Affinity purification with organic solvents at pH 7.5

[groupoliver]

Hi Guys,

I am working with membrane proteins which have high tendency to aggregate. My protein is soluble in 70% acetonitrile which is an organic solvent. I would like to know if Ni2+ column works in binding His-tagged proteins in these solvents.

regards
Oli

[danfive]

I searched and found a compatibility chart from qiagen, but no mention of acetonitrile.

Here's the chart [Only registered users see links. ]

Note that Nickel is a catalyst in nitrile hydrogenation.

[groupoliver]

Hi Dan,

Thanks for replying.....Ya I went through many company catalogs whoever supply the resin, but without success on information about the organic solvents. I assume that it is not compatible as acetonitrile has pH of ~1-2. But then my idea was to mix acetonitrile with 50% tris buffer so that pH can be adjusted to around 7.5-8. Just working conditions with the buffers what would happen by mixing them with and without protein.

As you mentioned, if Nickel is a catalyst in hydrogenation of of nitrile molecule, what do you think can happen ????

I understood that it doesn't change the Nickel ion bound to resin in any form (oxidation/reduction) by acting as a catalyst. And if acetonitrile is hydrogenated, I don't think, protein can be damaged. If you have more information, please let me know.

Regards
Oli

[danfive]

I think the big issue (my organic chem is fuzzy) is that nickel will catalyze hydrogenation of acetonitrile, (if under acidic conditions this will release CN, lethal cyanide). Another is that nickel is also used to produce primary amines, the literature I found is about mostly chemical compounds, so I'm guessing that the amine groups in your proteins are also vulnerable to being reduced into secondary and primary amines.

I'm not sure what the condtions are for producing these amines, may not happen due to high energy requirement, or may occur at benchtop conditions.