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Mitochondrial purification for western blot

Discover our guide to the isolation of mitochondria from cells for western blotting.

Last edited Mon 29 Aug 2022

You will need the following materials:

NKM buffer

Homogenization buffer

​Always add PMSF and DTT immediately before use.

Mitochondrial suspension buffer

Stage 1 - Mitochondrial purification for western blot

Steps

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

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

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

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

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

Electrophoresis.