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细菌膜囊泡提取纯化试剂盒
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为什么选择此级别
BioReagent,无菌 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
室温。常规运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
大多数细菌均可释放直径约 20-400 nm 的膜囊泡（Bacterial Membrane Vesicles，bMVs），这些囊泡内含有特定的功能分子，能够转运毒力因子、参与水平基因转移以及携带宿主防御因子等，在细菌与宿主间的信息交流中扮演重要角色，对细菌通讯、免疫逃逸和致病机制具有重要意义。
细菌膜囊泡的生物学功能研究中需要分离完整的囊泡颗粒，而传统超速离心方法硬件要求高、操作复杂且可能损害囊泡的完整性。为优化囊泡提取方法，阿拉丁生物自主开发的细菌膜囊泡提取纯化试剂盒，组分经过优化处理，适用于来自细菌培养上清中的细菌膜囊泡提取，并搭配纯化过滤装置，过程快速高效，获得的囊泡颗粒可用于电镜分析、NTA粒径分析、核酸分析、蛋白分析、细胞学实验和动物实验等。
组分与储存
| 组分货号 | 组分名称 | 物理外观 | 2T | 储存 |
| B1526235A | 细菌膜囊泡浓缩液 | 液体 | 12 mL | RT |
| B1526235B | 细菌膜囊泡纯化滤器 | - | 2 EA | RT |
自备材料：
高速离心机，涡旋振荡器，50 mL 离心管，1.5 mL 离心管，0.45μm 针头滤器，1×PBS 缓冲液（无菌）。
操作流程
一、 样品预处理
1、 取样：对于冻存的细菌培养上清，从冰箱取出后于 25℃水浴中进行解冻，将完全融化后的样品置于冰上；对于新鲜的细菌培养上清，收集样品后置于冰上；
2、 样品初始用量：单次提取时的样品量建议细菌培养上清液不低于 20 mL；
3、 离心去细菌碎片：将样品转移至离心管中，于 4℃以3,000 ×g（~5,200 rpm**）离心 10 min，去除样品中的细胞碎片（注：若沉淀较多，可 3,000 ×g，10 min离心多次至无明显沉淀，每次取离心上清液）；
**为约10 cm有效离心半径的大离心机换算（≥15 mL离心管），下同。
4、 离心去杂质碎片：将离心上清液转移至新的离心管中，于 4℃以 10,000 ×g（~9,500 rpm**）离心 10 min，去除样品中的杂质碎片；
5、 过膜去大颗粒杂质：收集去除杂质碎片后的上清液，将上清液过 0.45μm 针头滤器后，将滤过上清转移至新的离心管中。
二、 提取囊泡
1、 上清液预处理：在去除杂质的离心上清液中加入细菌膜囊泡浓缩液（BCS 试剂），具体的加入剂量为20 mL的细菌培养上清加入5 mL的BCS试剂（其他剂量请根据表中的用量等比例换算）。
2、 溶液混合：加入 BCS 试剂后将离心管盖紧，通过涡旋振荡器混匀 1 min，再放置于 4℃静置８ h 以上（注：增加静置时间可提高囊泡得率，但不可超过 24 h）；
3、 沉淀囊泡：取出装有混合液的离心管于 4℃以 10,000 ×g（~9,500 rpm**）离心 60 min，弃上清，沉淀中富含囊泡颗粒（注：尽可能吸尽上清液）；
4、 再次离心：将含有沉淀的离心管再次于 4℃以 10,000 ×g（~9,500 rpm**）离心 2 min，弃上清，以便去除管壁残留液体（注：尽可能吸尽上清液）；
5、 囊泡重悬：取合适量的 1×PBS 均匀吹打离心沉淀物，待其溶解后，将重悬液转移至新的 1.5 mL 离心管中（建议每 20 mL 细菌培养上清用 200 μL 左右 1×PBS 重悬）；
6、 收获囊泡颗粒：将含有重悬液的 1.5 mL 离心管于 4℃以 12,000 ×g（~12,400 rpm*）离心 2 min，保留上清液，上清液富含囊泡颗粒（注：若沉淀较多，可 12,000×g，2 min 离心多次至无明显沉淀，每次取上清液）。
*为约 7 cm 有效离心半径的小离心机换算（≤2 mL 离心管），下同。
三、 纯化细菌膜囊泡
1、 纯化囊泡：将收获的细菌膜囊泡颗粒粗品转入细菌膜囊泡纯化滤器（BPF 柱）上室中，于 4℃以 3,000 ×g（~6,200 rpm*）离心 10 min，离心后收集 BPF 柱管底的液体，此液体即为纯化后的细菌外囊泡颗粒（注：BPF 柱不可重复使用）；
2、 细菌膜囊泡的保存：纯化后的细菌膜囊泡以合适体积进行分装冻存于-80℃低温冰箱中，以备后续实验使用。
注意事项：
本产品仅用于生命科学研究，不得用于医学诊断及其他用途！
41、英文描述
Most bacteria can release membrane‑bound vesicles with a diameter of approximately 20‑400 nm, termed **bacterial Membrane Vesicles (bMVs). These vesicles contain specific functional molecules, which can transport virulence factors, mediate horizontal gene transfer and carry host‑defense factors. They play a critical role in bacterial‑host inter‑cellular communication, and are of great significance for bacterial communication, immune evasion and pathogenic mechanisms.
For biological‑function studies of bacterial membrane vesicles, intact vesicle particles need to be isolated. Conventional ultracentrifugation requires high‑end hardware, involves complicated operations, and may compromise vesicle integrity. To optimize vesicle isolation workflows, Aladdin Bio has independently developed the Bacterial Membrane Vesicles Isolation and Purification Kit. It is suitable for isolating bacterial membrane vesicles from bacterial culture supernatants. Equipped with supporting purification filter devices, the whole procedure is rapid and efficient. The obtained vesicle particles are compatible with electron‑microscopy analysis, nanoparticle‑tracking analysis (NTA), nucleic‑acid analysis, protein analysis, cytological assays and in‑vivo animal experiments.
Contents & Storage
| Component No. | Component Name | Appearance | 2T | Storage |
| B1526235A | bMVs Concentration Solution | Liquid | 12 mL | RT |
| B1526235B | bMVs Purification Filter | - | 2 EA | RT |
Materials to be supplied
High‑speed centrifuge, vortex mixer, 50 mL centrifuge tubes, 1.5 mL centrifuge tubes, 0.45 μm syringe filter, sterile 1×PBS buffer.
Procedure
I. Sample Pre‑treatment
1. Sample collection: For frozen bacterial culture supernatant, remove it from the refrigerator and thaw in a 25 °C water bath, then place the fully‑thawed sample on ice. For fresh bacterial culture supernatant, place the collected sample on ice.
2. Starting sample volume: A minimum of 20 mL bacterial culture supernatant is recommended per extraction.
3. Centrifugation to remove bacterial debris: Transfer the sample to a centrifuge tube and centrifuge at 3,000 × g (~5,200 rpm**) for 10 min at 4 °C to remove cellular debris. (Note: If a large pellet forms, repeat centrifugation at 3,000 × g for 10 min until no obvious pellet is observed, and collect the supernatant after each run.)
** Conversion for large‑format centrifuge with approximately 10 cm effective rotor radius (for ≥15 mL centrifuge tubes), applies hereinafter.
4. Centrifugation to remove impurity debris: Transfer the supernatant to a new centrifuge tube and centrifuge at 10,000 × g (~9,500 rpm**) for 10 min at 4 °C to remove impurity debris from the sample.
5. Filtration to remove large‑particle impurities: Collect the clarified supernatant, filter it through a 0.45 μm syringe filter, and transfer the filtered supernatant to a new centrifuge tube.
II. Vesicle Isolation
1. Supernatant conditioning: Add Bacterial Membrane Vesicle Concentrate (BCS Reagent) to the clarified supernatant. Add 5 mL of BCS Reagent for every 20 mL of bacterial culture supernatant. Scale proportionally for other sample volumes according to the table.
2. Solution mixing: Tighten the centrifuge‑tube cap after adding BCS Reagent, mix on a vortex mixer for 1 min, then incubate at 4 °C for at least 8 h. (Note: Longer incubation can improve vesicle yield; do not exceed 24 h.)
3. Vesicle precipitation: Centrifuge the tube containing the mixture at 10,000 × g (~9,500 rpm**) for 60 min at 4 °C and discard the supernatant. The pellet contains abundant vesicle particles. (Note: Remove the supernatant as completely as possible.)
4. Second centrifugation: Centrifuge the pellet‑containing tube again at 10,000 × g (~9,500 rpm**) for 2 min at 4 °C and discard the supernatant to eliminate residual liquid on the tube wall. (Note: Remove the supernatant as completely as possible.)
5. Vesicle resuspension: Pipette an appropriate volume of 1×PBS to gently resuspend the pellet. Once dissolved, transfer the resuspension to a new 1.5 mL centrifuge tube. (Approximately 200 μL of 1×PBS is recommended for resuspension per 20 mL starting bacterial culture supernatant.)
6. Harvest vesicle particles: Centrifuge the 1.5 mL tube with resuspension at 12,000 × g (~12,400 rpm*) for 2 min at 4 °C. Retain the supernatant, which is rich in vesicle particles. (Note: If substantial pellet remains, repeat centrifugation at 12,000 × g for 2 min until no obvious pellet is observed, and collect supernatant each time.)
*Conversion for small‑format centrifuge with approximately 7 cm effective rotor radius (for ≤2 mL centrifuge tubes), applies hereinafter.
III. Purification of Bacterial Membrane Vesicles
1. Vesicle purification: Load the crude bacterial‑membrane‑vesicle sample into the upper chamber of the Bacterial Membrane Vesicle Purification Filter (BPF Column). Centrifuge at 3,000 × g (~6,200 rpm*) for 10 min at 4 °C. Collect the liquid from the bottom receiver of the BPF column; this liquid contains purified bacterial membrane vesicles. (Note: BPF columns are non‑reusable.)
2. Storage of bacterial membrane vesicles: Aliquot purified bacterial membrane vesicles into suitable volumes and store at −80 °C for subsequent experiments.
Precautions
This product is for life‑science research only and must not be used for medical diagnosis or any other purposes.
规格
规格或纯度
BioReagent, 无菌, 无核酸酶
英文名称
Bacterial Membrane Vesicles Isolation and Purification Kit
应用
囊泡提取纯化
储存与运输
储存条件
室温
运输条件
常规运输
稳定性与储存
室温条件下保质期为24个月。
组分与储存
| 组分货号 | 组分名称 | 物理外观 | 2T | 储存 |
| B1526235A | 细菌膜囊泡浓缩液 | 液体 | 12 mL | RT |
| B1526235B | 细菌膜囊泡纯化滤器 | - | 2 EA | RT |
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高级数据
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技术文档和文章
溶液计算器
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