# Lysozyme crystals versus NaCl

If someone has some experience with lysozyme crystals I would be interested in knowing whether any of the images below seem likely to be lysozyme. Solutions were made with powdered lysozyme ($$\pu{50 mg/mL}$$) and $$\ce{NaCl}$$ ($$\pu{3 M}$$), except the lower left which is $$\ce{NaCl}$$ alone.

The top two images were made from a lysozyme solution with a small amount of $$\ce{NaCl}$$, then adding a piece of tissue paper. The square/cuboid crystals came out of solution immediately onto the fibers. That they are cubes suggest to me $$\ce{NaCl}$$ but the appearance is odd.

The middle two images have a higher ratio of $$\ce{NaCl}$$ to lysozyme--maybe close to 2:1 or 1:1. The crystals look OK to me but as a complete novice I am not sure. The crystals are all on the order of $$\pu{0.5 mm}$$.

A better image of the square crystals from lysozyme/$$\ce{NaCl}$$ + tissue paper.

If someone has some experience with lysozyme crystals I would be interested in knowing whether any of the images below seem likely to be lysozyme.

The bottom two have the typical shape of well-grown lysozyme crystals. Source: Jena Biosciences (https://www.jenabioscience.com/crystallography-cryo-em/get-started/crystallization-of-model-proteins/co-401-lysozyme-solution)

You can also add a dye. If it diffuses into the crystal, it can't be NaCl.

There is also a destructive method. If you try to squeeze the crystals with a hard object (like a needle), NaCl crystals will stay intact or break with smooth faces while the lysozyme crystals will look like smashing a watermelon with a baseball bat.

Here are some more ideas: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2864706/

And here a paper about dye penetration with some great photos of lysozyme in polarized light: https://www.semanticscholar.org/paper/Penetration-of-dyes-into-protein-crystals.-McPherson/5d79f2f257f85163d5c14ad0967de1d4354bd1c8

• So protein crystals don´t cleave well if tapped gently with a sharp blade of a safety razor? Based on own experience, it may take some training of the eye (comparison with other «broken» crystals in the vial), manual dexterity (angle along one of the cleavage planes) and check (Nicols for a hopefully still sharp extinction, later recording an orientation matrix or/and an early and quick $\phi$ scan on the diffractometer to interpret non-sharp diffraction peaks as an indicator for physical damage). For me, this worked well enough for some organic crystals, once too much in needle shape. Sep 22 at 4:56
• But it may be well a question of scale, too, with protein crystals often smaller than $\approx \pu{0.2 mm^3}$. Sep 22 at 5:02
• I was more or less shocked at how much the (Jena) crystals resembled mine. This is a very helpful answer. I left a comment yesterday but somehow today it's gone. Sep 23 at 0:50
• @daniel That’s great, you grew protein crystals! I did read your comment yesterday. Sep 23 at 3:11

The one in the top right and those large in the very last photo pretty much look like $$\ce{NaCl}$$. Those in the second row (left) and third row (both left and right) look different by the crystal habit's symmetry; and what could be birefringence (especially third row, right hand side) -- a plausible first evidence for protein crystals which typically crystallize in a lower than cubic space group symmetry.

However:

The habit of a crystal (outer shape) may vary on the crystal's condition of growth. Only the angles enclosed by the crystal's surfaces remain constant for the same polymorph of a compound (Steno's law). The presence of impurities the purification did not remove, intentionally added detergents, dyes and buffers, the applied technique (e.g., sitting drop vs. hanging drop), the age of the crystals while growing may influence which faces may be seen or not (more).

Remedy: A quick and easy check is to observe the sample put between two linear polarizers/Nicols. Check if there is a polarization microscope nearby and put the dish on its table (it very well may contain the solution including the crystals). Since there is there is no birefigence for cubic $$\ce{NaCl}$$, you may sort these out visually. If Nicols are crossed, crystals of $$\ce{NaCl}$$ appear uniformly black.

It may happen that accidentally such a salt crystal still ends on the X-ray diffractometer instead of your wanted protein crystal. But these will be recognized quickly by symmetry, $$d$$-spacing and brilliance (compared to a protein sample) of the diffraction pattern.

• The polarizer trick for NaCl looks very useful and I will be trying that. Very helpful answer, thanks. Sep 21 at 22:23