On Jun 27, 2020, at 3:15 AM, Umar Farook <umarfaroo...@gmail.com> wrote: > > > Dear All, > > Sorry for an offtopic question, your suggestions are highly appreciated. > > We have been working on iron sulfur cluster binding protein, which is usually > expressed as a nice soluble protein expressed in BL21 cells but aggregated in > the affinity column itself and unable to recover from it. We had made n > number of truncations and fused to soluble tags such as MBP, but always ended > up in large aggregates. Anyone has experience in working with iron-sulfur > cluster binding protein before, please let us know the critical steps in > purification of such proteins, whether you have completely done the > expression, purification and crystallization in anaerobic conditions? or else > changing the expression system to eukaryotic system such as Baculo or HEK > 293T would help? > > Please share your valuable experience, thank you. > > When you say “affinity column”, are you referring to a Ni2+-affinity column? -John
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