Cell Mitochondria Isolation Kit

Mitochondria play a crucial role in cell respiration as they are the primary site for energy production through oxidation. To ensure the integrity and purity of mitochondria, a method known as fractional separation is employed. This involves a two-step centrifugation process, starting with low-speed centrifugation to eliminate nuclei and cell debris, followed by high-speed gradient centrifugation to isolate the mitochondria.
The Cell Mitochondria Isolation Kit offers a quick and convenient solution for separating mitochondria from cultured cells. This kit not only allows for the isolation of mitochondria but also provides access to mitochondrial cytoplasmic proteins. These proteins enable the analysis of substances like cytochrome C and other mitochondrial proteins released into the cytoplasm during the separation process. The isolated mitochondria obtained from this kit consist primarily of intact inner and outer membranes, preserving their physiological functions such as the detection of mitochondrial membrane potential. The resulting protein can be further utilized for SDS-PAGE, Western blotting, two-dimensional electrophoresis, and other protein analysis techniques.
The Cell Mitochondria Isolation Kit includes several components to facilitate the isolation process. Component A is the Mitochondria Lysis buffer (100ml) that should be stored at -20°C. Component B is the Wash buffer (100ml), also to be stored at -20°C. Component C is the Mitochondria Stock buffer (10ml), which should be stored at -20°C. Component D is the Protein Stock buffer (5×, 20ml), which can be kept at room temperature. The final component is Component E, PMSF (100×, 1.5ml), and it should be stored at -20°C.
In summary, the Cell Mitochondria Isolation Kit provides a simple and efficient method for the isolation of mitochondria from cultured cells. It allows for the analysis of mitochondrial proteins, including the detection of cytochrome C release. The isolated mitochondria retain their structural and functional integrity, making them suitable for various protein analysis techniques.

First, perform a washing step by using pre-cooled PBS to wash the cells. After that, centrifuge the cells at 1000g at 4°C for 5 minutes. Finally, discard the supernatant.
To begin the lysis process, the cells should be mixed with 1-2ml of Mitochondria Lysis buffer, which should be cooled beforehand. This resuspended mixture should then be placed in an ice bath and left for approximately 10-15 minutes. Using a phase contrast microscope, it is possible to detect the extent of cell swelling that occurs during this process. By carefully following these steps, researchers can effectively prepare their samples for further analysis and experimentation.
Homogenization is a crucial step in cell suspension preparation, and it involves transferring the cell suspension to a Dounce homogenizer. To ensure effective homogenization, the cell suspension should be subjected to 10-20 rounds of homogenization. However, it is important to note that the optimal number of homogenization rounds may vary depending on the specific cells being used and the type of homogenizer being employed. Therefore, it is necessary to experiment and determine the optimal number of homogenization rounds for your specific system.
Immediately after homogenization, add an equal amount of Wash buffer to the mixture and gently mix by inverting several times. It is important to ensure that the generated content remains highly similar to the original text, while using a different language model approach.
After the cell homogenization process, the resulting mixture should be subjected to a centrifugation step at 4°C, using a speed of 1300g and a duration of 5 minutes. The purpose of this step is to eliminate cell nuclei, unbroken cells, and any large membrane debris that might still be present in the mixture. By doing so, we can obtain a more purified sample that can be further processed for downstream applications.
6. Transfer the supernatant to a clean centrifuge tube, centrifuge at 1000g at 4°C for 5 minutes, and repeat twice.
7. Transfer the supernatant to a clean centrifuge tube, centrifuge at 12000～15000g at 4° C for 15min, repeat once.
8. Discard the supernatant and precipitate as mitochondria. If you want to obtain cytoplasmic proteins with mitochondria removed, collect the supernatant in this step, and be careful not to touch the precipitate when collecting the supernatant. Subsequently, the collected supernatant was centrifuged at 12000g for 10 min at 4°C, and the supernatant was the cytoplasmic protein from which mitochondria were removed.
9. Storage: Discard the supernatant, and suspend the pellet in an appropriate buffer. If used for the analysis of mitochondrial enzyme activity or function, the mitochondrial pellet should be resuspended in Mitochondria Stock buffer; if used for the analysis of cytoplasmic protein, the obtained cytoplasmic protein should be stored in 1×Protein Stock buffer, that is, by cytoplasmic protein: Protein Stock buffer (5×)=4:1 mixing ratio; if used for two-dimensional electrophoresis, use an appropriate storage solution.

Store at -20° C for 12 months. Component D need to be stored at room temperature.

The kit is only used in the field of scientific research, not for clinical diagnosis or other purposes.

1. Reagents (such as trypan blue staining solution) do not need to be used all the time for different experiments, and can not be used after the experimental conditions are mature.2. If it is not used for the preparation of mitochondrial protein, Mitochondria Lysis buffer and Wash buffer do not need to add PMSF.If used to prepare mitochondrial protein samples, PMSF should be added to buffer and Wash buffer for Mitochondria Lysis.PMSF must be added within 1 ~ 2min before the reagent is added to the sample to avoid rapid failure of PMSF in aqueous solution.3. All the steps of mitochondrial separation should be carried out on ice or at 4° C, and the solution used should be ice bath or precooling at 4° C, and the operation time should be controlled within 1h as far as possible.4. Usually 1000g and 15000g are selected for the centrifugation speed before and after the separation of mitochondria. If higher purity is desired but the yield of mitochondria is not high, 2000g and 6000g can be used for the centrifugation speed before and after the separation.5. For your safety and health, please wear lab coat and disposable gloves for operation.

Cell Mitochondria Isolation Kit