Mitochondrial extraction kit instructions

Â Component

SM0020 -50

SM0020-100

Lysis Buffer

50 ml

100 ml

Mito-Wash Buffer

25 ml

50 ml

Store Buffer

5 ml

10 ml

Mitochondrial extraction kit

Item No.: SM0020

Specifications: 50T/100T

Storage: Store at 4 Â°C for 4 weeks and -20 Â°C for long-term storage .

Component

Product introduction:

The mitochondrial extraction kit is used to isolate intact and purified mitochondria from animal cells or tissues. Preparation of mitochondria suitable for animal soft tissue (liver or brain tissue) and hard tissue (muscle) as well as cultured cells. Its preparation yield is high and can be used for research on apoptosis, signaling, metabolism and proteomics.

Steps:

1. Sample processing a. Tissue homogenization: Weigh 100~200 mg fresh tissue such as liver, brain, heart muscle, etc., rinse with PBS or normal saline, wash blood, filter paper, dry, use scissors to cut into pieces and put into small capacity. Inside the glass homogenizer. Add 1.0 mL of ice-cold Lysis Buffer , and grind the tissue up and down 20 times in an ice bath at 0 Â°C ; b. Culture the cell homogenate: digest the cells, wash with PBS , centrifuge at 800 g for 5-10 min, collect the cells, and count. 5 Ã— 107 cells were needed for each extraction , and 1.0 mL of ice-cold Lysis Buffer was used to resuspend the cells. The cell suspension was transferred to a small-capacity glass homogenizer and ground in an ice bath at 0 Â°C for 30 to 40 times.

2. Transfer the tissue or cell homogenate to a centrifuge tube, centrifuge at 1000g for 5 min at 4 Â°C .

3. The supernatant was transferred to a new centrifuge tube, 4 â„ƒ, 1000g again centrifuged 5 min.

4. Remove the supernatant, transfer to a new centrifuge tube, centrifuge at 12,000 g for 10 min at 4 Â°C . The supernatant after centrifugation contains a cytosolic component from which cytosolic proteins can be extracted. The supernatant was transferred to a new centrifuge tube and the mitochondria were deposited at the bottom of the tube.

5. Add 0.5 mL Wash Buffer to the mitochondrial pellet and resuspend the mitochondrial pellet, and centrifuge at 1000 Â°C for 5 min at 4 Â°C .

6. Remove the supernatant, transfer to a new centrifuge tube, centrifuge at 12,000 g for 10 min at 4 Â°C . The supernatant is discarded and high purity mitochondria are deposited at the bottom of the tube.

7 with 50 -100 Î¼ L Store Buffer or a suitable reaction buffer mitochondrial pellet was resuspended used immediately or stored at -70 â„ƒ.

Note: 1. In order to ensure the complete mitochondria, it is necessary to: first, the whole process of low temperature operation; second, fast; third, breaking the cell without destroying the subcellular organelle, which is the most critical link in the preparation of mitochondria . Compared with the tissue block, the cultured cells, especially the adherent cultured cells, are more difficult to break when homogenized with a glass homogenizer. Therefore, the cells should be cultured up and down by using a small-capacity glass homogenizer and a well-stitched mortar. 2. Calculate the correct centrifugal speed by centrifugal force g . Different centrifuges can accurately calculate the centrifugal speed. 3. Perform Western Blot and 2D- gel electrophoresis, and directly add the loading buffer to lyse the mitochondria. Speed â€‹â€‹and centrifugal force conversion: G = 1.11 Ã— (10-5) Ã— R Ã— [rpm] 2 G is the centrifugal force, generally expressed as a multiple of g (gravity acceleration); [rpm] 2: the square of the rotational speed; R is the radius The unit is in centimeters.