This protocol begins by converting yeast cells into Sphero plats by digesting the cell wall with simul liase, the sphero plats are then homogenized using a glass Teflon homogenizer. The resulting homogenate is twice subjected to centrifugation, to pellet, unbroken cells, nuclei, and large debris. Next, the resulting supernatant is centrifuged for 15 minutes at 12, 000 Gs to sediment mitochondria.
Finally, the crude mitochondrial fraction that still contains other organelles is subjected to centrifugation for 60 minutes at 134, 000 Gs in a sucrose density gradient, pure mitochondria devoid of contamination by other organelles are recovered in the interface of 60%weight per volume and 32%weight per volume sucrose. Hi, I'm Dr.Christopher Greg from the lab of Dr.Vladimir tko in the biology department at Concordia University. Today I'm gonna show you how to isolate mitochondria from the yeast scro mysia.
We do this to study the dynamics and the age related changes that take place in the mitochondria. As the cells age, we're gonna look at changes in the proteome, lipid do, and the DNA of the mitochondria. So let's get started.
Begin this protocol by growing the yeast strain in ten two hundred and fifty milliliter flasks. Each containing 50 milliliters of YP medium at 30 degrees Celsius with rotational shaking for 48 hours. Next, pour the culture into 500 milliliter Nalgene centrifuge tubes.
Harvest the cells by centrifugation for five minutes At 3000 Gs at room temperature, decant the supernatant and resus resuspend the pelleted cells in 250 milliliters of distilled water centrifuge the cells for five minutes at 3000 Gs at room temperature. Repeat this. Step resuspend the pelleted cells in 250 milliliters of distilled water and centrifuge for five minutes.
At 3000 Gs at room temperature, weigh the resulting wet cell pellet, then resuspend the pelleted cells in DTT buffer transferred the cell suspension to a 250 milliliter flask. Rotate the flask at 70 RPMs in a 30 degrees Celsius for 20 minutes. After the 20 minutes, harvest the cells by centrifugation for five minutes At 3000 Gs at room temperature, resus suspend the pelleted cells in Xs buffer without Xs and centrifuge for five minutes.
At 3000 Gs at room temperature, resus suspend pelleted cells in Zy buffer without Xs again and transfer the cell suspension to a glass flask. Add the zy liase 100 T powder to the cell suspension and rotate at 70 RPMs in a 30 degrees Celsius shaker For 30 minutes, the Zy liase 100 T will digest the cell walls to form Sphero plats. All steps subsequent to the previously shown X digestion should be carried out at four degrees Celsius or on ice.
Suspensions of splats should be handled gently using pipettes with cut tips to avoid breaking organelles. Upon completion of the 30 minute XMO digestion, transfer the Sphero plats to 50 milliliter plastic centrifuge tubes centrifuge for eight minutes at 2200 Gs at four degrees Celsius reus suspend the pelleted S splats in ice cold homogenization buffer centrifuge. The S splats for eight minutes at 2200 Gs at four degrees Celsius.
Resus suspend the pelleted sphero plast in ice. Cold homogenization buffer and transfer cells to a pre chilled glass homogenizer. Using a tight pestle homogenize the cells by making 15 strokes of the pestle.
Add one equal volume of ice cold homogenization buffer to the homogenized S splats, and to transfer them to 50 milliliter plastic centrifuge tubes. Centrifuge the homogenized splats for five minutes at 1500 Gs at four degrees Celsius to pellet, unbroken cells, nuclei and large debris. Pour the resulting supernatant into new tubes and centrifuge for five minutes.
At 3000 Gs at four degrees Celsius. After that centrifuge, the resulting supernatant for 15 minutes At 12, 000 Gs at four degrees Celsius, decant the supernatant and resuspend the pellet in ice. Cold homogenization buffer centrifuge.
The resuspended pellet for five minutes at 3000 Gs at four degrees Celsius. Finally centrifuge the resulting supernatant for 15 minutes. At 12, 000 Gs at four degrees Celsius decant the supernatant and resus resuspend the pellet in three milliliters of ice cold.
SEM buffer. This suspension is the crude mitochondrial fraction. Although it is enriched in mitochondria, it also contains other organelles such as the endoplasmic reticulum or microsomes GOGI and VAs to get pure mitochondria.
The suspension will be subjected to further fractionation as described in the next section. To begin fractionation of the crude mitochondrial suspension to obtain pure mitochondria, place 1.5 milliliters of ice cold, 60%weight per volume. Sucrose in em buffer into a Beckman UltraClear centrifuge tube.
Next, gently overlay the 60%sucrose with four milliliters of 32%sucrose, 1.5 milliliters of 23%sucrose and 1.5 milliliters of 15%sucrose all weight per volume in em. Buffer place three milliliters of the crude mitochondrial fraction in SEM buffer on top of the 15%weight per volume. Sucrose centrifuge in a Beckman SW 41 TI swinging bucket rotor for one hour at 134, 000 Gs or 33, 000 RPMs at four degrees Celsius.
After centrifugation, the intact mitochondria will form a brown band at the 60%to 32%Sucrose interface. Carefully remove the sucrose until it reaches the mitochondrial band using a pipette with a cut tip, remove the mitochondrial band and place it into a Beckman centrifuge tube. For an MLS five rotor, fill the tube with SEM buffer pellet, the pure mitochondria by centrifugation in an MLS five rotor at 10, 000 GS or 31, 000 RPM for 30 minutes at four degrees Celsius decant the supernatant.
This pure mitochondria can now be used for the analyses of mitochondrial functions, mitochondrial proteome, and lipid do and mitochondrial DNA. The crude mitochondrial fraction in the left lanes and mitochondria purified by centrifugation in a sucrose density gradient in the right lanes were subjected to SDS page and immuno blotting with antibodies to protein markers of various organelles. The presence of mitochondria in both preparations was confirmed by PO one P and COX one p immunoreactivity immuno blotting indicated the presence of DP M1 P, FO 13 p and VPS 10 P, representing endoplasmic reticulum, ular, and GOGI proteins respectively in the crude mitochondrial fraction.
In contrast, these marker proteins were not detected in the purified mitochondria confirming that the protocol yielded mitochondria free of contamination by other organelles. So we've just shown you how to isolate mitochondria from the yeast sace visier. Just remember when you're running this experiment not to let the cells incubate in zy lies 100 T for longer than half an hour because you risk degrading your membrane proteins or organelle proteins that might be present in the cells.
This could be due to proteases present in the reagent. Also, when you're about to isolate the mitochondria and the sucrose gradient, you can freeze these mitochondria at minus 20 degrees before doing that and then thaw them before you're ready to actually do the centrifugation step. So that's it.
Good luck with your experiments.
