Hi All,
I was told that it is best practice to attempt growing protein crystals only after gel filtration column, but my protein is already quite pure after ion exchange column. I wonder have anyone try growing crystals after ion exchange + dialysis but not using gel filtration column?
Thank in advance for your advices!
Best,
Lyra
Proteins are highly idiosyncratic when it comes to crystallization, and there is no hard-and-fast-rule that holds in general for crystallizing any protein. So my advise is to try crystallizing your protein without the gel filtration step -- you might get lucky and get crystals right away. If not, at least you would've learned something about your protein.
I agree with Vicente, just give it a try if the protein is already pure. I have crystallized several proteins right after ion exchange. If you have access to a dynamic light scattering (DLS) instrument this can tell you if your sample is not only pure but also monodisperse (absence of aggregates) - we use this routinely as an extra quality assay.
Thank you so much Vicente & Tjaard! I will try it out then!
To Tjaard: Did you dialysis before you grow crystal from the ion exchange column? Is it hard to repeat the growing of crystal without the gel filtration step?
Hi, it depends on which salt concentration your protein eluted in the ion exchange step. If it is higher than 150 mM I would try to lower it to this value by dialysis or ultrafiltration/washing.
Reproducibility of growing crystals should not depend on which purification step was the last. It depends on the quality and stability of your sample.
Best, Tjaard
Thank you, Tjaard! I will try it out for sure!!
If we can grow crystal without gel filtration step, we can save lots of efforts & time!
Best,
Lyra
Quick update!
Yes! I can get protein crystals with Ni column --> cleave His-Tag --> another Ni column clean up (collect flow through).
But my protein is small (~20 kDa), single domain and expressed very well.
I guess that's why it works. :-)
Best,
Lyra
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