Consider a crystallization condition that readily gives salt crystals on addition of protein. Is there a logical way to increase the probability of protein crystallization against the undesired salt crystallization by making slight changes around the same condition? Or is it totally worthless to pursue any further optimizations of the condition that produces salt crystals as soon as few minutes?
It is rare but not unheard of to have salt and protein crystals appearing in the same condition. Just keep optimizing your protein crystals and ignore the salt if you can. If you can't distinguish both species morphologically, you need to pick all of them and shoot for protein with X-rays. Or use a "UV microscope" if your protein has aromatic residues.
Try flipping the ions if you want to try suppress the salt crystals or optimize the crystals in general. lithium for ammonium, magnesium for manganese, citrate for acetate, potassium for sodium, get creative. Crystal screening kits are not complete screens and never will be.
One of my proteins was really good in stimulating salt crystallization but gave no protein crystals. When I changed the construct I got ~10% protein crystals over all screens and no salt crystals.
So yes, if that is really the only condition your protein crystallizes in, stay happy, you got crystals, try to make them better. If it does not work out for you, keep working on your sample quality. A good protein sample should crystallize in several conditions.
Out of curiosity, was it a Fluoride, Iodine or Bromide salt?
Hey Stefan,
Thank you very much for your valuable advice.
1. To begin with, I did look at the crystal under the UV. But, it left me with more questions than answers. First of all, I am not sure if the crystal fluorescence is due to it being a protein crystal or due to inherent reflective property of the Corning plate. Also, the crystal that fluoresces under UV disappears soon due to radiation damage. Next time, I guess I would try Izit dye to confirm what I see is protein.
2. Flipping ions was a smart idea. Thanks. I will try that.
So, when you changed the construct, you tried protein crystallization on the optimization screen directly?
Hi Sagar, Can you provide more information about the conditions you wish to optimise? There might be something in your protein sample (ie counter ion / small molecule / buffer) that is promoting salt crystal formation that you can remove or replace. (ie. Phosphate buffers are notorious for producing salt crystals in broad screens. )
Kind Regards,
Mike
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins