In cases like this, I keep FAD around during all steps starting from cell lysis, purification, dialysis and even crystallization. In one case we added FAD to protein such that the protein: FAD ratio was 1 prior to setting up crystallization trials.
Hope that helps.
Harkewal
Sent from my iPad
> On Mar 13, 2014, at 5:40 PM, "Benini Stefano (P)" <[log in to unmask]> wrote:
>
> Dear All (those dealing with wetlab stuff..),
>
> While purifying a FAD containing protein we lose part of the FAD (on the gel filtration we clearly see two bands corresponding to holoprotein and free FAD).
>
> We obtain crystals but diffracting to only about 4 A despite their beautiful look. Our hypothesis is that the crystals contain a population of molecules with and without FAD (?).
>
> The questions are:
>
> 1) how to keep FAD bound to the protein during purification and crystallization?
>
> 2) how to completely remove FAD from the protein?
>
> Thank you very much for any help provided!
>
> Best regards
>
> Stefano (part-time wetlab person)
>
>
> Dr Stefano Benini, Ph.D.
> Assistant Professor
>
> First International workshop: "Molecular Basis of Fire Blight", Bolzano 15.10.2014
>
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>
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