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大型真菌囊泡提取纯化试剂盒
有货
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,-80℃储存,避免反复冻融。常规运输,特低温运输 。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
大型真菌囊泡在免疫调节、抗肿瘤及神经修复等治疗中具有重要作用，但其提取面临子实体组织成分复杂（如多糖、萜类等干扰物）和细胞壁结构致密的技术瓶颈，导致囊泡提取得率低、纯度不足。本试剂盒专为大型真菌设计，可高效释放真菌子实体组织中的囊泡，并去除子实体组织中的非囊泡成分，获得的囊泡可用于电镜分析、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
注：发货时各组分依据储存温度（RT/-20℃）独立包装，差异化温控运输以保障产品性能。同一订单含多温区物料可能分批次送达，请以实际收货为准。收货后按组分表标注温度分类存放，有效期自生产之日起 12 个月。 |
自备材料：
一、实验材料/耗材
1、 削皮刀、水果刀、玻璃砧板等（可以把待提取植物处理成方便榨汁的小块的用具）；
2、 200 mL 烧杯 1~2 个；
3、 50 mL 离心管、1.5mL EP 管若干；
4、 pH 试纸，1~10（广泛），5.5~8.5（精密）。
二、实验设备
1、 电子秤；
2、 碾磨榨汁机（推荐使用，可搜索原汁机购买）；
3、 4℃高速离心机；
4、 水浴锅；
5、 涡旋振荡器。
三、实验试剂
1×PBS 溶液（无菌）
操作规程：
一、 榨汁前准备
1、 榨汁机消毒：将榨汁机各部件拆分开，喷洒 75%酒精仔细擦拭后，放入生物安全柜紫外消毒至少 20 min；
2、 样品准备：2 T包装可处理20 mL的大型真菌汁液（加 PBS处理后）。
3、 样品处理：将新鲜的待提取大型真菌洗净外表皮后，用无尘布擦干，放入生物安全柜紫外消毒至少 20 min 后，将真菌子实体切成方便榨汁的薄片；
注：一般药用大型真菌含水量很少，榨汁阶段无法出汁，所以尽量将真菌子实体组织处理成尽可能小的薄片。
二、 榨汁取渣
1、 榨汁机准备：在生物安全柜下，将完成消毒的榨汁机部件按照榨汁机说明书组装；
2、 榨汁取渣：将处理好的真菌子实体小片投入榨汁机榨汁，注意少量多次，收集出渣口碾磨粉碎后的真菌渣；
注：a. 如需控制内毒素，榨汁取渣过程请在生物安全柜内进行；
b. 如果收集到的真菌渣中仍有未粉碎完全的子实体组织，建议将收集到的真菌渣重新榨汁粉碎。
3、 真菌渣分散：向收集好的真菌渣中加 PBS，边加边搅拌混匀，直至真菌碎渣完全分散在 PBS 中，且混匀后的液体有良好的流动性；
注：a. 如需控制内毒素，榨汁取渣、真菌渣分散过程请在生物安全柜内进行；
b. 分散所加的 PBS 体积需要根据真菌渣释放出内容物的粘稠程度而定，可以少量多次加，及时搅拌观察液体状态。
三、 提取囊泡
1、 汁液预处理：在分散好的真菌渣液体中加入混匀的溶液A 试剂，添加剂量为20 mL
的PBS 分散好的真菌渣液体加入200 μL 溶液A（其他剂量请等比例换算）。
注：溶液A试剂使用前需混匀。
2、 混合孵育：加入溶液A试剂后盖好管盖，颠倒混匀 8~10 次，将混合液 37℃水浴 16 h 左右（过夜）；
3、 调 pH：将孵育好的混合液取出，在生物安全柜下用试剂盒自带的1M 氢氧化钠和 1%柠檬酸将混合液 pH 调至 7.0；
4、 低速离心去杂：将调 pH 后的液体于 4℃，5,000 ×g，10 min 离心，收集上清；
5、 高速离心去杂：将低速去杂后的上清 4℃，10,000 ×g，20 min 离心后再次收集上清；
6、 过滤：将上步收集得到的上清依次经 1 μm 滤膜和0.45 μm 滤膜过滤；
注：如需获得 30-1000 nm 大型真菌囊泡，可只进行1 μm 滤膜过滤，不进行 0.45 μm 滤膜过滤。
7、 加提取试剂：取上步离心上清液加入溶液 B试剂，添加剂量为20 mL的离心过膜后的上清液加入5 mL的溶液 B（其他剂量请等比例换算）。
8、 二次混合孵育：加入溶液 B后拧紧管盖，通过涡旋振荡器混匀 1 min，将混合液 4℃静置 4 h 左右；
9、 沉淀大型真菌囊泡：取孵育好的混合液于 4℃，10,000×g，60 min 离心，弃上清，将含有沉淀的离心管再次于 4℃，10,000 g，2 min 离心后，吸尽上清；
注：沉淀中富含大型真菌囊泡，需尽可能吸尽上清液。
10、 大型真菌囊泡重悬：取合适量的 1×PBS 均匀吹打离心沉淀，待其溶解后，将重悬液转移至新的 1.5 mL EP管中（建议每 20 mL 植物汁液用 400 μL 左右 1×PBS重悬）；
11、 大型真菌囊泡收获：将含有重悬液的 1.5 mL EP 管于4℃，12,000 ×g 离心 2 min，保留上清液，其中富含大型真菌囊泡颗粒；
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。
四、 纯化和保存大型真菌囊泡
1、 囊泡纯化：将收获的大型真菌囊泡颗粒粗品转入外泌体纯化滤器（EPF 柱）上室中，于 4℃以 3,000 ×g 离心 10 min，离心后收集 EPF 柱管底
的液体，此液体即为纯化后的大型真菌囊泡颗粒；
注：a. EPF 柱不可重复使用；
b. 如需获得大颗粒囊泡，可不进行该步骤。
2、 囊泡保存：纯化后的大型真菌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
1、试剂盒内的溶液A，收到后尽快放-80℃保存，使用前 4℃解冻，解冻后尽快用完，禁止反复冻融。
2、溶液A试剂静置有棕黄色沉淀，属于正常现象，使用前需混匀。
3、纯化后的大型真菌囊泡可保存在-80℃冰箱中，避免反复冻融导致囊泡破裂。
Macro‑fungi‑derived extracellular vesicles exhibit promising therapeutic potential in immune regulation, anti‑tumor therapy, nerve repair and other fields. Nevertheless, their isolation is hampered by technical bottlenecks including complex matrix components (polysaccharides, terpenoids and other interfering impurities) and dense cell‑wall structures in fruiting‑body tissues, which usually result in low vesicle yield and poor purity. This kit is specially developed for macro‑fungi samples. It can efficiently release extracellular vesicles from fungal fruiting‑bodies and eliminate non‑vesicular contaminants. The harvested vesicles are suitable for transmission electron microscopy (TEM), nanoparticle‑tracking analysis (NTA), nucleic acid assay, protein analysis, cell‑based experiments and in‑vivo animal studies.
Contents & Storage
Note: Each component is individually packaged according to its storage temperature (RT/-20 °C) upon shipment, with differentiated temperature‑controlled transportation to guarantee product performance. Orders containing materials requiring multiple temperature zones may be delivered in separate batches; please refer to the actual received goods. Upon receipt, store components separately at the temperatures specified in the component list. The shelf‑life is 12 months from the date of manufacture. |
Materials to Be Prepared
1.Consumables & Supplies
(1) Peeling knife, paring knife, glass cutting board and other tools for cutting fungal samples into small pieces suitable for juicing.
(2) One to two 200 mL beakers.
(3) Multiple 50 mL centrifuge tubes and 1.5 mL EP tubes.
(4) Wide‑range pH test strips (pH 1‑10) and precision pH test strips (pH 5.5‑8.5).
2.Equipment
(1) Electronic balance.
(2) Grinder‑juicer (slow‑speed juicer is recommended).
(3) High‑speed refrigerated centrifuge (4 °C).
(4) Water‑bath incubator.
(5) Vortex mixer.
3.Self‑supplied Reagent
Sterile 1×PBS solution
Operating Protocol
Step 1. Pre‑juicing Preparation
1. Juicer disinfection: Disassemble all components of the juicer. Wipe each part thoroughly with 75 % ethanol, then perform UV‑sterilization inside a biosafety cabinet for at least 20 min.
2. Sample‑loading reference: One 2‑test kit is designed to process 20 mL of macro‑fungal homogenate after dilution with PBS.
3. Sample pretreatment: Rinse fresh macro‑fungal fruiting‑bodies and dry the surface with lint‑free wipes. Place samples in a biosafety cabinet for UV‑sterilization for no less than 20 min, then cut fruiting‑bodies into thin slices for juicing.
Note: Medicinal macro‑fungi usually contain very low water content and produce little free juice during juicing. Slice tissues into pieces as small as possible.
Step 2. Juicing and Tissue Homogenization
1. Assemble the disinfected juicer inside a biosafety cabinet following the manufacturer’s instructions.
2. Juice the sliced fruiting‑bodies in small batches. Collect the ground fungal residues from the pulp outlet.
Notes:
a. All juicing and residue‑collection operations should be carried out in a biosafety cabinet if low‑endotoxin vesicles are required.
b. If incompletely crushed tissue fragments remain in residues, re‑juice the sample for full homogenization.
3. Residue dispersion: Add 1×PBS to the collected fungal residues with continuous stirring until all tissue fragments are fully suspended and the mixture maintains good fluidity.
Notes:
a. Perform residue dispersion inside a biosafety cabinet for low‑endotoxin preparations.
b. Add PBS incrementally and observe the viscosity of suspension to determine the final working volume.
Step 3. Extraction of Extracellular Vesicles
1. Pre‑treatment of homogenate: Add well‑mixed Solution A to the fungal‑residue suspension. Add 200 μL of Solution A for every 20 mL of PBS‑suspended homogenate; scale the dosage proportionally for other sample volumes.
Note: Mix Solution A thoroughly before use.
2. Incubation: Seal the tube, invert 8‑10 times for mixing, then incubate the mixture in a 37 °C water‑bath for approximately 16 h (overnight).
3. pH adjustment: After incubation, adjust the pH of the mixture to 7.0 inside a biosafety cabinet using the kit‑supplied 1 M NaOH and 1 % citric acid solution.
4. Low‑speed centrifugation for debris removal: Centrifuge the pH‑adjusted mixture at 5 000 × g, 4 °C for 10 min and retain the supernatant.
5. High‑speed centrifugation for secondary clarification: Centrifuge the collected supernatant at 10 000 × g, 4 °C for 20 min and collect the supernatant again.
6. Filtration: Filter the supernatant sequentially through a 1‑μm filter membrane followed by a 0.45‑μm filter membrane.
Note: If vesicles with particle sizes of 30‑1000 nm are required, filter only with the 1‑μm membrane and skip the 0.45‑μm filtration step.
7. Addition of precipitation reagent: Add Solution B to the filtered supernatant. Add 5 mL of Solution B for every 20 mL of supernatant; scale proportionally for other volumes.
8. Secondary incubation: Tighten the cap, vortex‑mix for 1 min, then incubate the mixture at 4 °C for around 4 h.
9. Pelleting macro‑fungal extracellular vesicles: Centrifuge the incubated mixture at 10 000 × g, 4 °C for 60 min. Discard the supernatant. Re‑centrifuge the tube containing the pellet at 10 000 × g, 4 °C for 2 min, then aspirate residual supernatant completely.
Note: The pellet is rich in macro‑fungal extracellular vesicles; remove supernatant as thoroughly as possible.
10. Vesicle resuspension: Resuspend the pellet with an appropriate volume of 1×PBS by gentle pipetting. Transfer the resuspended solution into a new 1.5 mL EP tube. It is recommended to use approximately 400 μL of 1×PBS for resuspension of vesicles derived from 20 mL fungal homogenate.
11. Harvest crude vesicles: Centrifuge the resuspension at 12 000 × g, 4 °C for 2 min. Keep the supernatant which contains enriched macro‑fungal extracellular‑vesicle particles.
Note: If visible precipitate remains, repeat centrifugation (12 000 × g, 2 min) several times and collect supernatant each time.
Step 4. Purification and Storage of Extracellular Vesicles
1. Vesicle purification: Transfer the crude vesicle sample into the upper chamber of the EPF purification column. Centrifuge at 3 000 × g, 4 °C for 10 min. Collect the eluate at the bottom of the EPF column; this fraction contains purified macro‑fungal extracellular vesicles.
Notes:
a. EPF columns are single‑use and cannot be reused.
b. Skip this purification step if large‑size vesicles need to be preserved.
2. Storage: Aliquot purified extracellular‑vesicle suspensions and store at −80 °C for downstream experiments.
Precautions
1. Store Solution A at −80 °C immediately upon receipt. Thaw at 4 °C before use and consume quickly after thawing. Repeated freeze‑thaw cycles are prohibited.
2. A brown‑yellow precipitate may form in Solution A after standing, which is normal. Thoroughly mix before use.
3. Purified macro‑fungal extracellular‑vesicle samples should be preserved at −80 °C. Avoid repeated freeze‑thaw cycles, which may cause vesicle rupture.
规格
规格或纯度
无菌, BioReagent
英文名称
Macro-fungi extracellular vesicles Isolation and Purification Kit
应用
囊泡提取纯化
样品类型
真菌(Fungi)(复数)
储存与运输
储存条件
室温,-80℃储存,避免反复冻融
运输条件
常规运输,特低温运输
稳定性与储存
各组分在相应的储存条件下保质期12个月。
组分与储存
| 储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 2T | 储存温度 |
| M1528033NT | 大型真菌囊泡提取纯化试剂盒(常温组分) | M1528033A | 溶液B | 液体 | 6.5mL | 室温 |
| M1528033B | 1%柠檬酸 | 液体 | 2.5mL | 室温 | ||
| M1528033C | 1M 氢氧化钠 | 液体 | 5mL | 室温 | ||
| M1528033D | 1 μm 滤膜 | - | 2EA | 室温 | ||
| M1528033E | 0.45 μm 滤膜 | - | 2EA | 室温 | ||
| M1528033F | 外泌体纯化滤器 | - | 2EA | 室温 | ||
| M1528033UT | 大型真菌囊泡提取纯化试剂盒(-80℃组分) | M1528033G | 溶液A | 液体 | 2.5mL | -80℃.避免反复冻融 |
注：发货时各组分依据储存温度（RT/-20℃）独立包装，差异化温控运输以保障产品性能。同一订单含多温区物料可能分批次送达，请以实际收货为准。收货后按组分表标注温度分类存放，有效期自生产之日起 12 个月。
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