Pierce Mitochondria Isolation Kit for Cultured Cells

Only 40 minutes to isolate intact mitochondria with maximum yield

Isolation of mitochondria is typically a laborious process requiring singlesample processing with Dounce homogenization. The Mitochondria Isolation Kit uses a non-mechanical, reagent-based method that allows multiple samples (six) to be processed concurrently. Cultured mammalian cell pellets are gently lysed using a proprietary formulation that results in maximum yield of mitochondria with minimal damage to integrity (Figure 2). 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 more mitochondria recovery compared to the reagent-based method. Both methods use differential centrifugation to separate the mitochondrial and cytosolic fractions with a bench-top 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 2 x 10⁷ cells in approximately 40 minutes (post-cell harvest)
• Flexible – process several samples simultaneously using the reagentbased 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 2-D electrophoresis (Figure 3)

Figure 1. Procedure for the 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 3,000 x g instead of 12,000 x g.

Figure 2. Analysis of mitochondrial integrity. Mitochondria and cytosol fractions were prepared from C6 cells using the reagent-based method (A. and B.) or Dounce homogenization (C. and D.). Fractions were analyzed via Western blot for cytochrome C (A. and C.) or VDAC (B. and D.). SuperSignal West Pico Chemiluminescent Substrate was used for detection. Cytochrome C resides in the mitochondrial intermembrane space and VDAC is a membrane protein on the outer mitochondrial membrane. M: mitochondria, C: cytosol.

Figure 3. 2-D 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 2-D sample buffer (7 M urea, 2 M thiourea, 4% CHAPS, pH 3-10 carrier ampholytes, 50 mM 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.

* U.S. patent pending on Pierce Mitochondria Isolation Technology. SuperSignal^® Technology is protected by U.S. Patent # 6,432,662. BCA Technology is protected by U.S. Patent # 4,839,295

Ordering Information	Certificate of Analysis   Product Instructions   MSDS
Buy Product # Description Pkg. Size Files Price Add 89874 Mitochondria Isolation Kit for Cultured Cells Sufficient reagents for 50 applications. Kit contains: Mitochondria Isolation Kit Reagent A   50 ml Mitochondria Isolation Kit Reagent B 500 µl Mitochondria Isolation Kit Reagent C 70 ml Kit $199.00