Last edited Mon 29 Aug 2022

You will need the following materials:

NKM buffer

• 1 mM Tris HCl, pH 7.4
• 0.13 M NaCl
• 5 mM KCl
• ​7.5 mM MgCl₂

Homogenization buffer

• 10 mM Tris-HCl, pH 6.7
• 10 mM KCl
• 0.15 mM MgCl₂
• 1 mM PMSF
• 1 mM DTT

​Always add PMSF and DTT immediately before use.

Mitochondrial suspension buffer

• 10 mM Tris HCl, pH 6.7
• 0.15 mM MgCl₂
• 0.25 mM sucrose
• 1 mM PMSF
• 1 mM DTT

Stage 1 - Mitochondrial purification for western blot

Steps

Collect cells by centrifugation at approximately 370 x g for 10 min.

• Decant supernatant and re-suspend cells in 10 packed cell volumes of NKM buffer.

Pellet cells and decant supernatant, repeat this washing step 2 times.

• Resuspend cells in 6 packed cell volumes of homogenization buffer.

Transfer cells to a glass homogenizer and incubate for 10 min on ice.

• Using a tight pestle, homogenize the cells.
• Check under the microscope for cell breakage, the optimum is around 60%.
• This may require 30 strokes or so of the pestle.

Pour homogenate into a conical centrifuge tube containing 1 packed cell volume of 2 M sucrose solution and mix gently.

• Pellet unbroken cells, nuclei, and large debris at 1,200 g for 5 min and transfer the supernatant to another tube.
• This treatment is repeated twice, transferring the supernatant to a new tube each time, discarding the pellet.

Pellet the mitochondria by centrifuging at 7,000 x g for 10 min.

• Resuspend the mitochondrial pellet in 3 packed cell volumes of mitochondrial suspension buffer.
• Spin at 9,500 x g for 5 min to re-pellet the mitochondria.

Electrophoresis.

• At this point, you can add 1x protein gel loading buffer and run on a gel if a whole mitochondrial protein extract is needed, further purify the mitochondria on a sucrose gradient if you need very pure mitochondria, or purify a soluble (S-100) fraction of the mitochondria.
• See our soluble (S-100) mitochondrial fractionation protocol.