Q: Your ViraBind™ Adenovirus Purification Kit is listed as a 10 prep kit. Why does the kit only contain 5 filters?
A: The syringe-type filters found in this kit are designed to be used twice. Please see the product manual for the protocol to regenerate the filter for the second use, which will ensure there is no cross-contamination. Filters should not be used more than twice.
Q: How can I get the highest yield with this kit?
A: Here are a few tips to get the best yield:
1. Lysate volume: each filter can only handle up to 15 mL of cell lysate.
2. Lysate pH: after 2-3 days of infection, the medium may become yellow and too acidic for optimal viral binding. Either resuspend the cell pellet in fresh medium before freeze/thaw cycles, or if you want to keep the same medium, add 50 mM Tris pH7.5 after freeze/thaw to shift the pH back to neutral before applying to the filter.
3. Loading: please allow the lysate to pass through the filter by gravity (drop by drop) and reload once.
4. (Optional) Nuclease treatment: before loading lysate onto filter, you can treat lystae with nuclease such as RNase A, DNAse I or Benzonase for 30 min at 37ºC. This can help increase viral binding to the filter.
Q: How much benzonase should I add?
A: Typically 10-20 units of benzonase is added to each 1 mL of crude adenovirus lysate.
Q: What is the maximum volume of supernatant I can add to each filter?
A: The filter can handle up to 15 mL of viral supernatant. The virus is captured based on charge and size, rather than affinity binding, and the capture is affected by excess serum proteins when more than 15 mL of supernatant is applied. A concentration step can be performed prior to starting the purification kit to increase the titer. To concentrate, a concentrator unit with a 100,000 kDa molecular weight cut off membrane can be used.
Q: Will there be any cytotoxicity after using your ViraBind™ Adenovirus Purification Kit?
A: No, there is no cytotoxicity after using this kit. Purification of adenoviral lysates results in up to 90% virus recovery while removing cellular debris that can interfere with infection. Step 5 of the protocol gives the option to dialyze into a desired buffer, leaving you with purified virus in your choice of buffer.
The purified adenovirus contains just virus and buffer, and applying this to cells during an infection will not introduce any other toxic components.
Q: Can the filters provided in your ViraBind™ Adenovirus Purification Kit be re-used? How long can they be stored?
A: The syringe-type filters found in this kit are designed to be used twice. Please see the product manual for the protocol to regenerate the filter for the second use. The regenerated filter can be stored at 4ºC for one year. Be sure to pass some 1X Wash Buffer through the filter before the first loading of your viral supernatant. Each filter may only be used twice.