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基本描述
| 英文名称 | Nuclear and Cytoplasmic Extraction Kit | ||||||||||||||||||||||||||||||||||||||||||
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| 储存温度 | -20°C储存 | ||||||||||||||||||||||||||||||||||||||||||
| 运输条件 | 超低温冰袋运输 | ||||||||||||||||||||||||||||||||||||||||||
| 产品介绍 |
产品内容
产品简介
Nc-细胞核/浆蛋白抽提试剂盒能够简单、快速的提取来源于哺乳动物细胞及组织的细 胞核和细胞浆蛋白，所提取蛋白保持生物学活性。本试剂盒首先通过细胞浆蛋白抽提试剂 裂解细胞膜，释放细胞浆蛋白，然后通过离心得到细胞核沉淀。最后通过细胞核蛋白抽提 试剂抽提得到细胞核蛋白。抽提获得的核蛋白及浆蛋白纯度高，有效避免核/浆蛋白的交叉 污染，可以用于Western、Gel Shift、报告基因检测以及酶活力测定等后续操作。 注意事项 1．如需提取磷酸化蛋白请在抽提试剂中加入磷酸酶抑制剂。 2．所有样品操作请置于冰上进行。 3．可根据具体实验情况调整试剂用量，保证各试剂使用比例为Nc-Buffer A:Nc-Buffer B:Nc-Buffer C=100:5.5:50。 4．可以采用更高的速度来离心。 操作步骤 I 细胞中胞浆、胞核蛋白的提取 1．请在蛋白抽提前取出抽提试剂Nc-Buffer A和Nc-Buffer C进行预冷 2．收集细胞，计数。离心去上清。 3．1×107 细胞中加入1 ml Nc-Buffer A（按照1:99比例在蛋白抽提前2-3分钟内加入Protease Inhibitor Cocktail），涡旋5秒以充分混匀，冰上孵育20分钟。 注意：各种细胞的特性不同，需要根据不同细胞的特性调整Nc-Buffer A的用量，如果蛋白浓度小， 按比例减少Nc-Buffer A及后续Nc-Buffer B、Nc-Buffer C的用量。 4．加入55 μl Nc-Buffer B，涡旋5秒以充分混匀，冰上孵育 1分钟。 5．4℃ 12,000 rpm（~13,400×g）离心 15分钟，收集上清（尽量收集干净上清液）至新的 离心管中，-20℃保存（此提取液为胞浆蛋白）。 6．向上步所得的沉淀中加入500 μl Nc-Buffer C（使用前按照1:99比例加入Protease Inhibitor Cocktail），涡旋5秒以充分混匀，重悬沉淀，冰上孵育40分钟，每隔10分钟涡旋混匀一 次，每次约15-30秒。 7．4℃ 12,000 rpm离心15分钟，收集上清（尽量收集干净上清液）至新的离心管中，-20℃ 保存（此提取液为胞核蛋白）。 II 组织中胞浆、胞核蛋白的提取 1．取材，保存组织。 2．在蛋白抽提前取出抽提试剂Nc-Buffer A和Nc-Buffer C进行预冷。 3．称组织重量，每100 mg组织中加入1 ml Nc-Buffer A（按照1:99比例在蛋白抽提前2-3 分钟内加入Protease Inhibitor Cocktail），用匀浆器在冰上充分匀浆，放置在冰上孵 育20分钟。 注意：各种组织的特性不同，需要根据不同组织调整Nc-Buffer A的用量，如果蛋白浓度小，按比例 减少Nc-Buffer A及后续Nc-Buffer B、Nc-Buffer C的用量。 4．加入55 μl Nc-Buffer B，涡旋5秒以充分混匀，放置在冰上孵育 1分钟。 5．4℃ 12,000 rpm离心 15分钟，收集上清（尽量收集干净上清液）至新的离心管中， -20℃保存（此提取液为胞浆蛋白） 6．向上步所得的沉淀中加入500 μl Nc-Buffer C（使用前按照1:99比例加入Protease Inhibitor Cocktail），涡旋5秒以充分混匀，重悬沉淀，冰上孵育40分钟，每隔10分 钟涡旋混匀一次，每次约15-30秒。 7．4℃ 12,000 rpm离心15分钟，收集上清（尽量收集干净上清液）至新的离心管中，
-20℃保存（此提取液为胞核蛋白）。
Product content
Products The Nc-Nucleus/Plasma Protein Extraction Kit is a simple and rapid method for extracting nucleus and plasma proteins from mammalian cells and tissues, and the extracted proteins remain biologically active. The kit first cleaves the cell membrane and releases plasma proteins using the plasma protein extraction reagent, and then centrifuges the nucleus to obtain a nucleus precipitate. Finally, the nuclear proteins are extracted by the nuclear protein extraction reagent. The extracted nuclear and plasma proteins are of high purity, effectively avoiding cross-contamination of nuclear and plasma proteins, and can be used for subsequent operations such as Western, Gel Shift, reporter gene detection and enzyme activity determination. Caveat 1. If phosphorylated proteins are to be extracted, add a phosphatase inhibitor to the extraction reagent. 2. All sample handling should be done on ice. 3. The amount of reagents can be adjusted according to the specific experimental situation to ensure that the ratio of each reagent used is Nc-Buffer A:Nc-Buffer B:Nc-Buffer C = 100:5.5:50. 4. Higher speeds can be used for centrifugation. Procedure I Extraction of cytoplasmic and cytosolic proteins from cells 1. Please remove the extraction reagents Nc-Buffer A and Nc-Buffer C for pre-cooling before protein extraction. 2. Collect the cells and count them. Centrifuge to remove supernatant. 3. 1×107 cells were added with 1 ml of Nc-Buffer A (added to Protease Inhibitor Cocktail at a ratio of 1:99 within 2-3 minutes prior to protein pumping), vortexed for 5 seconds to mix well, and incubated on ice for 20 minutes. Note: The characteristics of various cells are different, and the amount of Nc-Buffer A needs to be adjusted according to the characteristics of different cells. If the protein concentration is small, reduce the amount of Nc-Buffer A and subsequent Nc-Buffer B and Nc-Buffer C proportionally. 4. Add 55 μl of Nc-Buffer B, vortex for 5 seconds to mix thoroughly, and incubate on ice for 1 minute. 5. Centrifuge at 12,000 rpm (~13,400 x g) for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytoplasmic protein). 6. Add 500 μl of Nc-Buffer C (add Protease Inhibitor Cocktail at a ratio of 1:99 before use) to the precipitate obtained in the previous step, vortex for 5 seconds to mix thoroughly, resuspend the precipitate and incubate on ice for 40 minutes, vortexing and mixing at 10-minute intervals for about 15-30 seconds each time. 7. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is for cytosolic proteins). II Extraction of cytoplasmic and cytosolic proteins from tissues 1. Sampling and preservation of tissues. 2. Remove the extraction reagents Nc-Buffer A and Nc-Buffer C for pre-cooling before protein extraction. 3. Weigh the tissue and add 1 ml of Nc-Buffer A per 100 mg of tissue (add Protease Inhibitor Cocktail 2-3 minutes before protein extraction at a ratio of 1:99), homogenize well on ice with a homogenizer, and incubate on ice for 20 minutes. Note: The characteristics of various tissues are different, and the amount of Nc-Buffer A needs to be adjusted according to different tissues. If the protein concentration is small, reduce the amount of Nc-Buffer A and subsequent Nc-Buffer B and Nc-Buffer C proportionally. 4. Add 55 μl of Nc-Buffer B, vortex for 5 seconds to mix thoroughly, and place on ice for 1 minute of incubation. 5. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytoplasmic protein). 6. Add 500 μl of Nc-Buffer C (add Protease Inhibitor Cocktail at a ratio of 1:99 before use) to the precipitate obtained in the previous step, vortex for 5 seconds to mix thoroughly, resuspend the precipitate and incubate on ice for 40 minutes, vortexing and mixing at 10-minute intervals at , each time for about 15-30 seconds. 7. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytosolic protein). |
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