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
christian201
October 26th, 2009 at 8:45 AM #
Hi Sean,
I really like your blog, fantastic work! Its great to know that theres someone out there fighting with exactly the same issues day by day.
Here’s a minor correction about birefringence:
Protein crystals, unless they are cubic, ARE birefringent (albeit weakly). Salt crystals are strongly birefringent under cross polarizers, again unless they are crystallizing in a cubic space group. Common cubics are sodium chloride almost all other alkali halides.
A nice explanation is also here:
http://www.hamptonresearch.com/documents/growth_101/20.pdf
Peter Nollert
October 26th, 2009 at 1:23 PM #
Just saw that christian201 corrected #2 on birefringence. Thanks, christian201! Two more points though:
1. if your crystallization experiment is prepared in birefringent plastic trays, you’ll see a lot of background birefringence. If this hampers observation, contrast can be increased by taking the crystals out and inspecting them on i.e. a glass surface (glass is non-birefringent). Alternatively you can use crystallization trays that are made of low-birefringent plastic.
2. the birefringence of crystals that you see by eye depends on the (i) orientation (of the xtal relative to the polarizers), (ii) crystal thickness and (iii) crystal material property. Most salt crystals have strong birefringence because of (iii), but keep in mind that there are other effects (i & ii) that generate the birefringence signal.
So, overall, I’d say that – for the ‘untrained eye – crystal birefringence is a somewhat weak differentiator between salt & protein crystal.
Having said that, observing the pattern of birefringence does give you good info. I’ve seen many detergent & PEG objects/crystals that display a characteristic birefringent cross pattern that rotates as you rotate the polarizers (or crystal).
BTW – at Emerald we’re developing a birefringence microscope (called DETECT-X) that de-convolutes orientation and intrinsic birefringence. The images have high bulk contrast because colorless protein crystals are displayed colored in DETECT-X images. Check out “DETECT-X Crystal Imaging Technology” in http://atcg3d.org/Pages/technologies.aspx for a few example images.
Sean
October 26th, 2009 at 2:16 PM #
Hello christian201,
Welcome to the 19th space group! Thanks for the support and comment. I updated the post to reflect your correction, thanks for help.
@Peter
Wow. Thanks again for insights – always amazed.
Jeff Christensen
October 26th, 2009 at 4:09 PM #
Hi Sean, great website! I followed a link from the ccp4bb and run into Peter, a colleague here a deCODE biostructures / Emerald Biosystems – small world.
One thing I would add to your “is it salt?” list would be “will it float?” Salt crystals are much denser than protein crystals, and unless the crystallization condition is very viscous or the crystals very small, they tend to drop through your loop and crash to the bottom of the well like little stones.
Sean
October 27th, 2009 at 9:34 AM #
Hello Jeff,
Thanks for visiting and the tip!
I had not heard of sinking rate as a method to differentiate between salt and protein crystals, thanks.
Happy to see the Seattle area coming through
I received my BS from Seattle University and lived in Tacoma/UP for a couple of years, love the area.
Yin
November 14th, 2009 at 5:55 AM #
The dyes don’t always work. Tried it with some crystals and they didn’t get absorbed, but when I chucked the crystals onto the detector they turned out to be protein.
Sean
November 14th, 2009 at 10:46 PM #
Hello Yin!
Thanks for the comment and welcome. Very true, dyes do not always work.
Chen
July 20th, 2010 at 6:34 AM #
Hey
UV lamp rays luminates only protein due to the aromatic residues. Were working with the formulatrix robot and it’s working like a charm.