Structural analysis of a purified protein

Protein Chemistry Protein Structure / Modeling Protein
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Hi, Is there a rule for how pure a protein must be (how many purification steps) in order to go ahead and do SAXS and obtain its crystal for x-ray Christalography ?

I’m asking this because I have a protein which is coming out quite pure only by doing 1 step affinity chromatography purification. This protein is fused to his tag which I was told does not interfere with structural analysis that much once it has a quite small molecular weight…

Besides my protein band I only see one or two nearly invisible bands in the purified lane in the sds-page gel from purification…

Also, I run a gel-filtration analysis and besides my protein peak in A280 absorbance I see 2 smaller peaks immediately before my protein peak, forming something like an aggregate…I don’t know if this is dimmer or just an aggregate… Any tips?

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A general rule is lesser the number of Steps, higher the yield is. But in your case, since you’ll be proceeding for X-ray Crystallography, You require high purity and those two bands adjacent to your protein are extra. Im presuming that you are staining with CBB and that is going to be an added caveat becuase its sensitivity is low. You can use a Silver staining method and then check number of Bands and eventually Optimize your Chromatography to get purified yield or adding another layer of Purification Step.

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