Fe3+ is insoluble in water, so if you keep it as Fe3+, you should be able to spin it out. However, it seems that you are reducing it to Fe2+ with ascorbic acid, and chelating it with EDTA, which keeps the Fe in solution, but apparently does not inactivate it for RNA degradation, despite its being chelated. I suppose the ascorbic acid is necessary to allow the virus to be resuspended after flocculation. So maybe you need to find a different method of collecting the virus that doesn't rely on Fe3+, or find an Fe2+ chelating agent that prevents the Fe2+ from degrading the RNA. There are many chemicals that bind Fe2+ very tightly. You could also try using EDTA to chelate the Fe3+ without ascorbic acid reduction. Also, leave out the MgCl2, since it competes with Fe for binding to EDTA. Make sure the pH is not basic, because RNA is unstable at basic pH. Use a strong neutral buffer, like 100 mM HEPES pH 7.0.

Did you have tried already to use magnetic racks, as those we used to extract DNA from samples? Maybe it helps you?

I don't know, I thought about it, because in your explanation I saw that you only tried chemical separation, maybe if you use chemical and physical separation it will helps you.

The best way to get rid of iron is to raise the pH and make the solution slightly basic. This precipitates iron out as Fe(OH)3 which can be filtered off. If you cannot make the solution basic, you can try cation-exchange columns instead.

Why not use dialysis? Resuspend in the buffer you are currently using, spin briefly to get rid of insoluble Fe3+, add EDDHA to chelate/solubilize any remaining Fe3+ and dialyze against an appropriate buffer.

Hi all!

thank you very much for your kind answers!

Fe(III) from FeCl3 forms Fe(OH)3 which makes precipitate the viruses in flocculates. So the process of the buffer I add is the following: Fe(III) is reduced by ascorbic acid to Fe(II), so the viruses can be realeased from the flocculate. Now the solubilized Fe(II) is chelated by EDTA.

I don't know about Fe(III), but Fe(II) for sure cleavages the RNA..., so it means that the disodium-EDTA added to the buffer does not capture all the iron..., or maybe it's just not enough quantity. Maybe it's better theEDTA(-4) free?

Now, I don't know really which are the quantities of Fe(III) and Fe(II) in the solution after the resuspension.

How can I get rid of the Fe?

Ferrozine apparently binds Fe2+ quite tightly.

I read that enterobactin is the most powerful chelant for Fe3+.

I'm not sure about the options here. Maybe it's not about adding a ligand for iron, and it's about simply osmosis strategy by dialysis..., or cationic exchage...

just an idea: by electrophoresis? the method should consist on add a protein dissolved in a polar solvent(s), then apply and electrical field to collect the iron onto a metallic Surface. Inconvenient; polar solvents and electricity could kill viruses.

Guillermo Domínguez Huerta , Hello, I have a very similar situation with the same protocol. Did you ever solve this situation or move on to a different method entirely?