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Exosome Isolation Kit (from plasma, for protein detection) (EZB-exo102)
北京百奥创新科技有限公司
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Plasma + PBS |
Reagent |
200 µl + 100 µl |
60 µl |
1 ml + 0.5 ml |
300 µl |
3. 通过涡旋或倒置充分混合血浆/试剂混合物,直至溶液均匀。将样品在 2 ~ 8°C 下孵育 2 小时。
注意:如果需要,此沉淀步骤可以延长至过夜。
4. 孵育后,将样品在 20°C 下以 10000 × g 离心 30 分钟。 注意:对于小鼠血浆,在 4 °C 下离心 30 分钟。
5. 通过移液器小心吸出上清液并丢弃。 注意:外泌体包含在试管底部的颗粒中。
6. (可选)以 10000 × g 离心管 30 秒以收集任何残留试剂。用吸管小心吸出任何残留的上清液。
重悬外泌体
1. 向沉淀中加入 1× PBS 或类似缓冲液,上下涡旋或移液器重悬外泌体。
Starting Plasma Volume |
Resuspension Volume |
200 µl |
20 µl |
1 ml |
100 µl |
Introduction
The EZBioscience® Exosome Isolation Kit (from plasma, for protein detection) provides a simple, reliable, and rapid method for isolating high-quality total exosome from human and animal plasma, without the need for ultracentrifugation.
Exosomes are small vesicles (30–120 nm) containing RNA and protein that are secreted by various types of cells in culture, and found in abundance in body fluids, such as blood, saliva, urine, and breast milk. Exosomes were reported to function as intercellular messengers, delivering their cargo of effector or signaling macromolecules between specific cells; however, their formation, the makeup of the cargo, and biological pathways in which they are involved remain unclear.
The biological & medical study of exosome function and trafficking requires the isolation of intact exosomes, but the current methods used are tedious and difficult. The EZBioscience® Exosome Isolation Kit (from plasma, for protein detection) provides a simple and reliable method of concentrating intact exosomes from human and animal plasma samples. By tying up water molecules, the Exosome Isolation reagent forces less-soluble components (i.e. exosomes) out of solution, allowing them to be collected with a brief and relatively low-speed centrifugation.
The exosome could be isolated from the plasma within 4 hours by using this Kit.
After Isolation, the exosome can be used for further experiments, auch as Immuno-blotting and RNA purification, RNA sequencing, and RT-qPCR etc. .
Protocol
General Guidelines
a) The Exosome Precipitation Reagent (EPR) is not recommended for isolation of exosomes from any other body fluids or cell culture media. Specialized Exosome Isolation reagents are available for serum, urine, cell culture media, and other body fluids, each optimized for its specific type of biological sample.
b) For sample from blood, if downstream RNA expression profiling is planned, do not use hemolyzed samples. Even traces of red blood cells in serum or plasma will affect the RNA profile. Instead, we recommend using serum or citrate/EDTA plasma and discourage using heparin plasma (RNA isolated from heparin plasma can reduce PCR performance).
Prepare Sample
1. Remove the plasma sample from storage and place on ice. If the sample is frozen, the sample in a 25°C to 37°C water bath until it is completely thawed, and place on ice until needed.
2. Centrifuge the plasma sample at 3000 × g for 10 minutes at 20°C to remove cells and debris.
3. Transfer the supernatant containing the partially clarified plasma to a new tube without disturbing the pellet.
4. Centrifuge the new tube at 10000 × g for 20 minutes at 20°C to remove debris.
5. Transfer the supernatant containing the clarified plasma to a new tube without disturbing the pellet, and place it on ice until ready to perform the isolation.
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