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Practical Issues: Sample Preparation

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

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  1. 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)

  2. Practical Issues:Solution Conditions • Variables: buffer, ionic strength, pH, T • Binding studies: co-factors, ligands • H20/D20 exchange- requires unfolding • NO CRYSTALS!

  3. 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)

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

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