I want to make a cryoprotectant solution for my protein crystal. I don't have many crystal to check several cryogenic conditions. My crystallization buffer contain: 50% MPD, 0.1M Tris pH 8.6, 0.1M ammonium phosphate, 0.12M Li2SO4, 0.3M NaCl, 1mM EDTA. What is the best cryoprotectant according to this condition? In the other world, when there is MPD, PEG400 or PEG 600 in crystallization buffer, is there any need to make further cryoprotectant solution?
As Chinar pointed out, there is no need to further protect your crystals, a concentration of 30% MPD should be cryoprotectant. But if you want to make sure, put a drop of your crystallization solution directly into liquid nitrogen, if the drop becomes white is because the water in the solution is forming ice, if it remains clear, is a good cryoprotectant already. That way you don't need to waste your crystals.
I think It will be OK..go ahead without CRYO... or if required try more MPD only
In general, 50% MPD should be a cryoprotectant. I would recomment a large cryoloop, dipped into the reservior solution, then flash frozen in liquid nitrogen. If the drop in the loop is glassy with no white color (it would look a bit like a snow globe, depending how far off cryo the condition is). Now, based on general stock concetrations of your starting material, there appears to be about 22.8% water in the condition. This means you can increase MPD concentration or try 10-15% ethylene glycol, glycerol or P400 as an additional cryoagent. Lastly, there are a number of good cryo oils that work very well. I can recomment Paratone N (Hampton Research) or LVCO (Mitegen). Good Luck!
Your xtals should be fine without any further cryoprotection, and if you need one just increase the MPD concentration in the mother liquor and let them sit for a minute ... you my want to adjust the soaking time.
The most common cryoprotection is achieved by soaking crystals in mother liquor to which you added glycerol (10, 20 or 30 %) . Ususaly in these cases the crystallization buffer doesn't contain any other cryoprotectiing element. If your xtals grow in PEG usually use PEG of smaller molecular weight and increase the concentration.
Hope this helps
Dear Zaiddodine Pashandi,
I hope you already collected your best data set. But for your future crystals ... One thing was missing in this discussion - a time frame. If your crystals appeared the next day after you set your plates, than conditions in the drop are far away from listed conditions. And even 50% of MPD, which is cryo condition for most cases, could be not enough. In the cases with fast growing crystals, the best way would be to use reservoir (well) solution as a cryo protector. Just before the freezing putting your crystals in the small drop of well solution ( 10-30 seconds) could make a job.
Best wishes.
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