product description
This reagent kit is based on the principle of molecular exclusion chromatography (SEC method), which separates extracellular vesicles through differences in molecular size. It has the advantages of easy operation, high purity, and high recovery rate, and is particularly suitable for extracellular vesicle separation of large volume samples. The isolated extracellular vesicles can be used for WB analysis, NTA or nanoflow cytometry particle size analysis, electron microscopy detection, omics research, and functional studies in cell and animal experiments. This kit is most commonly used for large volume samples such as cell culture supernatants and urine, and is also suitable for samples such as serum and plasma.
ordering information
product name | Item number | specifications |
Extracellular vesicle isolation kit (SEC method) | AC25L434 | 4T |
Product components
components | specifications | storage condition |
A. Exosome Purification Column（10mL） | 4个 | 4℃ |
B. Column Adapter（10mL） | 4个 | normal temperature |
Transportation and storage
Transport at room temperature. Component A is stored at 4 ℃, while component B is stored at room temperature with a shelf life of 12 months.
Usage
Self provided instruments, reagents, and consumables: high-speed freeze centrifuge, centrifuge tube, ultrafiltration tube (MWCO: 50 kDa), PBS (freshly prepared and used, 0.2um filtration, ultrasonic or vacuum degassing), 20% ethanol (freshly prepared and used, 0.2um filtration, ultrasonic or vacuum degassing), purification column fixator (self purchased or suitable fixed test tube iron stand to avoid shaking during operation)
1. Sample pretreatment
(1) Remove cells: Centrifuge the sample at 4 ℃ and 300g for 5 minutes, transfer the supernatant to a new centrifuge tube; Cell free samples can skip this step.
(2) Remove cells and debris: Centrifuge at 4 ℃ and 2000g for 10 minutes, transfer the supernatant to a new centrifuge tube.
(3) Remove large volume particles: Centrifuge the supernatant obtained in step (2) at 4 ℃ and 14000g for 30 minutes, and transfer the supernatant to a new centrifuge tube.
2. Purification column pretreatment
(1) Install the Exosome Purification Column (10mL) onto the purification column holder and place the waste liquid tank below. Allow the purification column to equilibrate at room temperature (18-25 ℃) for at least 30 minutes; Low or high temperature can affect the separation efficiency.
(2) Check if the lower end of the purification column is filled with liquid. If there is no air, skip this step; If there is air, invert the purification column, open the lower sealing cap, inject water or 20% ethanol with a pipette or syringe to push out the air, fill the sealing cap with liquid, then cover it again and place it back in the holder.
(3) Open the top cover of the purification column, then open the lower sealing cap, discard the sealing solution (which can be poured out or aspirated with a pipette), connect the purification column adapter (10 mL) to the purification column, and add 2 times the column volume (20 mL) PBS from the top for equilibrium until no solution flows out from the lower end. Ensure that the top sieve plate remains moist throughout the cleaning process. After completion, cover the bottom sealing cap, disconnect the adapter, and add 1 mL of PBS for later use.
Note:
① Before each operation, the upper cover of the purification column should be opened first, and then the lower sealing cap should be opened to prevent air from entering and affecting the separation efficiency.
② Ensure that the top sieve plate of the purification column is always moist during the separation process to maintain optimal separation efficiency.
③ When adding a large volume of solution to the purification column, the adapter can be connected to the purification column and the solution can be added to the adapter.
④ It is recommended to freshly prepare PBS and filter it through a 0.2 μ m membrane, or use commercially sterile PBS to prevent microbial or particle contamination. PBS must be equilibrated to room temperature before use to avoid bubbles in the purification column affecting the separation efficiency.
3. Separate extracellular vesicles
(1) Sample loading: Remove PBS from the purification column and add 1 mL of sample above; If the sample is less than 1 mL, PBS can be used to make up to 1 mL, mix well, and load the sample. Remove the bottom sealing cap of the purification column and add PBS after all samples have entered the purification column.
Note:
① If the sample volume exceeds 1 mL, it is recommended to concentrate the sample to 1 mL using a 50 kDa ultrafiltration tube, with a concentration factor not exceeding 20 times (see the ultrafiltration tube user manual for specific instructions).
② For high viscosity samples (such as plasma, serum, high viscosity pleural and peritoneal fluid, etc.), 0.5 mL of the sample can be diluted with PBS to 1 mL, mixed well, and loaded onto the sample.
③ Add PBS after the sample has completely entered the sieve plate to avoid dilution and affecting the separation efficiency.
(2) Extracellular vesicle separation: Place a 1.5 mL centrifuge tube below the purification column, add 0.5 mL PBS upward, and wait for the 0.5 mL fraction to be collected below. Continue to add 0.5 mL PBS and replace with a new 1.5 mL centrifuge tube for collection. Mark each distillation tube number in the order of outflow.
(3) Extracellular vesicle collection: Collect tubes 4, 5, 6, 7, and 8 to obtain extracellular vesicles, with tubes 5, 6, and 7 having higher concentrations of extracellular vesicles. The number of extracellular vesicle particles and protein concentration can be directly measured in the collected solution.
(4) Extracellular vesicle concentration: After measuring the concentration of extracellular vesicle particles and proteins, concentration can be selected based on subsequent experimental needs. If concentration is required, it is recommended to use a MWCO 50 kDa ultrafiltration tube and centrifuge at 4000g to the desired volume.
4. Purification column maintenance
(1) After collecting all fractions, connect the adapter to the purification column and wash the column with at least twice the column volume (20 mL) of PBS from the top until no solution flows out from below.
(2) Continue to add 1.5 times the column volume (15 mL) of 20% ethanol for cleaning until no solution flows out below.
(3) Finally, remove the adapter and add 3 mL of 20% ethanol blocking solution to the purification column. Install the top cover of the purification column and fill the sealing cap with 20% ethanol. Cover the bottom outlet of the purification column and store it upright at 4 ℃.
matters needing attention
1. This product is only for scientific experimental research and should not be used in clinical diagnosis, treatment, or other fields.
2. For your safety and health, please wear lab clothes and disposable gloves when operating.
3. There may be a certain gap between the upper sieve plate and the white agarose microsphere of the newly purchased or used purification column, which is caused by gel sedimentation during storage and use, and does not affect the performance of the purification column. Push the upper sieve plate down to no gap before normal use.
4. This kit is most commonly used for large volume samples such as cell culture supernatants and urine, and is also suitable for samples such as serum and plasma. For other trace precious samples, please consult our technical personnel.
5. The purification column is stored in a closed liquid, which is 20% ethanol. 20% ethanol is recommended to be prepared and used immediately, and filtered through a 0.2um filter membrane, ultrasonically or vacuum degassed to avoid causing a large number of bubbles in the purification column, which may affect the separation efficiency.
6. Before each use of the purification column, sterile and equilibrated PBS should be used for cleaning. PBS is recommended to be prepared and used immediately, and filtered through a 0.2um filter membrane, ultrasonically or vacuum degassed to avoid causing a large number of bubbles in the purification column, which may affect the separation efficiency.
7. There should be no bubbles in the middle of the purification column, and careful inspection should be carried out before and after use to avoid affecting the experiment.
8. Purification columns can be reused repeatedly, but multiple uses can affect the effectiveness. It is recommended to reuse them no more than 5 times.
9. When performing NGS or other omics analysis on isolated exosomes, it is recommended to use a new purification column for each sample to avoid cross contamination.
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Extracellular vesicle isolation kit (SEC method) (item number: AC25L434)
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S-Buffer Replacement Kit (Product Code: AC25L491)
Extracellular vesicle purification fixator (item number: AE25L134)
This product is only for scientific experimental research and should not be used in clinical diagnosis, treatment, or other fields.
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