How to Get Started on an NMR Project

1. Expression System/Cell types
   * BL21(DE3)
   * HMS174(DE3)
   *** We typically use pET expression vectors***
2. Appropriate Buffers
   * Tris
   * Hepes
   * Phosphate
   *** if you have questions about a buffer, just give us a call***
3. Sample Conditions
   * Protein Concentration: We recommend a protein concentation of between 0.2mM - 2mM (less than 0.2mM is possible for some experiments if necessary)
   * Sample Volume: Samples need to be 600ul if using a standard NMR tube, or no less than 300ul if using a Shigemi tube
   * D2O: The final NMR sample must contain between 5% and 10% D2O
   * Salt Concentrations: Try to keep this LOW (<100mM total ionic strength)
4. Things to buy:
   * Sample Tubes:
        - Shigemi
        - Wilmad
   * Labeled Isotopes:
        - CIL for 13C-glucose
        - CIL for 15N-ammomium sulfate
   * CIL for D20

The above image illustrates typical preliminary data collected at the start of a protein NMR project. The above spectrum takes approximately 15 minutes to collect and tells much about the feasibility of a given NMR project. NMR structural work is carried out on proteins grown with nutrients labeled with both 15N and 13C.