Does Tween-20 have any effect on 6xHIS tag

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louhazosc
louhazosc's picture
Does Tween-20 have any effect on 6xHIS tag

This is a linked question to a huge post I have on here.
I have an insoluble protein with a 6xHIS tag
My first lysis buffer was simply a 20mM Tris, 250mM NaCl pH 8.0. Then we switched to 50mM HEPES, 250mM NaCl, 1mM Betamercaptoethanol, 1% Tween-20.
Since the switch I have affinity for my protein gone from bumping off with 250mM Imidazole with the Tris/salt lysis buffer to 20mM with the HEPES buffer and now I am getting everything coming out in my flowthrough.
If you read my other post you can see that I hit this many ways including re-charging the resin so my only question is....
Does Tween-20 at 1% have an effect on the integrity of a 6xHIS tag?
I honestly can not see it being 1mM BetaMercaptoethanol but at this point what the hell do I know.

Chin Fen Teo
Chin Fen Teo's picture
 Hi louhazosc,

 Hi louhazosc,
I have not used Tween 20 for protein purification before... However, in western blotting, 1% Tween20 will block antibody binding to the membrane whereas 0.1% is the maximal concentration that allows antibody binding...
Given that protein-protein interaction is the basic requirement for both cases, and that your proteins ended up in the flow through with the addition of 1% Tween20, it may happen that the detergent concentration is too high and prevent the binding of your protein to the resin...  
Can you use 10% glycerol and 0.1%NP-40 to soluble your protein? At least I know this combination is compatible with Ni-NTA resin...
Really hope you can get around your problem very soon!