Dear All, Thank you for the replies to my post. I received more than 14 response. The summary is given below.
The suggestions were - add another purification step. * Ni-column * remove His-tag * Ni-column * gel filtration if you have done that: add another step to test for better crystals - additive screens - room temperature data set to check if freezing hampers the crystal - seeding - crystallisation at different temperatures - removal of the His-tag (or leave it on if it is cleaved) Dehydration. Reductive methylation Add PEG400 (or set your PEG concentration ~5% higher) into the cryosolution. Cryoprotection with DMSO. Grew the crystals in the presence of 5% glycerol. Crosslink the crystals with glutaraldehyde by vapor diffusion. Adding 20% peg200 as cryoprotectant. Using the Hampton Research Crystal Screen HT as additive screen (adding 5% into the mother liquor). Slowly increase the PEG concentration to 28 - 30% Add 1-2% Glycerol or MPD or Ethylene Glycol or other cryo-agent to your protein buffer you may find similar or even identical crystallization condition in which your crystal may tolerate higher concentration of cryo-agent. To include any salts in the cryo (at least half of the concentration) that may be already in the protein solution.
