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Mitochondria Isolation Kit

AR0156

1 kit

Enabled

Mitochondria Isolation Kit

Western Blotting Reagents, Sample Preparation

No

Mitochondria Isolation Kit is a ready-to-use Western blot related reagent solution used for efficient isolation of mitochondria from animal homogenized animal tissues and cell lysates utilizing a dialyzable mild detergent at low concentration, for maximal protein solubilization and minimal interference with protein biological activity enabling direct use in downstream analysis.

Mitochondria Isolation Kit is a ready-to-use Western blot related reagent solution used for efficient isolation of mitochondria from animal homogenized animal tissues and cell lysates utilizing a dialyzable mild detergent at low concentration, for maximal protein solubilization and minimal interference with protein biological activity enabling direct use in downstream analysis.

List of Components Components Size Catalog Numbe Mitochondria Isolation Reagent A 100mL AR0156-A Mitochondria Isolation Reagent B (Mitochondria lysis buffer) 50mL AR0156-B Mitochondria Isolation Reagent C (Trypan Blue Solution) 10mL AR0156-C PMSF(100mM) 1mL AR0156-D Overview Pack Size 1 kit Assays per kit 100 assays for 2.0x10 7 cells. 100 assays for 0.1g tissue Storage & Expiration Upon receipt store at -20°C. It is stable for one year. Product is shipped on ice. Reagent Type Universal tissue extraction buffer, detergent solution, cell lysis buffer Usage Separation of Mitochondria from eukaryotic cells Equivalent Thermofisher (Product No. 89801, 89874), Millipore Sigma (Product No. MITOISO1,MITOISO2) Cite This Product Mitochondria Isolation Kit (Boster Biological Technology, Pleasanton CA, USA, Catalog # AR0156) Application SDS-PAGE, IP, Western Blotting, Dimensional Electrophoresis, Co-Immunoprecipitation Analysis and Enzymatic Determination . *Our Boster Guarantee covers the use of this product in the above tested applications. Description Mitochondria Isolation Kit is a ready-to-use Western blot related reagent solution used for efficient isolation of mitochondria from animal homogenized animal tissues and cell lysates utilizing a dialyzable mild detergent at low concentration, for maximal protein solubilization and minimal interference with protein biological activity enabling direct use in downstream analysis. Assay Principle . The Mitochondria Isolation Kit enables isolation of intact mitochondria from mammalian cells and fresh tissues. Highly purified mitochondria protein will be subsequently yielded after applying lysis buffer containing protease inhibitor. The kit uses traditional Dounce homogenization and the isolation relies on differential centrifugation to separate the mitochondrial and cytosolic fractions with microcentrifuge. Once isolated, the obtained mitochondria can be used for a number of downstream applications, including SDS-PAGE, IP, Western Blotting, Dimensional Electrophoresis, Co-Immunoprecipitation Analysis and Enzymatic Determination. Assay Protocol . Ⅰ. Mitochondria Isolation . 1. Reagent preparation . Equilibrate all reagents to room temperature and then place them on ice. Immediately before use, add PMSF to Reagent A and Reagent B in proportion and the final concentration of PMSF will be 1mM. . 2. Sample Preparation . a. Tissues . Rinse the fresh tissue (100-200 mg) with pre-cooling PBS or physiological saline to remove the blood. Then dry the tissue with filter paper. Mince the tissue into small pieces using scissors and place minced tissue in a small glass homogenizer. Add 1-2ml of pre-cooling Mitochondria Isolation Reagent A into the tissue and homogenize on ice until no tissue chunks remain. . b. Cultured cells . Suspending cell: Centrifuge directly to collect cells. Adherent cell: Scrape cells with scraper and then centrifuge to collect cells. Pellet 1×10 7 cells by centrifugation at ~600 × g for 5 minutes at 4°C. Carefully remove and discard the supernatant. Add 1 ml of pre-cooling Mitochondria Isolation Reagent A. Mix thoroughly and incubate on ice for 10-15 minutes. Vortex several times during incubation. (Add Mitochondria Isolation Reagent C to a small volume of cell suspension in the ratio of 1:1. Observe under microscopy. Homogenate is unnecessary if 80% of cells are blue. Homogenize cell suspension appropriately if less than 80% of cells are blue.) Please do not homogenize excessively to avoid mechanical injury to the mitochondria. . 3. Transfer tissue or cell homogenate to a tube, centrifuge at~600 × g for 5 minutes at 4°C. (To obtain a more purified fraction of mitochondria, centrifuge at ~1000 × g for 5 minutes at 4°C.) . 4. Discard precipitate and collect the supernatant in a separate tube. . 5. Centrifuge supernatant at ~12,000 × g for 10 min at 4°C. (To obtain a more purified fraction of mitochondria, centrifuge at ~16,000 × g for 10 min at 4°C.) . 6. Remove the supernatant from the mitochondrial precipitate carefully. . Ⅱ. Protein Extraction . 1. Add 50-70uL Mitochondria Isolation Reagent B to 10uL of isolated Mitochondria. . 2. Incubate for 10-15 minutes at 4°C. Vortex several times during incubation. . 3. Centrifuge at 10,000× g for 10 min at 4°C. Collect the supernatant (Mitochondria protein extracts). . Notes: . 1. For intact mitochondria, keep the temperature at 0-4°C and pH at 7.0. Process the whole mitochondria isolation in a short time. . 2. The number of Dounce homogenization strokes will vary depending upon homogenizer elasticity. Insufficient cell lysis with small number of strokes will result in low yield of mitochondria. . 3. If Mitochondria Isolation Kit is used in Dimensional Electrophoresis assays, Dimensional Electrophoresis lysis buffer should be used to lyse mitochondria. . 4. Mitochondrial protein can be analyzed by Boster BCA Protein Assay kit (AR0146).

1 kit

No

Integrity of mitochondria extracted from cultured cells . Hela cells were extracted with Boster Mitochondria Isolation Kit using Dounce homogenization. Mitochondrial(Lane 1) and cytosolic fractions( Lane 2) were analyzed via Western blot for Cytochrome C. Results indicate that mitochondria remained intact. Mitochondria Isolation Kit Components . Mitochondria Isolation Reagent A 100 ml. Mitochondria Isolation Reagent B (Mitochondria lysis buffer) 50 ml. Mitochondria Isolation Reagent C (Trypan Blue Solution) 10ml. PMSF(100mM) 1ml

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