1. Optimum Solubility Screen
To optimize buffer conditions for homogeneity and crystallization of proteins
Jarmila Jancarik, Ramona Pufan, Connie Hong and Rosalind Kim
Lawrence Berkeley National Laboratory
PPCW, March 29-31, 2004
The work described here was supported by the National Institutes of Health GM 62412

2. Step 1 Optimium Solubility Screen
Aggregated protein in purification buffer
1 µl
Buffer
1 µl
Protein
Set up protein using 24-well Linbro plates,
use 0.5 ml buffer (24) in reservoir; 1 µl buffer + 1 µl protein
on the cover slip (vapor diffusion method)
Incubate overnight over the same buffer
buffer
Perform Dynamic Light Scattering (DLS) on clear drops, pick best condition.

3. List of optimization buffers
Linbro Plate
List of optimization buffers
Version 1

4. Additive Screen Step 2 Test additives using best buffer:
25, 50, 100 mM NaCl
5 and 10% glycerol
2 mM CHAPS (CMC=6 to 10 mM)
0.1%, 1% Octylglucoside (CMC=0.53%)
0.1%, 1% Dodecyl Maltoside (CMC=0.0087%)
30mM TCEP, 5mM DTT,10 mM β-mercaptoethanol
Two hour incubation
Perform DLS
Pick best additive, exchange protein into optimal buffer/additive
Set up Crystal Screens

5. DLS in Different Buffers
1371B in Buffer #16
100 mM Na K Phosphate, pH 7.0
1371B in Buffer #21
100 mM Tris, pH 8.5

6. 1371B 1371B in 100 mM Tris, pH 8.5 1% Octylglucoside Crystals of 1371B
Structure solved

7. Results * Radius in nanometers, % polydispersity
**Crystals appeared in Optimium Screen

8. Summary
This assay provides an opportunity to find an optimum buffer/additive for crystallization of a given protein.
Screen is fast and requires a small amount of protein.
Out of 14 targets, protein monodispersity improved greatly for 11 targets and 9 of these proteins crystallized.
This screen is being implemented into a high throughput protocol.