Hello, I am new to Crystallization and I am looking for a trick to identify protein crystals or salts in my samples. Does anyone know a dye that I can use for this kind of screening? This has to be easily shipped to UK.
My lab used to have Izit dye (methylene blue; see Hapmton Research), but I need another option if it's available.
Thanks!
Dear Irene Feliciotti many thanks for posting this very interesting technical question. Izit Crystal Dye from Hampton Research appears to be very popular. For some possible alternatives, please have a look at the homepages of the following companies:
https://www.jenabioscience.com/crystallography-cryo-em/screening/oils-dyes/crystal-dyes
and
https://www.mitegen.com/product/jbs-rainbow-crystal-dye-copy/
Also please see the following potentially useful reference in which the use of Texas Red dyes is described:
https://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.591.5412&rep=rep1&type=pdf
(see attached pdf file)
A rather surprising method for distinguishing between salt crystals and protein crystals without the use of any dyes has been reported in the following very interesting article:
Trial by fire: are the crystals macromolecules?
Article Trial by fire: Are the crystals macromolecules?
This paper has been posted by the authors a public full text on RG. Thus you can freely download it as pdf file.
Good luck with your work and best wishes, Frank Edelmann
Dear Irene Feliciotti , in addition to the previous post by Frank T. Edelmann , you could also consider the use of your protein autofluorescence for distinguishing from salt crystals. Please, have a look to the following research paper dealing with this technique:
Article Latest methods of fluorescence-based protein crystal identification
Hope it helps. Good luck with your research.
The thing about dye absorption to proteins is that a green dye doesn't necessarily absorb as easily as a violet dye as a blue dye. Absorption of dyes into proteins is based on specific interactions of the proteins with the dye. Dyes are like small molecules. Some small molecules bind to a protein with high affinity some dyes straight-up don't bind to the protein.
The Izit dye is a common dye for protein crystallization. That's just a fancy sales word for methylene blue. Apparently salts don't soak up the dye because their crystals do not possess solvent channels like protein crystals do.
As the size of the dye increases, changing color is irrrevelevent. Crystal violet is a much larger dye than methylene blue so it is not easily absorbed by protein crystals. However, some proteins do absorb the dye meaning that there are specific interactions between the protein and the crystal violet molecules.
A few methods to confirm protein crystals are ...
- diffract the crystals. Do they spots at low resolution? Then they protein.
- Pass the coverslip containing the crystal through a flame. Does it get destroyed? If yes then that was a protein crystal. If no then it is a strong salt crystal.
- Try to crush the crystal under a microscope with very small tools. If the crystal is breakable then you just destroyed your precious protein crystal. If not then it was a salt crystal.
- Change the temperature of the crystallization plates. If at some probably higher temperature the crystals un-crystallize (redissolve) or transform into yucky precipitate then those were protein crystals. It is also possible to change the crystal form at certain temperatures. Some protein crystals tolerate this. Some do not tolerate this.
- Soak a dye like methylene blue into the protein crystal. Use a concentration of dye less than 200 uM or you won't be able to see anything but the dye in the droplet.
These are all destructive methods.
A nondestructive method is to attach a polarizer to your microscope. Twist it around. If you see a shower of colors then you are probably looking at salt crystals. However, not all salts behave this way. This is called birefringence. Proteins are either weakly birefringent or not.
If you are crystalllizing your protein with a phosphate salt then your crystals are 90% probably salt. If you purified your protein in Sodium Phosphate buffer and crystallized your protein in that buffer then it is salt, too. Phosphate is bad news especially if you got got magnesium present.
McPherson, A. (2019). Penetration of dyes into protein crystals. Acta Crystallographica Section F: Structural Biology Communications, 75(2), 132-140.
- Fix the crystal with a crystal ring, expose it to the air for a long time, the protein crystal is destroyed by dehydration while the salt crystal does not change.
- Remove the crystal and place it on the crystal droplet, and the protein crystals can float on the surface for a short time while the salt crystal sinks rapidly.
- Under a low-power optical microscope, the crystal is touched with the tip of a needle in the crystal solution. The protein crystal is soft and easy to be broken into powder while the salt crystal is hard and easy to be broken into pieces.
- Touching the crystal with wet filter paper, the protein crystal dissolves quickly, and the salt crystal takes a long time to dissolve.
- Remove the crystal and place it on the coverslip. The flame burns the bottom of the coverslip. Under the microscope to observe the crystal, the protein crystal will change from translucent to pure black powder, and also have the unique burnt smell of the hair.
- Remove the crystals, dip them in the buffer, and determine whether there are target dye bands after electrophoresis. (Recommended for priority).
- Utilize X-ray single-crystal diffraction method to obtain the diffraction pattern, analyzes the structure, and further determine whether it is a protein crystal.
- Badges
- Science topic
- Similar topics
- Chemistry
- Sulfites
- Inorganic Chemistry
- Inorganic Chemicals
- Salts