Sample Preparation

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Sample preparation for ITC

  • One day before - extensively dialyze titrant and receptor samples at 4oC in same container overnight to avoid artifacts from dilution heats with non-equivalent buffers.  Resuspend small molecules in same dialysis buffer the day of the experiment.
  • All buffers should be made fresh and filtered 0.2 µm.
  • Purity: Protein samples should be >95% pure by SDS page.  Oligonucleotides used should be gel or HPLC purified.
  • For one experiment, 300 µLs of sample and 80 µLs of ligand is required for proper instrument loading. Samples will be spun at 14,000RPM for 10m at 4oC before loading and the experiment. 
  • What concentration? That will be dictated by the association constant and the derived “c value.” See Malvern lecture notes. Typically these values are in the tens of micromolar regime in the cell and >100 µM-1mM in the syringe.
  • No Reducing agents or glycerol (TCEP is generally ok)

Sample Preparation for DLS

  • All buffers should be made fresh and filtered 0.2 µm.
  • Purity: Protein samples should be >95% pure by SDS page.  Oligonucleotides used should be gel or HPLC purified.
  • For best results, it is best practice to syringe filter straight into cuvette immediately before initiation of the experiment.
  • Multiple concentrations should be tested for optimal results, ideally >0.5 mg/mL average macromolecules.

Sample Preparation for SEC-MALS

  • All buffers should be made fresh and filtered 0.1 µm by the core before use.
  • Purity: Protein samples should be >95% pure by SDS page.  Oligonucleotides used should be gel or HPLC purified.
  • 1 Day before - Core will wash SEC-MALS system and column extensively in running buffer to achieve a low scattering background. 
  • We suggest injecting 100 uLs at ~3-5 mg/mL for the best results; higher concentrations for small macromolecules, and lower concentrations possible with larger macromolecules (as the intensity of scattering varies as the square of the volume of the particle).  Samples are spun at high speed for 5-10 min at 14,000 RPM before injection, to pellet and large aggregates.
  • No glycerol or sucrose in running buffer - perturbs the RI measurements.

Sample Preparation for AUC

  • All buffers should be made fresh and filtered 0.2 µm.
  • Purity: Protein samples should be >95% pure by SDS page.  Oligonucleotides used should be gel or HPLC purified.
  • Ideally, samples (whether they are freshly purified or from frozen aliquots) should be processed through size-exclusion chromatography, in the buffer to be used for analysis.  Half-peak fractions should be selected and concentrated at 4oC to approximately 100 µLs in volume.
  • This sample should then be dialyzed 4-16 hours at 4oC.  About 10 mLs of dialysate should be saved, as it will be used as the match/blank buffer for both AUC and SAXS analyses.
  • Ideally, these steps would occur 1-2 days before the measurements are made. (even samples sitting at 4oC can develop aggregates!)
  • To further ensure that no aggregates are present, we would give it an additional high speed spin in a table top centrifuge at 4oC, or filtered through a 0.2 or 0.02 µm syringe filter (20 µL dead volume) just before data collection.

Material amount needed for AUC:

Sedimentation Equilibrium:

This analysis can yield information about molecular weight of single species, oligomeric behaviors, heteromeric associations, and Kds of association. Usually SE is most useful in distinguishing between possible distinct models of solution behavior.

  • Typically, one sample is analyzed at three different concentrations, ranging from an ABS280 or ABS260 of 0.8 or less (ie: 0.8, 0.4, 0.2), in 100 µLs of volume for each concentration. An equal volume of match buffer is needed for each concentration.
  • The sample should be stable for approximately ~60 hours at the temperature chosen for the analysis (whether it is at 4oC or 25oC).

Sedimentation Velocity:

This analysis can yield information about the hydrodynamic properties of particles in solution, including size, shape, and distribution.  Very useful in distinguishing heterocomplexes from their component parts.

  • Typically, one sample is analyzed at an ABS280 or ABS260 of 0.5-1.0, in 400 µLs of volume. An equal volume of match buffer is needed for each sample.
  • A typical experimental design for the analysis of a two-component heterocomplex would be to analyze Component A alone in Cell 1, Component B alone in Cell 2, and Complex AB in Cell 3.
  • The sample should be stable for at least ~6 hours at the temperature chosen for the analysis (whether it is at 4oC or 25oC).

Buffer Limitations for AUC

  • No glycerol or sucrose greater than ~1%
  • No detergents, if possible; if a membrane protein please consult with core director on experimental design.
  • Buffer components such as HEPES and TRIS can absorb strongly in the UV region, so it is important that low concentrations (ca. 10-50 mM) are used and that match buffers are as close as possible to the sample buffer.

-KG 04/19