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?The ligand concentration will depend somewhat on how tightly your protein normally binds to the protein, whether the pH, salts, PEG, etc that may be found in your crystallization conditions will affect this binding interaction, whether the active site is blocked and, as you have mentioned, whether the cryo used may bind better and bump your ligand of choice out. I think the standard response to your question is generally "as high as you can get the concentration without cracking/ dissolving your crystals". A good place to start might be 1 mM. cheers, tom Tom Peat Proteins Group Biomedical Program, CSIRO 343 Royal Parade Parkville, VIC, 3052 +613 9662 7304 +614 57 539 419 [log in to unmask] ________________________________ From: CCP4 bulletin board <[log in to unmask]> on behalf of chemocev marker <[log in to unmask]> Sent: Monday, May 16, 2016 8:41 PM To: [log in to unmask] Subject: [ccp4bb] any advice on soaking the crystals Dear all I am planning some soaking experiments of my crystal with the ligand. Can someone share his experience, tips and tricks for that. What concentration has work for you. Is there is any chance that the cryo can wash out the ligand while one is freezing the crystals. Best