mitochondrial extraction kit

Product Introduction: Mitochondrial extraction kits are used to isolate complete and purified mitochondria from animal cells or tissues. Suitable for the preparation of animal soft tissues (liver or brain tissue) and hard tissues (muscle) as well as mitochondria of cultured cells. It has a high yield and can be used for the study of apoptosis, signaling, metabolism and proteomics. Operation steps: A. Tissue homogenate: Weigh 100~200 mg of fresh tissue such as liver, brain, myocardium, etc. , rinse with PBS or normal saline, wash off blood water, blot dry with filter paper, cut into pieces with scissors and put them into a small-capacity glass homogenizer. Lysis Buffer with 1.0 mL ice was added and the lysis was grinded up and down for 20 times in a 0℃ ice bath. b. Cultured cell homogenate: digested cells, washed with PBS, centrifuged at 800g for 5-10 min, collected cells and counted them. The Lysis Buffer of 5×10 7 cells was added to 1.0 mL of suspended cells pre-cooled by ice for each extraction. The cell suspension was transferred to a small-capacity glass homogenizer and ground in a 0℃ ice bath for 30~40 times. 2. Transfer the tissue or cell homogenate to a centrifuge tube and centrifuge at 1000g at 4℃ for 5 min. 3. Remove the supernatant and transfer it to a new centrifuge tube. Centrifuge again at 1000g at 4℃ for 5 min. 4. Remove the supernatant and transfer it to a new centrifuge tube, centrifuge at 4℃ at 12,000 g for 10 min. The supernatant after centrifugation contains cytoplasmic components from which cytoplasmic proteins can be extracted. The supernatant is transferred to a new centrifuge tube, and the mitochondria are deposited on the bottom of the tube. 5. 0.5mL Wash Buffer was added to the mitochondrial precipitation to suspend the mitochondrial precipitation, and centrifuged at 1000 g at 4℃ for 5 min. 6. Take the supernatant and transfer it to a new centrifuge tube, centrifuge at 4℃ at 12,000 g for 10 min. After the supernatant is discarded, high purity mitochondria are deposited on the bottom of the tube. 7. Re-suspend mitochondrial precipitation with 50-100 μL Store Buffer or appropriate reaction buffer for immediate use or -70℃ preservation. Note: 1. In order to obtain complete mitochondria, it is necessary to: first, the whole process of low temperature operation; Second, fast; Third, breaking up cells without destroying suborganelles is the most critical step in making mitochondria. Compared with tissue blocks, cultured cells, especially adherent cells, are more difficult to break the wall when homogenized by glass homogenizer, so small capacity glass homogenizer and tight gap pestle should be used to grind the cultured cells up and down. 2. Calculate the correct centrifugal speed with centrifugal force g, and different centrifuges can accurately calculate the centrifugal speed accordingly. 3. For Western Blot and 2D gel electrophoresis, mitochondria can be directly lysed by adding loading buffer and boiling at 100 °C for 5–10 minutes. Non-denatured proteins do not need to be boiled. Rotational speed and centrifugal force conversion: G = 1.11×(10-5)×R×[rpm] 2 G is the centrifugal force, generally expressed as a multiple of g (acceleration of gravity); [rpm] 2: the square of the speed; R is the radius in centimeters. Note: Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.