Hi all,

I am currently working on 14-3-3 sigma protein. I have obtained good diffraction (2.15 A) from a sitting drop Bis-tris propane (BTP) screening plate. The conditions are 26% PEG400, 5% Glycerol, 0.1 M BTP (pH =6), 0.2 M sodium nitrate.

Translation of these conditions to hanging drop has not successful.

I do not see any crystallization or protein precipitations after 3 weeks at 4 C.

The drop volume was 2 and 3 uL, with 1:1or 2:1 Buffer to complex (at 10, 12 and 14 mg/ml of complex). The complex is incubated for 16 h at 4 C before addition.

So far I have checked the pH of the buffer, it is a bit high (pH = 6.4) although I did see crystals in the screening plate at concentrations as high as pH 8.

I have also tried running a nylon loop through the well to encourage nucleation with no result. I also tried adding 5% PEG400 to the buffer solution to encourage further diffusion.

Currently, I am looking at doing a small screen in as sitting drops format at differing drop volumes to see if the volume of the drop or the sitting has any effect on nucleation.

Does anyone have experience with trouble shooting hanging drop crystallography?

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