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Practical Issues: Sample Preparation. 1D 100 m M 10 m M Concentration: Cryoprobe! nD 400 m M 50 m M Volume: 500 m L, 200 m L (microprobes soon) Quantity: @ 20kDa  100 m M = 1 mg Purity > 95%, buffers

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practical issues sample preparation
Practical Issues:Sample Preparation

1D 100 mM 10 mM

  • Concentration: Cryoprobe!

nD 400 mM 50 mM

  • Volume: 500mL, 200 mL (microprobes soon)
  • Quantity: @ 20kDa  100 mM = 1 mg
  • Purity > 95%, buffers
  • Sensitivity () isotope enrichment (15N, 13C)
practical issues solution conditions
Practical Issues:Solution Conditions
  • Variables: buffer, ionic strength, pH, T
  • Binding studies: co-factors, ligands
  • H20/D20 exchange- requires unfolding
  • NO CRYSTALS!
practical issues molecular weight
Practical Issues:Molecular Weight

*Symmetry reduces complexity*

8 x 10 kDa ≠ 80 kDa

  • 30-40 kDa for 3D structure  domains
  • 40-100 kDa: residue-, site-, and atom-specific labeling, uniform deuteration
  • 1 MDa: TROSY (Transverse Relaxation Optimized [NMR] Spectroscopy)

Fernandez & Wider, Curr. Opin. Struct. Biol. 13, 570-580 (2003)

practical issues hardware
Practical Issues: Hardware
  • Magnet: homogeneous, high field
  • Electronics: stable, tunable
  • Environment: temperature, pressure, humidity
  • Environmental: stray fields