Sample Requirements

Samples of biomolecules or biofluids submitted to the NMR facility or staff should be safe and nonhazardous

Task: NMR Sample Preparation

Equipment Used: Centrifuge, pH meter, NMR spectrometers, fume hood, fridges, freezers, tube cleaner

Location: Rooms G14, G16, G23 and G24 in the Henry Wellcome Building for Biomolecular NMR Spectroscopy, University of Birmingham

Sample requirements table
HazardsRisk  (‘high’, ‘medium’ or ‘low’):
Radiation: danger to people and equipment due to inappropriate submission of radioactive samples. Low risk: All Users must understand and agree that no samples are allowed that require any form of radiological monitoring
Chemical: danger to people and equipment due to submission of carcinogenic, corrosive, flammable or toxic samples. Medium: Use of hazardous chemicals is discouraged. Users must follow all Control of Substances Hazardous to Health (COSHH) regulations, providing forms where appropriate. Hazardous chemicals brought into the building must be reported and approved by HWB•NMR staff, and labelled and stored in a safe manner at all times. Excluded Chemical Weapons Convention Scheduled Chemicals are listed in Appendix 2. COSHH Assessment NMR002 ‘Protein NMR Sample Preparation’ on the Institute of Cancer & Genomic Sciences COSHH database covers a number of chemicals commonly used in biomolecular NMR. Copies are available from the NMR Lab Manager.
Biological: danger to people due to submission of biological hazards such as toxins, infectious agents, viruses, pathogenic bacteria. Low: Use of any biological hazard is discouraged and must follow all COSHH regulations. Some of the disallowed biological agents are listed in Appendix 1.  All samples must be labelled and stored in a safe manner at all times. BIological risk assessments must be provided, where appropriate.
Ethical: samples submitted may be from human or animal origins, requiring consent. Low: No testing of human or animal subjects shall be conducted on the premises.  Ethical approvals for human or animal tissues or biofluids must be obtained from the appropriate ethical review body and submitted to HWB•NMR staff prior to initiation of NMR analysis.
Physical: Inappropriate use of manual centrifuge can cause sample breakage and debris, and involves spinning an exposed rotor. The insertion of samples into NMR magnets requires climbing access platform near the magnets. Low: Use of equipment in the laboratory and NMR chambers requires appropriate user induction and appropriate access systems.
Financial (e.g. damage to NMR systems): NMR sample tubes can break and release their contents onto and damage probes. Probes can be damaged by insertion of hazardous samples, stripping the probe tuning rods by forced overturning, or use of excessive RF pulse power or probe temperatures.  These probes can cost over £250,000, and full insurance cover is not affordable. Medium: NMR samples can only be inserted and experiments initiated by fully trained users and NMR staff who understand the risks and accept their responsibilities.  Users are not permitted to modify the NMR hardware or control software or attempt new pulse sequences unless these actions have been authorised by the NMR staff.

Safety Pre-Requisites:

  1. Ensure compliance with COSHH regulations
  2. Submit completed access form for NMR time, including sample characteristics, indicating all hazards.  Provide MSDS datasheets for any hazardous chemicals, and biological risk assessments where appropriate.
  3. All Users must obtain approval from the Operations Manager before initiating NMR experiments, including the completion of an orientation for the safe use of the NMR systems.

Key Points:

  1. In case of any accident, concern or question regarding these protocols please notify HWB•NMR staff.
  2. It is the user’s responsibility to be familiar with and comply with these procedures.
  3. Negligence or non-compliance can result in barring from future use of HWB•NMR resources.
  4. Disputes will be handled by the Health and Safety Committee.
  5. Approved chemicals (e.g. D2O) and glassware (e.g. NMR tubes) may be stocked in the HWB•NMR sample preparation laboratory,  and can be requested (subject to availability) from the NMR Lab Manager at reasonable cost.

Considerations for Protein NMR Sample Preparation

  1. Molecular weight: Larger molecules have longer correlation times, leading to faster relaxation and increased line-widths.  NMR is best suited to characterising folded proteins in the 5-40 kDa range, larger proteins and complexes require special isotope-labelling schemes.  Tags used for affinity purification (e.g. glutathione S transferase or thioredoxin) should be cleaved and removed unless they are small (under 10 residues, e.g. His-tags).
  2. Concentration: Protein concentrations should ideally be between 0.2 and 1 mM for structural analysis (1mM = 10mg/ml for a 10kDa protein), noting that doubling the concentration requires approximately a quarter of the acquisition time to obtain a given signal to noise ratio, but increases sample viscosity. Proteins should be purified to >90% homogeneity, i.e. a single clean band on a SDS-PAGE gel and, ideally, a single peak on a size exclusion chromatography column.  The sequence and exact size of the construct should be verified by DNA or protein sequencing and mass spectrometry, respectively. Ideally the monomeric (or oligomeric) state should be demonstrated by size exclusion chromatography, multi-angle static light scattering, analytical ultracentrifugation or other analytical measure.
  3. Stability: The sample may be in the NMR spectrometer for many hours or days, with temperatures for data collection ranging from 4 - 40ºC.  The standard operating temperature is 25ºC. Highly concentrated proteins tend to aggregate and precipitate, yielding poor NMR spectra.
  4. Buffer: Ideally 20 mM, although 0-50 mM is common. Phosphate buffer is an economical non-protonated buffer, and there also are a variety of perdeuterated buffers (d-Tris, d-HEPES) available that do not obscure protein NMR signals.
  5. pH: Usually acidic pH is required (typically 5-7) because many NMR experiments require observation of exchangeable amide protons which are difficult to observe at higher pH.  Protein structure and interactions can be pH sensitive, so the final sample pH should be verified.
  6. Ionic Strength: Salt (e.g. KCl) can increase protein solubility. However, high ionic strength (>150mM) demands longer 90º pulses, increases sample heating, reduces signal to noise (especially using cryogenic probes) and makes it more difficult to tune the probe. 
  7. Paramagnetics: Paramagnetic metals such as Cu(II), Mn(II), Cr(III), Fe(III) and Co(II) lead to NMR line broadening, and should typically be avoided.
  8. Volume: The final sample volume required in the NMR tube will depend on the tube diameter used with a particular probe. Using a standard 5mm tube, each NMR sample should consist of a final volume of 600 uL (i.e. 40mm sample depth), including all additives. Standard 3mm tubes require a final volume of 200 uL. 1.7mm tubes require a final sample volume of 35 uL, transferred from a minimum stock volume of 50 uL, to allow for inevitable transfer losses when working with such small volumes. Shigemi tubes reduce the sample volume required for e.g. mass-limited samples, with 5mm shigemi tubes requiring >250 uL final sample volume (i.e. 17-18mm sample depth) and 3mm shigemi tubes requiring 100 uL final sample volume. So-called "shaped" tubes are useful for high-salt samples, and require final sample volumes of 350 uL.
  9. Additives: 2H2O is added to 5% for locking on the NMR signal frequencies, unless a 100% 2H2O solution is being used to clearly observe nonexchangeable protons. Sodium azide (usually 1-3mM) may be added to prevent protein degradation due to microbial contamination. A reducing agent such as TCEP (0.5mM, or higher concentration if perdeuterated TCEP) should be added to prevent cysteine oxidation and multimerisation, for any protein containing exposed cysteines.  The internal standard 2,2-dimethylsilapentane-5-sulfonic acid (DSS) can be added (typically 50 uM) to reference the chemical shifts.
  10. Sample Tubes: At HWB•NMR, typical sample tube diameters include 5mm (standard & shigemi tubes), 3mm, and 1.7mm, depending on probe used and/or sample composition. For example, a 5mm tube is best in 5mm probes for non-lossy protein samples where high protein concentrations can be achieved. However, if the sample contains high salt concentrations (>250 mM NaCl), 3mm tubes typically provide better sensitivity in 5mm cryogenic probes (and with significantly shorter pulse lengths, which becomes important for UHF probes). 3mm tubes are optimal for mass-limited samples when not concentration limited, especially at high salt concentration (but also at zero salt). Shigemi tubes (3mm & 5mm) can also be used to boost protein concentration for constant mass, low volume samples, but require special care to eliminate bubbles, and must be suitable for use with HWB•NMR's Bruker probes (i.e. the shigemi tubes must have the 'Bruker' length chamfered base). Even more sensitive than 3mm tubes in high salt conditions are 'shaped' tubes, which even at relatively low salt concentrations (>50 mM) win at constant protein concentration. Shaped tubes are recommended for samples where high concentrations are not possible. 7-inch tubes are standard for protein NMR of single samples, and can also be used with the sample changers at HWB•NMR. New NMR tubes are not 'analytically clean' when delivered, but usually have organic or inorganic residues. Ensure tubes are clean (a rinse with water or buffer is advisable) and not chipped or warped by excessive heat.  Tubes should be capped and the cap parafilmed to avoid evaporative loss. For high-throughput work, 96-sample racks are ideal, and come in 5mm, 3mm and 1.7mm tube diameters. The 1.7mm tube racks are frequently used at HWB•NMR for cell extracts, natural products, and other mass-limited samples, in combination with a 1.7mm cryogenic probe for optimal sensitivity.
  11. Isotope labelling: Although proteins can initially be assessed for suitability for structural analysis in unlabelled forms, detailed studies require labelling with 15N for small (~50-100 residues), 15N and 13C for medium (~100-150 residues), and 15N, 13C and 2H for large (>~150 residues) proteins. Specialised labelling of individual amino acids may also be required for the study of large proteins and complexes.

Procedure – Operational Notes

  1. Use of concentrators: Proteins are often exchanged into their final solution conditions using concentrators with molecular weight cut off filters. The filters are stored with glycerol, which must be removed by at least three washes or spins of the concentrator with deionized water.
  2. Washing NMR tubes: NMR tube washers or Solvent Jet Cleaners can be purchased from GPE Scientific Limited, Merck and Wilmad-LabGlass. An economical 9 tube washer unit can be built by the School of Chemistry glassblowing shop, and will be provided by the HWB•NMR wet-lab. Strong acids such as Nochromix (now supplied under the name AlNochromix by Alconox, Inc) are available to remove adhered materials and deposits by overnight soaking, followed by washes with water or buffer.
  3. Transport of NMR tubes: Individual NMR tubes can be safely transported in NMR tube racks made by, for example, Kimble-Kontes or Wilmad-LabGlass, in graduated cylinders, or using inverted and taped 15 mL Falcon tubes.
  4. Short term storage of NMR tubes: use specialised NMR tube racks at the consoles and refrigerator to minimise risks of breakage of the delicate NMR tubes.
  5. Long term storage of NMR tubes: Long term storage depends on the nature of the sample. For example, some protein samples can be flash-frozen using liquid nitrogen for storage at -80ºC, while others must be stored at fridge temperature because they don't tolerate the freeze-thaw cycle. Users must determine what is optimal for their samples. Ideally, samples should be transferred from the NMR tube into a suitable container e.g. an eppendorf for long term storage.

Sources of NMR reagents and consumables:

  1. Cambridge Isotope Laboratories - sells isotope labelled reagents for NMR
  2. Isotec -  (a division of Merck) sells isotope labelled reagents for NMR
  3. Silantes GmbH - sells isotope labelled reagents for NMR
  4. CDN ISOTOPES - sells isotope labelled reagents for NMR
  5. Medical Isotopes, Inc. - sells isotope labelled reagents for NMR
  6. Wilmad Glass Company - standard NMR tubes from Wilmad include product 5mm 535-PP 7 (at 600 MHz), 5mm 541-PP 7 (at 800 MHz), or 5mm 542-PP 7 (at 900-1000 MHz)
  7. New Era and Norell - supply NMR tubes
  8. Bruker - sells racks of 96 sample tubes for use with Bruker SampleJet autosamplers.
  9. Merck or CortecNet - for low volume samples use 5 mm tubes from Shigemi, Inc. Ensure that tubes are matched to the appropriate solvent. Sample racks for Bruker SampleJet autosamplers can also be purchased from CortecNet.
  10. GPE Scientific Limited - sells glassware for NMR
  11. Alconox, Inc -  sells Alconox (previously known as Nochromix) for cleaning tubes

Articles which may provide useful information:

  1. Chemical shift standards: DS Wishart et al. (1995) 1H, 13C and 15N chemical shift referencing in biomolecular NMR, J Biomol NMR 6, 135-140.
  2. NMR Buffers: CH Schein (1990) Solubility as a function of protein structure and solvent components, Biotechnology 8, 308-316
  3. J Freund and HR Kalbitzer (1995) Physiological buffers for NMR spectroscopy, J Biomol NMR 5, 321-322.
  4. Three Methods in Enzymology volumes (176, 177 and 239) are dedicated to biomolecular NMR. See Norman Oppenheimer's article "Sample Preparation" pp 78-89, Vol 176 for useful hints on preparing NMR samples.
  5. Protein solubility: AP Golovanov et al. A simple method for improving protein solubility and long-term stability. J Am Chem Soc. 2004 126:8933-9.

Excluded Infectious Agents, Bacteria, Viruses and Toxins:

Note: The published list of excluded substances is not necessarily exhaustive. Please contact NMR staff if you are unsure about a particular substance.

ANTI-TERRORISM, CRIME AND SECURITY ACT 2001

Part 7 of the Anti-Terrorism, Crime and Security Act 2001 is concerned with the security of dangerous substances that may be targeted or used by terrorists. These substances are listed in Schedule 5 of the Act. The Schedule was amended in 2013. The current list is shown in the link above and includes viruses, rickettsiae, fungi, bacteria and toxins.

The provisions set out in Part 7 (and Schedules 5 and 6) place an obligation on managers of laboratories and other premises holding stocks of specified disease-causing micro-organisms and toxins to notify their holdings, and to comply with any reasonable security requirements which the police may impose.

It also requires managers of laboratories and other premises, on request, to furnish the police with details of people with access to the dangerous substances held there. The Secretary of State has given power to direct that a named individual must be allowed access to such disease strains or to the premises in which they are held.

Reporting etc is co-ordinated through the University Health and Safety Unit.