Technical Resources

Sample Preparation

Crystallization guidelines: The X-ray Crystallography Core Laboratory utilizes multiple commercial screens containing 96 crystallization reagents each. Screens may be set up for proteins of interest to identify initial crystallization conditions at incubation temperatures of 22°C, 18°C and/or 4°C. X-ray Core Users will typically provide 400+ microliters of a 2-10 mg/ml protein preparation to complete a screen. Avoiding phosphate buffer is recommended in order to reduce false positives. HEPES, MES, MOPS, PIPES, CHES, Tris, acetate, citrate and bicine are suitable protein buffers for crystallization. NaCl or KCl up to 150 mM concentration is acceptable. Reducing agents DTT, BME, and TCEP are also compatible with most of the crystallization reagents.

NMR sample guidelines: All samples for protein-based experiments must be labeled with stable isotopes during expression. For assignment experiments this labeling with require 15N and 13C (plus deuterated water for large proteins). For chemical shift perturbations, only 15N labeling is required. The preferred buffer is sodium phosphate, due to its lack of nonlabile protons and amide-like protons, with salt concentrations less than 150 mM. Other buffers can be used. The concentration for most experiments should be 300 micromolar to 1 millimolar.

Circular Dichrosim sample guidelines: A ballpark starting concentration would be 100 uM protein; centrifuge or filter the sample to remove aggregates. Avoid buffers, detergents or other molecules that contain aromatic groups, thiol reducing agents (βME, DTT), imidazole, metal ions, and chloride salts. The best buffer for CD is usually low-concentration (5-50 mM) phosphate or acetate with no salt (or less than 300 mM).