Glycerol as a Cryoprotectant
Glycerol is the most common cryoprotectant used in macromolecular crystallography. If you are considering using glyercol as a cryoprotectant for your protein crystals it is worth looking into what has worked for others.
Obviously, your mileage will vary depending on your protein, however, there are insights that we can gain by examining the literature. I am fond of the notion that an hour in the library can save you a week in the lab.
1) Only papers that used the crystallization reservoir (mother liquor) with glycerol alone (not mixed cryoprotectants such as PEG 200 10 % with 20 % glycerol) were selected.
2) Papers that contained a range of minutes, such as 5-10 min, were not included.
3) Proteins were not listed more than once
In total, 27 papers met the above criteria and listed the soaking time in either minutes or a few seconds.
The % glycerol is presented along the top row while the 1st column is the soaking time:
What percentage are crystallographers using?
The most popular percentage is 20-25 % glycerol.
How long are they soaking their crystals?
A soaking time of a few seconds is dominant as it was described in 15 out of the 27 papers.
Finally, I also noticed step soaking was used quite often (in 7 papers) which, in general, is performed in 5 or 10 % steps up to about 30 % glycerol with 1-2 minutes per step.