产品简介：
线粒体是细胞呼吸的主要场所，细胞活动所需的能量主要由在线粒体内进行的氧化所产生的能量来供应，制备线粒体的关键是保持线粒体的完整性和纯度，可通过分级分离法获得，即先低俗出去细胞核以及细胞碎片，再进行高速梯度离心分离线粒体。
组织线粒体分离试剂盒 (Tissue Mitochondria Isolation Kit)是快速便捷分离动物组织中的线粒体的试剂盒，分离线粒体的同时可以获得去除线粒体的细胞浆蛋白，可用于分析细胞色素C等线粒体蛋白向胞浆的释放，大部分获得的线粒体都含有完整的内膜和外膜，并具有线粒体的生理功能 (如检测线粒体膜电位)，获得的蛋白可用于SDS-PAGE、Western、双向电泳等蛋白分析。该试剂盒可用于从动物软组织 (如脑、肝脏)和硬组织 (心肌、骨骼肌)中提取线粒体。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
产品组成：
| O1509533 | Component | 50T | Storage |
| O1509533A | Mitochondria Lysis Buffer | 100 mL | -20℃. |
| O1509533B | Mitochondria Stock Buffer | 10 mL | -20℃. |
| O1509533C | Protein Stock Buffer (5×) | 10 mL | RT. |
| O1509533D | PMSF (100×) | 1.5 mL | -20℃. |
自备材料：1、低温离心机、匀浆器
2、PBS
操作步骤 (仅供参考)：
1、清洗：取新鲜组织 (不宜采用冻存的组织)，迅速称重50-100 mg，用预冷的PBS清洗1次，冰上剪成3 mm²大小的组织碎片。
2、匀浆裂解：加入10倍体积的预冷的Mitochondria Lysis Buffer (如需获得细胞浆蛋白，应提前加入PMSF，至PMSF浓度为1×)，置于冰浴上Dounce匀浆器中，匀浆10-20次；不同组织或不同匀浆器所需的匀浆次数有所不同，需自行优化。
3、离心：4 ℃ 600 g离心5 min以去除细胞核、未破碎的细胞和大的膜碎片。注：如需获得纯度更高的线粒体，可以将此步骤的离心速度改为2000 g离心3 min，其缺点是相同数量细胞的线粒体抽提得率会下降。
4、上清液转移至一干净离心管，4 ℃ 12000 g离心10 min。注：如需获得纯度更高的线粒体，可以将此步骤的离心速度改为6000 g离心10 min，其缺点是相同数量细胞的线粒体抽提得率会下降。
5、弃上清，沉淀为线粒体，如果希望获得去除线粒体的细胞浆蛋白，应在本步骤中收集上清，并且在收集上清时注意勿触及沉淀。随后把收集的上清12000 g 4 ℃离心10 min，上清即为去除线粒体的细胞浆蛋白。
6、保存：弃上清，用适当缓冲液悬浮沉淀。如果用于线粒体酶活性或功能的分析，线粒体沉淀应重悬于Mitochondria Stock Buffer；如果用于线粒体蛋白的分析，获得的细胞浆蛋白应保存于1×Protein Stock Buffer，即按细胞浆蛋白：Protein Stock Buffer (5×)=1: 4比例混合；如果用于双向电泳，应使用恰当的保存液。
注意事项：
1、试剂 (如PMSF)对于不同实验不必全部使用，在实验条件成熟后可以不必使用。
2、如果不是用于制备线粒体蛋白，Mitochondria Lysis Buffer不必加入PMSF；如果用于制备线粒体蛋白样品，Mitochondria Lysis Buffer需添加PMSF；PMSF一定要在试剂加入到样品中前1-2 min内加入，以免PMSF在水溶液中很快失效。
3、分离线粒体的所有步骤均需在冰上或4 ℃进行，所用溶液需冰浴或4 ℃预冷，全部操作时间尽量控制在1h以内。
4、通常在分离线粒体时前后两次离心速度选取1000 g和12000 g，如果希望纯度更高，但对线粒体的得率要求不高，前后两次离心速度可用2000 g和6000 g。
5、试剂开封后请尽快使用，以防影响后续实验效果。
6、为了您的安全和健康，请穿实验服并戴一次性手套操作。
Product Description
Mitochondria are the main site of cellular respiration. The energy required for cellular activities is mainly supplied by oxidation occurring within mitochondria. The key to mitochondrial isolation is to maintain mitochondrial integrity and purity, which can be achieved by differential centrifugation: low‑speed centrifugation to remove nuclei and cell debris first, followed by high‑speed gradient centrifugation to isolate mitochondria.
Tissue Mitochondria Isolation Kit is designed for the rapid and convenient isolation of mitochondria from animal tissues. During isolation, cytosolic proteins depleted of mitochondria can also be obtained, which can be used to analyze the release of mitochondrial proteins (such as cytochrome C) into the cytoplasm. Most isolated mitochondria maintain intact inner and outer membranes and physiological functions (e.g., for mitochondrial membrane potential detection). The isolated proteins can be used for protein analysis including SDS‑PAGE, Western blotting, and two‑dimensional electrophoresis. This kit is suitable for extracting mitochondria from soft animal tissues (e.g., brain, liver) and hard tissues (e.g., heart, skeletal muscle).This kit is for research use only and is not intended for clinical diagnosis or other purposes.
Components
| O1509533 | Component | 50T | Storage |
| O1509533A | Mitochondria Lysis Buffer | 100 mL | -20℃. |
| O1509533B | Mitochondria Stock Buffer | 10 mL | -20℃. |
| O1509533C | Protein Stock Buffer (5×) | 10 mL | RT. |
| O1509533D | PMSF (100×) | 1.5 mL | -20℃. |
Materials Provided by User
1. Refrigerated centrifuge, homogenizer
2. PBS
Protocol (For Reference Only)
1. Washing: Take fresh tissue (frozen tissue is not recommended). Weigh 50–100 mg rapidly, wash once with pre-cooled PBS, and cut into tissue fragments of approximately 3 mm² on ice.
2. Homogenization and Lysis: Add 10 volumes of pre-cooled Mitochondria Lysis Buffer (if cytosolic protein is required, add PMSF in advance to a final concentration of 1×). Place in a Dounce homogenizer on ice and homogenize 10–20 times. The number of homogenization cycles varies with different tissues or homogenizers and should be optimized by the user.
3. Centrifugation: Centrifuge at 600×g, 4 ℃ for 5 min to remove nuclei, unbroken cells and large membrane debris.Note: For higher mitochondrial purity, centrifuge at 2000×g for 3 min, but the mitochondrial yield from the same amount of cells will be reduced.
4. Transfer the supernatant to a clean centrifuge tube. Centrifuge at 12000×g, 4 ℃ for 10 min. Note: For higher mitochondrial purity, centrifuge at 6000×g for 10 min, but the mitochondrial yield from the same amount of cells will be reduced.
5. Discard the supernatant, the pellet is mitochondria.If cytosolic protein depleted of mitochondria is desired, collect the supernatant in this step without disturbing the pellet. Then centrifuge the collected supernatant at 12000×g, 4 ℃ for 10 min. The resulting supernatant is cytosolic protein without mitochondria.
6. Storage
Discard the supernatant and resuspend the pellet in an appropriate buffer. For mitochondrial enzyme activity or functional analysis: resuspend the mitochondrial pellet in Mitochondria Stock Buffer. For mitochondrial protein analysis: store the obtained cytosolic protein in 1× Protein Stock Buffer by mixing cytosolic protein: Protein Stock Buffer (5×) at a ratio of 1: 4. For two-dimensional electrophoresis: use an appropriate storage solution.
ZJ26F0434146
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