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Selecting a buffer to use for a SAXS experiment

  • Here's some information taken from various websites (referenced)
  • From ESRF BM29
    • Buffer requirements
      • To obtain the scattering data from your macromolecule of interest, the scattering from the buffer in which your protein is dissolved must be measured (both before and after the sample). We have found that most of biological buffers are compatible with SAXS studies at the beamline (Table 1). Excess volumes of buffer should be prepared for these measurements (we recommand at least 1 mL for 3 sample concentrations).
        • Table 1. Most frequent used buffers (salts) of biological samples at the BioSAXS beamline at the ESRF. ESRF BM29 Table 1
      • The buffer composition should exactly match the protein solvent. Any DTT, glycerol, detergent (DDM or SDS), small molecules (ATP/ADP) or salts added into samples, must be also put into buffers!
      • Pay also attention to the quantity of salts or surfactants added into samples.
        • It is recommended to use:
          • Ionic strength of salts (NaCl, HCl, Na2SO4, K2HPO4 ...) < 1M
          • Concentration of small proteins (ADP or ATP) < 10 mM
          • Glycerol percentage < 30% (usually, in the range [5-10%])
          • Detergent concentration < Critical Micelle Concentration (CMC)
          • If this is Unavoidable due to the behavior of your macromolecule, please consult us in advance for advice.
      • Particular Case of Detergents
        • Detergents are used with membrane lipids and membrane proteins to increase their solubility and stability in solution. However, we recommend not using detergents in the entire protein preparation for SAXS measurements because they affect the protein signal by micelles’ formation.
  • From SIBLYS beamline info
      • We recommend to not using detergents in entire protein preparation. We observed only a few cases (~1%) where the detergent ( bellow CMC concentration ) did not affect the protein signal.
    • BUFFER:
      • Salt increases the background, but we've gotten good signal with up to 1 M salt. Concentration of the macromolecule has more of an impact on signal than the buffer, so if the sample is monodisperse in high salt, put it in high salt. 1-5% glycerol cuts down quite a bit on radiation damage, although we've also seen oligomerization induced by addition of glycerol. If you have the quantity available, trying several different buffers is recommended. For a start, ideal buffer might contain 100-200 mM salt with 5% glycerol. Please send at least 15 mL of buffer with your sample.
      • This happens, maybe 1 in 10 samples we observe changes in the SAXS scattering curve due to radiation damage. We've found that complexes do seem more stable, so if you have a complex, then throw that in as well. Glycerol is a pretty good radical scavenger, so 5% glycerol is good.
  • From OU medicine
    • Avoid inorganic buffers like phosphate and cacodylate. Use MOPS, MES, or TRIS which scavenge free radicals generated by X-ray damage. 1-5 mM DTT, TCEP, and 2-mercaptoethanol also protect proteins from excessive radiation damage. So does glycerol but you must have a matching concentration in your buffer sample.
    • Take care with detergents. Some detergents scatter X-rays very strongly.
  • From
    • Composition of the buffer
      • Salts are often useful to suppress long-range electrostatic interactions between solutes (structure factor). They also increase background and decrease solute/solvent contrast but these effects are often negligible up to ~500 mM salt. High-Z elements should be avoided in the buffer. They decrease contrast and promote radiation damage by increasing photo-electron production. Due to RNA/DNA’s higher surface charge relative to proteins, structure factor suppression by salts is usually weaker than for proteins at a given salt concentration.
      • Free radical scavengers should be included in the buffer when preparing for a synchrotron data collection as they help to minimize the radiation damage. Common choices are DTT (2-10 mM), TCEP (1-2 mM), or glycerol (~5%). In cases when these cannot be used, organic buffers containing TRIS or HEPES can also act as radical scavengers. When using DTT, keep in mind that it is oxidized by air. Degassing the dialysis buffer and keeping both sample and buffer under nitrogen throughout the entire preparation up till data collection will suppress this.
      • Detergents are best avoided unless absolutely necessary (membrane proteins). Their signal can be comparable to or exceeding the protein signal complicating data interpretation.
Last modified 5 years ago Last modified on Oct 11, 2013 5:12:26 PM

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