Hello, have you tried making a matrix where you change your conditions one by one, first try changing PEG concentrations, mataining everything else constant. Try the same with the ionic strength of your buffer and protein concentration, leaving everything else constant. Another thing you can try is lowering the temperture to 4°C, in the literature it is reported that crystallizing at 4°C can make your crystals bigger.

You can also try using hanging drop technique to make your matrix.

Also the purity, as well as your purification process may influence in the obtention of bigger crystals.

Hope this helps you.

You could also try the Additive Screen from Hampton to see if any of the compounds in the screen can increase the size of your crystals or improve/change their morphology.

Dear Pachineella,

There are a lot of optimization you can do. For examples, grid screenings to vary the protein concentration, PEG3350 concentration, different buffer, pH grid (change one parameter at a time), also temperature. Hampton additive screen can be surprisingly nice. 

Now your goal would be to increase the diffraction, not the size as it is not always true that bigger the crystal, the better the diffraction.

How do you know if it's protein crystal without obtaining diffraction pattern?

Best of luck.

 we have tried diffraction and seen different spots not at the corner , near the beam center , i have tried  at 4 degree but they are not reproducible, even i have  varied pH ,PEG . presently iam optimising at room temperature only. i have varied PGA also.not much change, if size doesnt matter plz tell me  what factors may influence, with what steps i can go forward. plz tell me