Hello at all,
I am trying to crystallize a protein by using standard sitting drop vapor diffusion setup and crystallization screens.
After few days, spherulites or big needles aggregates appear in many conditions. By checking carefully with uv-vis microscope in fluorescence setup, I found the typical sign of protein: the objects become white on dark background.
However, diffraction experiments, sds-page on crystals, and crushing test don't show me or are not in agreement with the presence of protein. I get diffraction (powder) but it clearly comes from small molecule, sds-page doesn't show protein band, and crystal are hard to crush.
According to your experience, how can his opposite results be exploited ?
Is Uv-vis signal due of protein absorbed on salt crystal?
Thanks for your suggestions.
What's the crystallizing condition? Did you wash your crystals completely before Uv-vis test?
Conditions contain
Precipitants: pegs,
Buffertris: tris or hepes or cacodylate
Additives: calcium chloride or acetate
All chemicals that don't fluoresce in the used conditions.
No, crystals haven't been washed... This is a good suggestion.
I don't know if protein molecules would absorb on the surface of salt crystals. I do know that having Ca2+ in your condition in combination with some counterions are prone to yielding salt crystals - I think CaAc2 can easily be formed. What about the protein solution, does it contain any salt as well ? Do "blanc" experiments (without protein) give the same type of crystals ?
Thanks Tjaard,
Calcium based salt was the first thing I though, also considering the pH >7. I didn't performed a blanck but, even if they are salt crystals, this doesn't explain the strong uv-vis signal.
They could of course still be protein crystals that just don't diffract... If they grow fast, slowing down the growth may give better order - and diffraction. And, did you try to replace Ca2+ with other divalent cations ?
Also, I have had cases where protein crystals are hard to dissolve.
Dissolve a few crystals in a very small volume of buffer and add some Bradford reagent. Blue colour indicates protein. I would use mother well solution as a control.
Yes, you can certainly get protein that precipitates on top of salt crystals. The protein fluoresces and you get a false positive. I attach examples of verified salt crystals (at the synchrotron) with protein on top of them. These are calcium crystals. Notice how the UV signal looks funny: it is only on the edges of these salt crystals. However the protein can also cover the entire crystal and be very misleading. The pictures are generously provided by my office mate.
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