A better alternative to Dounce homogenization for small-scale isolation of intact mitochondria from cells.

The Thermo Scientific Mitochondria Isolation Kit for Cultured Cells provides a versatile, microcentrifuge-tube method for fractionating intact mitochondria from cultured mammalian cell samples in about 40 minutes.

Isolation of mitochondria is typically a laborious process requiring single-sample processing with Dounce homogenization. The Mitochondria Isolation Kit uses a non-mechanical,reagent-based method that allows multiple (six) cultured cell samplesto be processed concurrently in about 40 minutes. Cultured mammalian cell pellets are gently lysed using a proprietary formulation that results in maximum yield of mitochondria with minimal damage to integrity. The product instructions describe guidelines for optimizing purity vs. yield parameters. The kit also offers an optimized Dounce homogenization procedure, which results in two-fold greater mitochondria recovery compared to the reagent-based method. Both methods use differential centrifugation to separate the mitochondrial and cytosolic fractions with a benchtop microcentrifuge and are completed in approximately 40 minutes (post-cell harvest). Once isolated, the mitochondria can be used in downstream applications such as apoptosis, signal transduction and metabolic studies, as well as to facilitate mitochondrial proteomics efforts.

Highlights:

• Fast – isolate intact mitochondria from 20 million cells in approximately 40 minutes (post-cell harvest)
• Flexible – process several samples simultaneously using the reagent-based method, or obtain highest possible yield with the Dounce homogenization method
• Optimizable – guidelines provided for optimizing purity vs. yield with the reagent-based method
• Benchtop-easy – isolation performed in a microcentrifuge tube
• Compatible – isolated mitochondria may be lysed with detergent or processed for almost any downstream application, including 2D electrophoresis

Protocol summary for isolation of mitochondria from cultured mammalian cells. Option A. Reagent-based method Option B. Dounce homogenization method. A more purified preparation of mitochondria can be obtained by centrifuging at 3000 x g instead of 12,000 x g.

Product Details:

Integrity of mitochondria extracted from cultured cells. C6 cells were extracted with Thermo Scientific Mitochondria Isolation Kit using the reagent-based method (A and B) or Dounce homogenization (C and D). Mitochondrial (M) and cytosolic (C) fractions were analyzed via Western blot for cytochrome C (A and C) or voltage-dependent anion channel (VDAC) (B and D). Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080) was used for detection. Results indicate that mitochondria remained intact.

2D Western blot of superoxide dismutase (Mn-SOD) in isolated mitochondria. Mitochondria were isolated from NIH3T3 cells using the Dounce homogenization protocol and re-suspended in 2D sample buffer (7M urea, 2M thiourea, 4% CHAPS, pH 3-10 carrier ampholytes, 50mM DTT). Proteins were resolved on a pH 3-10 IPG strip followed by 8-16% SDS-PAGE and then analyzed by Western blot for superoxide dismutase.