SAXS Sample Prep

Checklist for SAXS sample preparation 1.0

  1. Buffer components
    • 1.1: 20-100 mM Tris or HEPES or phosphate buffer etc.
    • 1.2: Salt concentration <1M, typically 150-200 mM
    • 1.3: Reducing agents (1-5 mM DTT or 1-2 mM TCEP) - good free radical scavengers.
    • 1.4: Glycerol (1-5%, avoid greater than 10%)
    • 1.5: Avoid detergents (use them below CMC)
  2. Buffer should be perfectly MATCHED
    • 2.1: SEC-SAXS - make enough buffer for overnight equilibration for SEC-MALS-SAXS (2L) and at least 2 column volumes for SEC-SAXS (> 500 mL).
    • 2.2: Batch-mode SAXS - use dialysis buffer

  3. Sample (Protein or DNA/RNA) concentration and amount
    • 3.1: concentration: >5 mg/ml for proteins; 1-2 mg/ml for MALS only, Batch-mode SAXS - dilution series leading up to a maximum concentration of ~ 2mg/ml (could be higher for especially small proteins).
    • 3.2: minimum volume: SEC-MALS-SAXS / SEC-SAXS - 350 uL, Batch-mode SAXS - 140ul per concentration (at least three concentrations in the dilution series)

  4. Quality control: Pre-examination before X-ray scattering (Monodispersity)
    • 4.1: Gel filtration (single and symmetric peak)
    • 4.2: SDS-PAGE or Native GEL (single band)
    • 4.3: centrifugation or filter (> 15,000g * 10 mins)
    • 4.4: Dynamic light scattering
    • 4.5: Others

  5. Preparation of sample data-sheet