The Exosome Purification Column Kit is suitable for the isolation and purification of exosome vesicles from clarified, non-complex biological samples (such as cell culture supernatant, clarified milk solution, etc.). The purification column utilizes size exclusion chromatography (SEC) technology to separate particles based on their size as they pass through a column filled with porous polysaccharide resin. When the sample passes through the purification column via gravity flow, larger particles elute first, while smaller particles enter the pores of the resin during their descent, causing delayed elution. The column volume is composed of solid resin (stationary phase) and liquid buffer (mobile phase). Particles do not chemically bind to the resin, ensuring separation stability and reproducibility. This kit is characterized by rapidity, high efficiency, and high purity of the obtained exosomes, making it suitable for downstream applications such as electron microscopy analysis, NTA particle size analysis, nucleic acid analysis, protein analysis, cell-based experiments, and animal experiments.

Special Note: This kit is solely a purification kit, not a concentration kit. Users must purchase an exosome extraction kit or use other methods to perform a concentration pre-treatment on the sample.

Product Information

Components

Storage

Ship at room temperature. Store the kit at room temperature for up to 12 months.

Instructions

1. Sample Preparation

Load volume per run: 0.5 mL. Equilibrate the sample to room temperature before use.

1) Concentration: It is recommended to use the Cell Culture Supernatant Exosome Rapid Extraction Kit or the Milk/Emulsion Exosome Rapid Extraction Kit to concentrate the sample more than 100-fold. Alternatively, use ultracentrifugation, tangential flow filtration, or 100 kD ultrafiltration to concentrate the sample more than 50-fold.

[Note]: Use ultracentrifugation with caution, as it may cause protein aggregation affecting resolution.

2) Recommended BCA Concentration: Crude exosome BCA concentration from cell culture supernatant containing 10% exosome-depleted serum should be greater than 3 μg/μL; crude exosome BCA concentration from serum-free cell culture supernatant should be greater than 1 μg/μL; crude exosome BCA concentration from milk/emulsion should be greater than 3 μg/μL.

[Note]: The primary function of the purification column is to remove small particles such as proteins and free nucleic acids. The purification process will dilute the sample to some extent; therefore, ensure the sample contains a sufficient number of vesicles. For electron microscopy, the particle concentration of the original sample is recommended to be greater than 8.0E+10 particles/mL.

2. Sample Pretreatment

1) Concentrate the sample using the recommended method.

2) Filtration: Use a 0.22 μm filter to remove large particles from the crude exosome preparation.

[Note]: The sample purified by the Exosome Purification Column (EP Column) does not need to be filtered again with a 0.22 μm filter.

3) Prepare PBS: Use freshly filtered PBS (0.22 μm) to avoid contamination and introduction of large particles. Ensure the PBS temperature is the same as the column temperature (18 - 25°C).

[Note]: Use freshly prepared 0.22 μm filtered PBS or commercially available filtered PBS to avoid microbial and particulate contamination. If using PBS stored at 4°C, it must be warmed to room temperature before use; otherwise, excessive air bubbles may form in the column, affecting separation.

3. Column Equilibration and Washing

1) Ensure the purification column is within the operating temperature range of 18 - 25°C before the experiment. Do not remove the cap until this temperature is reached.

2) Remove the black rubber stopper from the top port to balance air pressure, then remove the top cap.

3) Install the purification column vertically onto a laboratory stand. Place a waste collection tube (centrifuge tube or beaker) beneath the column for use.

4) Discard the storage solution above the frit (remove the bottom cap and let the storage solution drain naturally, or aspirate it).

5) After the storage solution has drained, wash the upper chamber of the column with PBS 2-3 times. Then, add 20 mL of PBS to wash the resin bed (can be added in batches or connected to the adapter; flow rate approx. 1.5 - 2.5 min/mL). The liquid will stop flowing once all PBS has entered the column.

[Note]: Adapter usage: Connect the green adapter to a 20 mL syringe barrel..

6) After the PBS has drained, load the sample immediately, or add 2 mL of PBS, cap the bottom port, and store for later use.

[Note]: If using the adapter, once the PBS in the syringe has flowed through, approx. 2-3 mL buffer remains in the column. Remove the adapter and wait for the remaining PBS to drain before loading the sample or adding 2 mL PBS to store.

4. Exosome Collection

1) Preparation: Concentrate and filter the sample using the recommended method. Prepare labeled 1.5 mL microcentrifuge tubes.

2) Load 0.5 mL Sample: Immediately after the PBS stops flowing, load the equilibrated 0.5 mL sample onto the frit of the purification column (if the sample volume is less than 0.5 mL, adjust to volume with PBS).

[Note]: Avoid stopping the column flow for long periods to ensure optimal vesicle separation.

3) Flow-through 2.87 mL Void Volume: After all the sample has entered the frit and no liquid is flowing from the bottom, add 2.37 mL of PBS and wait for it to flow through until the flow stops. During this process, a total of 2.87 mL of eluent (0.5 mL sample volume + 2.37 mL buffer) will have flowed through.

[Note]: To accurately determine the void volume, add a fixed volume of solution and wait for it to flow through completely.

4) Collect Exosome Fractions: Immediately prepare new microcentrifuge tubes to collect the purified fractions. Add 0.5 mL of PBS to the top of the frit in batches and collect 2-3 fractions (0.5 mL each). The highest purity is found in the first 2 fractions (1 mL total), but it is recommended to merge the first 3 fractions (1.5 mL total). Wait for each volume to flow through until the flow stops.

5) Filter Exosomes: Transfer the collected exosome fractions to the upper chamber of an Exosome Purification Column (EP Column). Centrifuge at 4°C, 3,000×g for 10 min. The liquid collected at the bottom of the EP column tube is the filtered exosome (Note: The EP column is for single use only).

6) Measure the particle concentration and protein concentration of the collected product. If necessary, use a 100 kD ultrafiltration unit to further concentrate the collected product (optional).

5. Column Regeneration and Storage

1) Column Regeneration: After collecting the desired volume, wash the column with 10 mL of Regeneration Solution, followed by 20 mL of PBS. The column is then ready for a second use.

2) Column Storage: If storing for future use, wash the column with 10 mL of Regeneration Solution, followed sequentially by 20 mL of PBS, 20 mL of deionized water, and 20 mL of 20% ethanol. After washing, cap the bottom port, pour in the storage solution (20% ethanol), and seal with the top cap.

[Note]:

The regeneration solution is alkaline. Do not add deionized water or 20% ethanol directly after washing with regeneration solution to prevent salt precipitation within the resin bed. The deionized water wash removes residual salts, preventing crystallization when ethanol is added.

Estimate cleaning times at approx. 1.5 - 2.5 min per mL of liquid flow-through to prevent the resin bed from drying out for extended periods.

Notes

1. When exosome fractions are intended for downstream high-throughput sequencing or other omics analyses, it is recommended to use a new column to avoid cross-contamination.

2. Salts are inevitably generated during regeneration. Salt precipitation affects resin performance; it is recommended to reuse each column no more than 5 times.

3. Handle the column with care to avoid dropping or impacting it, which could fracture the resin bed.

4. The regeneration solution is alkaline and corrosive. Handle with extreme care!

5. For your safety and health, please wear a lab coat and disposable gloves during operation.

6. This product is for scientific research use only!

Documents:

Safety Data Sheet

41218_MSDS_HB260525

Manuals:

41218_Manual_HB20260518