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| Product Name | Exosome Purification Column Kit (Size Exclusion Chromatography) |
| Catalog No. | RESK-0621 |
| Description | This kit is designed for the isolation and purification of exosome vesicles from clarified, non-complex biological samples such as cell culture supernatant and clarified milk. The purification column uses size exclusion technology: particles are separated by size as they pass through a column packed with porous polysaccharide resin. As the sample flows through the column under gravity, large particles elute first, while small particles enter the resin pores and elute later. The column volume is occupied by solid resin (stationary phase) and liquid buffer (mobile phase); particles do not chemically bind to the resin, ensuring separation stability and reproducibility. The kit is fast and efficient and delivers high-purity exosomes suitable for electron microscopy, NTA particle size analysis, nucleic acid analysis, protein analysis, cell-based experiments, and animal experiments. Note: This is a purification kit, not a concentration kit; samples must be pre-concentrated using an exosome extraction kit or other concentration methods. |
| Intended Use | Isolation and purification of exosome vesicles from clarified, non-complex biological samples (e.g., cell culture supernatant, clarified milk). Suitable for a universal range of species. This kit purifies but does not concentrate samples; a pre-concentration step is required. |
| Principle / Technology | Size exclusion chromatography (SEC): particles are separated by size as they pass under gravity through a column packed with porous polysaccharide resin. Large particles elute first; small particles enter the resin pores and elute later. Particles do not chemically bind to the resin, ensuring stable and reproducible separation without ultracentrifugation. |
| Detection Method | Electron microscopy (morphological observation), NTA particle size analysis (particle size and concentration). |
| Sample Type | Cell culture supernatant, clarified milk; samples must be clarified and pre-concentrated before loading. |
| Performance Range / Specifications | High-efficiency purification: optimized for exosome separation and markedly improves sample purity. Reusable: each column supports up to 5 uses with stable performance. Gentle process: no ultracentrifugation required, effectively preserving exosome activity. Single load volume: 0.5 mL. Void volume: 2.87 mL. Fraction volume: 0.5 mL per fraction; pooling the first 3 fractions (1.5 mL) is recommended, with the highest purity in the first 2 fractions (1 mL). Operating temperature: 18-25 °C. Flow rate reference: approximately 1.5-2.5 min per mL of liquid flow-through. |
| Reaction Conditions / Protocol | 1. Sample preparation - Single load volume is 0.5 mL; equilibrate the sample to room temperature before loading. - Concentrate samples at least 100-fold using an exosome extraction kit (cell culture supernatant or milk), or at least 50-fold by ultracentrifugation, tangential flow filtration, or 100 kD ultrafiltration. Use ultracentrifugation with caution, as protein aggregation may affect resolution. - Recommended BCA protein concentration of crude exosomes: >3 ug/uL for cell supernatant containing 10% exosome-depleted serum; >1 ug/uL for serum-free cell supernatant; >3 ug/uL for milk. For electron microscopy, a particle concentration of >8.0E+10 particles/mL in the original sample is recommended. 2. Sample pretreatment - Filter the crude exosomes through a 0.22 um membrane to remove large particles (samples purified with the Exosome Purification Filter column do not require further 0.22 um filtration). - Use freshly 0.22 um-filtered PBS and ensure the PBS temperature matches the column (18-25 °C); PBS stored at 4 °C must be equilibrated to room temperature to avoid bubble formation in the column. 3. Column equilibration and washing - Ensure the column is within the operating temperature range of 18-25 °C before removing the caps. - Remove the black rubber cap on the top cover to equalize pressure, then remove the top cover. - Mount the column vertically on a stand and place a waste collection tube underneath. - Discard the packing storage solution above the frit (remove the bottom cap and let it drain naturally or aspirate). - Wash the upper chamber 2-3 times with PBS, then add 20 mL of PBS to wash the column bed (in portions, or via an adapter connected to a 20 mL syringe). - Load the sample directly after the PBS drains, or add 2 mL of PBS and cap the bottom for standby. 4. Exosome collection - Immediately load 0.5 mL of room-temperature-equilibrated sample onto the column frit once PBS stops flowing (make up to 0.5 mL with PBS if necessary). - Flow through 2.87 mL of buffer (void volume): after all sample enters the frit, add 2.37 mL of PBS and let it flow until no liquid exits (total flow-through: 0.5 mL sample + 2.37 mL buffer = 2.87 mL). - Collect exosome fractions: add 0.5 mL of PBS at a time and collect 2-3 fractions at 0.5 mL per fraction; the first 2 fractions (1 mL) give the best purity, and pooling the first 3 fractions (1.5 mL) is recommended. - Transfer the collected fractions into the upper chamber of the Exosome Purification Filter and centrifuge at 3,000 x g for 10 min at 4 °C; the liquid at the bottom is the filtered exosomes (the filter is single-use). - Measure the particle concentration and protein concentration of the collected product; if needed, further concentrate with a 100 kD ultrafiltration device (optional). 5. Column regeneration and storage - Regeneration: wash with 10 mL of regeneration solution, then with 20 mL of PBS before the next loading. - Storage: wash sequentially with 10 mL of regeneration solution, 20 mL of PBS, 20 mL of deionized water, and 20 mL of 20% ethanol; then fill with storage solution (20% ethanol), cap, and seal. |
| Storage Conditions | Store at room temperature; store the column in 20% ethanol storage solution after use. |
| Shelf Life | Stable for 12 months at room temperature. |
| Package Specifications | 5 T |
| Shipping Conditions | Shipped at room temperature. |
| Regulatory / Compliance | For research use only. |
| Recommended Buffer System | Freshly 0.22 um-filtered PBS, equilibrated to the column operating temperature (18-25 °C). |
| Application Notes / Precautions | This kit is a purification kit, not a concentration kit; pre-concentrate samples using an exosome extraction kit, ultracentrifugation, tangential flow filtration, or 100 kD ultrafiltration. Each column can be reused up to 5 times; salt generated during regeneration can precipitate and impair resin performance, so do not exceed 5 uses. Avoid prolonged interruption of column flow during the procedure to ensure vesicle separation performance. The purification process dilutes the sample; ensure the sample contains sufficient vesicles before loading. The regeneration solution is alkaline; do not add deionized water or 20% ethanol directly after regeneration washing, to prevent salt precipitation in the resin bed and column damage. Wash with deionized water before introducing 20% ethanol to avoid salt crystallization. The Exosome Purification Filter (EPF column) is single-use. Use freshly prepared, 0.22 um-filtered PBS to avoid microbial and particulate contamination; estimate each washing step at 1.5-2.5 min per mL and avoid leaving the resin dry for extended periods. |
For research use only, not for clinical use.
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