How to prepare samples for equilibrium SAXS

Checklist for SAXS sample preparation

Buffer components

  • 20-100 mM PBS, Tris, HEPES, etc. PBS is preferred if possible.
  • Salt concentration < 1M, typically 150-200 mM
  • Avoid radical scavengers (glycerol, DTT, etc) unless needed for the stability of your protein. With BioCAT’s coflow setup these actually increase the likelyhood of seeing radiation damage.

Buffer matching

  • SEC-SAXS/SEC-MALS-SAXS - Use dialysis buffer or a matched buffer from the final SEC purification step.
  • Batch-mode SAXS - use dialysis buffer
  • SEC-SAXS - make enough buffer for both the experiments and equilibration. For SEC-MALS-SAXS > 1.5 L, for SEC-SAXS > 500 mL.
  • If mailing concentrated buffer for SEC-SAXS or SEC-MALS-SAXS (e.g. 10x), we recommend you prepare the concentrated solution, create the final dilution in your lab, exchange your samples into that, and then mail us the remaining concentrated stock for dilution on site. While this doesn’t perfectly match buffer, it gets close, and the buffer exchange on the column will guarantee a perfect match.

Sample (Protein or DNA/RNA) concentration and amount

  • Concentration:
    • SEC-MALS-SAXS/SEC-SAXS: 240/(MW in kDa) = concentration mg/ml
    • Batch mode SAXS: 60/(MW in kDa) = concentration in mg/ml. Use a dilution series with a least 2 other concentrations bracketed around this. E.g. for a 20 kDa protein, measure at 6, 3, and 1.5 mg/ml.
  • Minimum volume:
    • SEC-MALS-SAXS / SEC-SAXS - 250 µL
    • Batch-mode SAXS - 50 µl per concentration

Note: If mail-in samples are shipped frozen, they will be fast thawed (e.g. in hand/water bath) unless you specify that the samples should be thawed on ice.

Quality control: Pre-examination before X-ray scattering (Monodispersity)

  • Gel filtration (single and symmetric peak)
  • SDS-PAGE or Native GEL (single band)
  • Centrifugation or filter (> 15,000g * 10 mins)
  • Dynamic light scattering
  • Others

More details

In the following video from the 2019 Everything BioSAXS 5 workshop, Dr. Kushol Gupta discusses what you need to know about sample preparation for SAXS (get slides)