I have purified adenoviruses on a caesium chloride gradient and then subjected selected bluish white band to dialysis using a 10mM Tris pH8 2mM MgCl2 5 % sucrose buffer ( 200x viral sample volume) with 3 changes.

However i notice that there is a precipitated fraction....
If this is my virus, what is the best way to resuspend it, and get over this precipitation problem

thanks

Does your supernatant contain enough of Adenovirus? If so, you might not worry about the precipitate.

Hi.

I am trying to do a virus purification. But I don't have MgCl2 aviable to do the dialysis Buffer . Can I change it for another reagment?

Hi.

I am trying to do a virus purification. But I don't have MgCl2 aviable to do the dialysis Buffer . Can I change it for another reagment?

Hi veritzaihc -

I'm not sure if there is a substitute for MgCl2 but you may find this reference helpful...

S Zolotukhin, B J Byrne, E Mason, I Zolotukhin, M Potter, K Chesnut, C Summerford, R J Samulski, and N Muzyczka. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Nature Gene Therapy. June 1999, Volume 6, Number 6, Pages 973-985.

Abstract -
Conventional methods for rAAV purification that are based on cesium chloride ultracentrifugation have often produced vector preparations of variable quality and resulted in significant loss of particle infectivity. We report here several novel purification strategies that involve the use of non-ionic iodixanol gradients followed by ion exchange or heparin affinity chromatography by either conventional or HPLC columns. These methods result in more than 50% recovery of rAAV from a crude lysate and routinely produce vector that is more than 99% pure. More importantly, the new purification procedures consistently produce rAAV stocks with particle-to-infectivity ratios of less than 100, which is significantly better than conventional methods. The new protocol increases the overall yield of infectious rAAV by at least 10-fold and allows for the complete purification of rAAV in 1 working day. Several of these methods should also be useful for large-scale production.

bluechampagne said:

I have purified adenoviruses on a caesium chloride gradient and then subjected selected bluish white band to dialysis using a 10mM Tris pH8 2mM MgCl2 5 % sucrose buffer ( 200x viral sample volume) with 3 changes. However i notice that there is a precipitated fraction.... If this is my virus, what is the best way to resuspend it, and get over this precipitation problem thanks

As Tracy suggested the precipitate may not be a problem if you can just pellet and dispose of it without loosing your infectious virus.

CsCl has been shown by some labs to lead to a reduction in infectious titre of AAV, but I don't think there is a problem with adenovirus preps. In fact I don't think there really is a problem with AAV either.

There are a lot of Adenovirus purification kits available now that are based on affinity chromatography and although they usually do not allow the same high yields of virus that you can get with CsCl gradients, they are a lot faster and do not require any dialysis (which is always going to lead to a loss of virus).

I personally tend to still favour the CsCl prep over affinity preps because they allow you to select for virus particles that contain DNA, since these are more dense. Affinity methods will give you a lot more empty Ad capsids. (Incidentally, the iodixanol based method that Tony suggested is very good for selecting the DNA-containing particles from an AAV prep in the same way if you use it to create a true gradient rather than the discontinuous "cushion" described in the paper.)

When I have removed my Ad virus from the second round of density centrifugation I just desalt it over a PD-10 column. You collect the fractions coming off of the column and OD spec them (260nm). Voila. No nasty loss of virus during dialysis.

Hope this helps