Hello
I am currently working with recombinant AAV-2 vectors. Also, I have the experience of Lentiviral vectors. The problem was that in my new laboratory settings, I do not have an ultra-centrifuge which can provide me with high speed. I have a Sigma Ultracentrifuge with a maximum speed of about 63000 x g and this way I can't use the Iodixanol gradient method at its typical centrifugation rate. My question is that is there any method for purification of AAV vectors which could be done with these settings (lower centrifugation rates)?
(I want the final concentration to be about 10^7 per microliter)
Yes, and its actually quite simple. Each rotor has a pelleting efficiency k, it should be specified in the rotor manual, if not, ask the manufacturer. Once you have the k-factors of both rotors, k1/t1 = k2/t2.
If you can't get the k-factor, it's calculated by k = t/S = ln(rmax/rmin) * 253303 / (rpm/1000)2. This is also useful for calculating the time after a rotor has been de-rated.
Consider ditching the centrifuge altogether, and look at methods typically used in protein purification: Affinity chromatography, size-exclusion chromatography and spin concentrators. Thermofisher, Cytiva and others have kits for that.
Thank you, @Engelbert Buxbaum
I will try it, I think I could compensate the lower RPM with a longer duration of centrifugation.
Ultra-centrifuge methods are some of the best for AAV, but you do need to utilize the proper methods. HPLC methods have limitations too. *The best method will depend on your exact application needs plus the available equipment/training to use them.
In our lab, we do not have an ultracentrifuge. We try to work around this by centrifuging for 16 h at 18.000g at 4 degrees Celsius. For lentivirus, a titer of 10^8 IU/mL can be achieved. For Sendai virus, around 10^7 IU/mL (estimated).
I guess this is not helpful for you since you need a 1000-fold higher titer and because AAV is much smaller. It depends on the application.
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