Determine if you have a Salt or Protein Crystal
Crystal! Sweet.
The next step is to determine whether the crystal is comprised of salt or protein. Here is a list of methods to help you determine whether you are dealing with a salt or protein crystal.
1) Prediction Tools - can be used to determine if the drop components have the possibility of forming a salt crystal.
2) Birefringence – is a sign of salt crystals although it is not visible in protein crystals that are cubic. A set of polarized lens are needed to check for to see if the crystals are birefringent.
Look for salt crystals to have strong birefringence while proteins are weaker
3) Dyes - such as methylene blue are able to enter large solvent channels of proteins that are not present in salt crystals
Look for dye to be absorbed into the protein crystals and not salt
4) Dehydrate - by removing the crystal from the drop.
Look for salt crystal to maintain its structure while protein to turn to mush
5) Crush - the crystals using a small tool. Protein crystals are softer than salt crystals due to their crystal packing. It is helpful to try this technique with a number of known samples to gain experience. Tip: Wick away excess solvent to save time
Look for protein crystals to collapse when compared to that of salt
6) Glutaraldehyde - can be added to your drops (~1 %), if protein is present the drops will turn a yellowish color. Since glutaraldehyde is quite volatile it may also be added to the reservoir, if you decide to go this route then use ~2 % concentration. Any free amines will turn yellowish so watch out for the presence of other protein or components in your buffer such as TRIS.
Look for a protein crystal to turn a yellowish color
7) SDS-PAGE - can be used by dissolving your crystal (or the entire drop)
Look for: the correct electrophoretic pattern
Control experiment - can be done (this is science) by setting up the exact same condition without your protein.
Look for motivation
9) Crystal shape - alone can be very misleading between salt and protein crystals. However, I have noticed the wings of death crystals (magnesium phosphate, in this case) appear quite distinct.
Look for wings of death
10) X-ray diffraction - although obvious, it is not possible for everyone. If you have easy access then this is the only definitive method out of all the methods mentioned.
Look for salt crystals have fewer reflections (larger lunes) compared to protein crystals