1 / 4

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

rowland
Download Presentation

Practical Issues: Sample Preparation

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related