tips remove white fat floating on the top of supernatant.
?. [Tissue Prepration]
·Discard supernatant.
?. [Tissue Prepration]
·Resuspend pellet in 25 mL of Tissue Lysis Buffer.
?. [Tissue Prepration]
·Transfer into fresh tube. Label tube “Pellet 2”
?. [Tissue Prepration]
·Adjust balance volume to new sample volume.
?. [Tissue Prepration]
·Put 50 mL tube with homogenate and balance in Rotor F-35-6-30.
?. [Tissue Prepration]
2st slow centrifugation: Close rotor lid tightly,  Close centrifuge.Set centrifuge: 2000 rpm, 10 min,
4 °C
?. [Tissue Prepration]
·After centrifugation finished, take tube from centrifuge and put on ice.
?. [Tissue Prepration]
·Transfer supernatant into fresh tube. Label tube “Supernatant 2”
?. [Tissue Prepration]
·Discard pellet.
?. [Tissue Prepration]
·Adjust balance volume to new sample volume.
?. [Tissue Prepration]
2st fast centrifugation: Close rotor lid tightly, close centrifuge.Set centrifuge: 7830 rpm, 20 min,
4 °C
?. [Tissue Prepration]
·After centrifugation finished, take tube from centrifuge and put on ice.
?. [Tissue Prepration]
·Change rotor for Rotor FA-45-48-11 for 48 2 mL tubes.
?. [Tissue Prepration]
·With q-tips carefully remove fat floating on top of supernatant.
?. [Tissue Prepration]
·Discard supernatant.
?. [Tissue Prepration]
·Resuspend pellet in 12 mL of Tissue Lysis Buffer.
?. [Tissue Prepration]
·Transfer for 2 mL into fresh 2 mL microcentrifuge tubes.
?. [Tissue Prepration]
·Load rotor distributing tubes equally.
?. [Tissue Prepration]
Wash centrifugation: Close rotor lid tightly, close centrifuge, Set centrifugre: 12700 rpm, 20 min,
4 °C
?. [Tissue Prepration]
·After centrifugation finished, take tube from centrifuge and put on ice.
?. [Tissue Prepration]
·Discard supernatant.
?. [Tissue Prepration]
·Pellets resuspend in 5-6 mL of Tissue Lysis Buffer and transfer into 15 mL conical tube.
?. [Tissue Prepration]
·Put back Rotor F-35-6-30 for 15 and 50 mL tubes.
?. [Tissue Prepration]
·Set centrifuge: 7830 rpm, 20 min,
4 °C
?. [Tissue Prepration]
·After centrifugation finished, take tube from centrifuge and put on ice.
?. [Tissue Prepration]
·Discard supernatant.
?. [Tissue Prepration]
3. Mitochondria Protein Extraction
?. [Tissue Prepration]
Prepare 10 mL of Cell Lysis Buffer with 1 tablet of Proteinase Inhibitor (PI) cocktail.
?. [Tissue Prepration]
·Label tube with preparation date.
?. [Tissue Prepration]
Using 5mL serological pipette add to mitochondria pellet up to 5 mL of Cell Lysis Buffer.
?. [Tissue Prepration]
·Using
?. [Tissue Prepration]
Using   pipettor with  pipette tip add  of Triton X-100.
200 µl
200 µl
50 µl
?. [Tissue Prepration]
·Pipette up and down 20 times to make sure all Triton X-100 was washed from the tip.
?. [Tissue Prepration]
·Resuspend the pellet.
?. [Tissue Prepration]
Incubate 15 min  (in cold room) with rotation.
4 °C
?. [Tissue Prepration]
·Transfer 2 mL into fresh 2 mL microcentrifuge tubes.
?. [Tissue Prepration]
·Change rotor for Rotor FA-45-48-11 for 48 x 2 mL tubes.
?. [Tissue Prepration]
·Load rotor distributing tubes equally.
?. [Tissue Prepration]
·Close rotor lid tight.
?. [Tissue Prepration]
·Close centrifuge.
?. [Tissue Prepration]
·Set centrifuge: 12,700 rpm, 20 min
4 °C
?. [Tissue Prepration]
·After centrifugation finished, take tube from centrifuge and put on ice.
?. [Tissue Prepration]
·Transfer supernatant into 5 mL tube.
?. [Tissue Prepration]
Next step – protein concentration measurement. Summar Lab SOP #4 “Protein Quantification by Bradford Assay”