I am looking for a kit to purify AAV2 instead of using ultracentrifuge. I have looked at the protocols of cellbiolabs, Millipore and virapur companies. Does anyone use these kits and how was the titer? I would like to inject 10E13 GU in rat brain.
For purification, we generally run a iodixanol gradient, followed by a column purification and concentration using a concentrator. These methods give us good yield of vectors which have used for in vivo administration. However, the titers you are looking for is stiff. We have realised that some serotypes give good yields (e.g. AAV8~1-4x10e13vg/ml) while others do not (e.g. AAV2~2-8x10e12vg/ml). You might want to start with lot of culture to get this dose and most importantly concentrate the vectors to a volume which can be administered. Dose fractionation might help, but that would depend on your experimental set up.
Thank you Nishanth and Henning. both answers are very helpful. I have tried Iodixanol gradient method which works very good but I have to optimize it a bit better. Nishanth I have never done any dose fractionation. If you have a protocol for that could you please send me. That would be very helpful. Many thanks to both of you.
There is no protocol Goutham.
All you have to do is check the titres of the vectors you obtain after purification and know the volume which can be injected to the part of rat brain. You will have to inject multiple times at set interval apart to reach the final dose of 10^13. But then I do not know if that can be done in brain.
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