Cell Mitochondria Isolation Kit

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Catalogue No: abx090630
Price: US$217.50
(Size: 50-100 tests)

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Datasheet
Abbexa's Cell Mitochondria Isolation kit is a quick, convenient, and sensitive method for the isolation of enriched and pure mitochondrial fraction from cells. This kit can also be used to obtain the mitochondria-free cytoplasmic protein which can be used for studying the release of mitochondrial protein and cytoplasm. As most of the isolated mitochondria will contain intact inner and outer membranes, after treating it with Mitochondria Lysis Buffer, the isolated mitochondria can be used for SDS-PAGE, Western blot and Di-electrophoresis assays. This kit can be used to perform 50-100 assays (2-5 × 107 cells) for the isolation of enriched mitochondrial fraction from cells.

Tested Applications WB
Storage Store all reagents at -20 °C for up to one year.
Kit Components
  • Mitochondria Isolaftion Reagent: 60 ml
  • Mitochondria lysis Buffer : 20 ml
  • PMSF (100 mM): 1 ml
Availability Shipped within 5-10 working days.
Note This product is for research use only.
Directions for use

Before carrying out the procedure, thaw all reagents, then place on ice. Gently shake each reagent bottle to ensure thorough mixing.  The working solutions should be prepared 2-3 minutes before the carrying out the protocol below.

Collection of cells

  1. Adherent cells: Wash the cells with PBS. Trypsinize the cells and then centrifuge to collect the cells. 
  2. Suspension cells: Centrifuge directly to collect the cells

Preperation of reagents

  1. Mitochondria isolation reagent working solution: Add 1 µl of PMSF to 99 µl of Mitochondria isolation reagent to make final concentration of working solution to 1 mM.
  2. Mitochondria lyis buffer working solution: Add 1 µl of PMSF to 99 µl of Mitochondria lysis buffer to make final concentration of working solution to 1 mM.

Assay procedure

  1. Wash the cells with PBS, count the cells and then centrifuge at 600 × g at 4 °C for 5 mins.
  2. Discard the supernatant and collect the cell pellets for carrying out the assay.
  3. Add 1-2.5 ml of prepared Mitochondria isolation reagent working solution per 2-5 × 107 cells. Incubate on ice for 10-15 mins.
  4. Gradually homogenize the cells on ice (around 10-30 strokes).
  5. Take 2 µl of cell homogenate and add 30-50 µl of Trypan blue staining buffer (optional) after 10 strokes of homogenization. 
  6. Count the cells under microscope.
  7. Repeat the homogenization step (additional 5 strokes each time) in case of <50% damaged cells (blue cells). Avoid excessive homogenization to avoid mitochondrial breakage.
  8. Centrifuge the cell homogenate at 600 × g at 4 °C for 10 mins and collect the supernatant. 
  9. Centrifuge the supernatant at 11,000 × g at 4 °C for 10 mins. 
  10. Discard the supernatant and the pellet is the mitochondria.

Usage of Mitochondria: 

  1. For applications requiring intact mitochondria: Resuspend the mitochondria with 150 - 200 µl PBS (0.01 M, pH 7.4) for measurement of citrate synthase activity or cytochrome c oxidase activity.
  2. For mitochondria protein characterization or functional assays: Allow the lysis of mitochondria with 150- 200 µl of prepared Mitochondria lysis buffer working solution for performing SDS-PAGE, Western blot and Immunoprecipitation assays.
  3. For Di-electrophoresis assay: Allow the lysis of mitochondria with Di-electrophoresis specific lysis buffer.

Notes

  • Sequentially homogenize the cells until there is at least 50% damaged cells (blue cells).
  • For isolating higher purfied mitochondria (change assay procedure step 8 & 9 with this), centrifuge the cell homogenate at 1000 × g at 4 °C for 10 mins. to collect the supernatant. Centrifuge the supernatant at 3500 × g at 4 °C for 10 mins, the pellet thus obtained is a higher purified mitochondria but the yield of the mitochondria of the same cells will be decreased accordingly.
  • For mitochondria-free cytoplasmic protein, collect the final supernatant and centrifuge at 1200 × g at 4 °C for 10 mins.
  • All isolation steps should be carried out on ice or at 4 °C.
  • This kit is intended for use with cultured cell and fresh tissue samples. It is not suitable for use with frozen tissue samples.
  • Personal Protective Equipment (PPE), such as lab coats, disposable gloves and lab glasses, should be worn when using this kit.
Research Articles on Cell Mitochondria Isolation Kit


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