Hi Scott, I'd try and set up drops at 20% PEG, then back off by dilution to 15% PEG at different time points (15 min, 30 min 1 hour...)
Since equilibration of PEG in hanging drops is much slower than this, you actually need to dilute the drops. You can also consider microbatch. Flip -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Scott Berry Sent: Monday, August 11, 2008 07:57 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein/DNA microcrystals Hello All, I am trying to crystallise a heterodimeric transcription factor complex bound to DNA. At the moment I have a construct which crystallises in many different conditions, all containing a PEG (3350, 4K, 6K, 8K) as precipitant along with various buffers and/or salts. When fine screening (in hanging drops) around these conditions I commonly get a shower of microcrystals at 20% (or higher) PEG. At lower PEG concentrations the crystals become more round and eventually the drops progress to phase separation (around 15% PEG). Lowering the protein concentration in the drops results in similar numbers of crystals forming, which end up smaller in size. I have tried seeding with the microcrystals from higher to lower PEG concentrations which results in crystal melting/phase separation. Also, the best crystals seem to grow on the glass slide and when I try to move them or pick them up, they are quite soft and hard to handle. Any advice on how to optimise conditions for growth of larger crystals and also tips for handling these soft crystals would be great. Thanks, Scott