Product Description

The Ad5 Purification Kit is designed for rapid and efficient purification and concentration of recombinant adenoviruses. The Ad5 Purification Kit is not only faster than CsCl methods, it is also easier, safer, and just as effective. It can purify viral particles from 15–18 × 10 cm culture dishes (1.0E+12 VP). Processing time: 60–90 min.

The purification protocol

Harvesting Adenovirus

1. When ~50% of cells exhibit cytopathic effect (CPE) (rounded, clustered, floating in medium; adherent cells intact), transfer cells and supernatant to a 50 mL centrifuge tube.
Note: Adenovirus Rapid Titer Kit can monitor CPE progression.

2. Centrifuge at 4°C, 3,000 × g for 5 min. Discard supernatant; retain cell pellet.

3. Resuspend pellet in 1 mL Storing Buffer per 10 × 10 cm dish.

4. Perform three freeze-thaw cycles (liquid nitrogen/-80°C freezer → 37°C water bath) or use a cell disruptor.

5. After final thaw, centrifuge lysate (4°C, 10,000 × g, 5 min) to remove debris. Collect supernatant.

6. Filter supernatant through a 0.45 μm membrane. Filtered virus may be purified or stored at -80°C.
Note: Optional addition of Benzonase Nuclease (37°C, 1 h) reduces viscosity and improves column loading.

II. Column Equilibration

            7. Place Ad5 Purification Column in a 50 mL tube. Centrifuge (4°C, 50 × g, 1 min) to remove air bubbles.
            8. Remove bottom cap; allow storage solution to flow by gravity or centrifuge (4°C, 50 × g, 1 min).
            9. Equilibrate column with 15 mL Buffer A twice (gravity flow or centrifugation: 4°C, 50 × g, 1 min) until no liquid remains.

III. Loading Virus Supernatant
            10. Dilute virus solution with Buffer A to 1 mL final volume per 1 × 10 cm dish.

            11. Load diluted virus onto the column. Allow flow-through by gravity or centrifugation (4°C, 20 × g, 1 min). Reload flow-through once to maximize binding.

IV. Washing & Elution
            12. Wash column with 15 mL Buffer B (centrifuge: 4°C, 20 × g, 1 min) until no liquid remains.
            13. Elute virus with 15 mL Buffer C (gravity flow or centrifuge: 4°C, 20 × g, 1 min). Collect eluate.
Note: Repeat elution once. Pool eluates after confirming titer/purity.

V. Desalting & Buffer Exchange
            14. Exchange eluate into Storing Buffer using the 100 kDa Ultrafiltration Concentrator:
            - Transfer 15 mL eluate to concentrator. Centrifuge at 4°C, 5,000 rpm until volume ≈1 mL. Discard filtrate.
            - Add 9 mL Storing Buffer; repeat centrifugation to ≈1 mL.
            - Pipette-mix on membrane to resuspend virus. Collect purified virus.
            Note: Aliquot and store at -80°C; avoid freeze-thaw cycles.

Ad5 Purification Workflow Diagram

1. Harvest & Clarify: Centrifuge to pellet adenovirus-infected cells.

Filter cell lysate through 0.45 μm membrane; store at -80°C or purify.

2. Column Equilibration: Equilibrate column with 15 mL Buffer A twice , Centrifuge (4°C, 50 × g, 1 min)

3. Purify:

 Dilute virus with Buffer A → Load onto equilibrated column → Wash with Buffer B → Elute with Buffer C.

4. Concentrate & Store:

Desalt using ultrafiltration concentrator → Collect virus → Store at -80°C.