Frequently Asked Questions (FAQs)
How much sample do I need?
NMR is not a particularly sensitive technique; however, recent advances in the technology have improved the ability to detect signals from samples at fairly low concentrations. NMRFAM makes use of cryogenic probe technology and modern consoles to provide state-of-the-art NMR sensitivity. To obtain a 3D structure of a biomacromolecule generally requires at least 300Î¼M (e.g., ~1.5 mg of a 15,000 Da protein) in 300 µl solvent. For other experiments, such as ligand binding or pKa determinations, concentrations of 10-50 μM in 300 μl solvent can be used. In general, the more sample, the better with the caveat that if is the molecule dimerizes or oligomerizes at high concentration, it may be better to go with a lower concentration.
The lowest practical concentration for the simplest experiments on small molecules (< 1000 Da) is about 0.1 μM in 300 μl solvent. For a structure determination of a small molecule (e.g., 260 Da), as little as 5μg in 300 μl can been used. however with this amount of material, structure determination is far from routine.
What do I need to take into consideration when preparing my sample?
Initial steps. The first step is to be sure that you can synthesize and purify a sufficient quantity of protein (or other biomacromolecule) and that it can be solubilized. Many different solvent systems are acceptable, but you should be aware that NMR signal-to-noise is degraded by high salt concentrations and that NMR lines become broad if the solvent viscosity is high. The NMR instrumentation at NMRFAM collects data only on liquid samples. Owing to the low natural abundance of 13C and 15N, observation of these nuclei usually requires that they be enriched. In some limited cases, natural abundance levels of the isotopes are sufficient.
Testing. NMR studies of biomolecules usually take at least one day; a week or two of data collection may be required for a structure determination of a protein or nucleic acid. The first criterion for success is that the sample must remain stable over this time period. The spectrometers have variable temperature capabilities, and the temperature of the sample can be adjusted between 0oC and 50oC to optimize the stability of your system. Raising the temperature above ambient typically gives higher quality spectra (because molecules tumble more rapidly) while lowering it may lead to lowered resolution. A second criterion for success is the spectral quality achieved with the sample under the solution conditions used, such as pH, buffer, additives, temperature.
Although it may be possible to determine the stability and spectral quality of a sample without labeling, more definitive results can be obtained with a sample labeled uniformly with 15N. A solution of a few milligrams of protein is prepared, and an initial test spectrum (a HSQC spectrum that takes 0.5-24 hr) is collected. This spectrum generally is diagnostic of further success. It may be necessary to adjust the sample conditions, such as pH, salt, buffers, to improve the quality of the spectrum. Sometimes, a redesigned molecule (protein or nucleic acid with altered sequence) will yield better quality spectra. Once a high quality spectrum is obtained, the sample is retested a week or other suitable time later to establish the stability of the sample. Obviously, the length of time that the sample must remain stable must be long enough to collect the data. Therefore, if you want to determine the structure of a large biomacromolecule, it must remain stable for a week or longer.
Once it is determined that the protein is suitable for 3D structure determination, a 13C and 15N labeled sample (double labeled) is prepared for data collection. If you have a large (>20-30 kDa) biomacromolecule or a large (>20-30 kDa) biomacromolecular complex, you may need to also replace carbon bound hydrogens with deuterium in addition to 13C and 15N labeling (triple labeling).
Special labeling schemes (selective, 13C, 15N, or both) may be used for assignments, dynamics, ligand binding, pH titration, and other studies. If you contact the facility, our experienced staff can help you determine the best labeling scheme for your experiment.
How do I label my protein?
There are several ways to label the proteins. If you are expressing proteins in E. coli, you can grow the bugs in minimal media supplemented with labeled glucose, ammonium chloride, and/or amino acids. Labeled samples also can be prepared in yeast, baculovirus, and insect cells. Some biomacromolecules can also be chemically synthesized with labeled compounds. NMRFAM also has the capability of preparing labeled protein samples by cell free synthesis.
There are also more specialized ways of labeling your protein: selective amino acid labeling, segmental labeling, or SAIL labeling (specially labeled amino acids) with cell free synthesis. If you contact the facility, we can help you determine the best approach for labeling your proteins.
I see the facility has spectrometers ranging from 400 to 900 MHz. How do I know which one to use?
The fundamental reason for choosing a higher frequency spectrometer is to obtain increased resolution (linear increase with frequency) and higher sensitivity (~frequency 3/2). NMRFAM staff will help you choose the proper spectrometer for your project. We recommend that you contact us before beginning a project for a consultation on the best experimental strategy for your project.
How long does it take to run an experiment?
The amount of time it takes to run an experiment depends upon the particular problem that you are studying, the amount of data that must be collected, and the concentration of the sample. For a simple 1D spectrum of a small molecule, the experimental time is usually less than 15 minutes. To collect all of the data for the determination of a protein structure may take 2-3 weeks and this does not include the time required to analyze the data. One core project at NMRFAM is an attempt to reduce both the required spectrometer and data analysis time for structure determinations.
Is there someone who can train me to use NMR and to analyze data?
Yes, our staff consists of many NMR experts who can help you not only use the spectrometers and analyze your data but can also help you design and set up your experiment. Because analyzing the data from a protein or nucleic acid is fairly time consuming, you need to have a scientist dedicated to this project and we can help train that person. NMRFAM staff members also collaborate with outside users, especially if the project is compatible with one of the NMRFAM core research projects. Hands-on training is encouraged. Contact Dave Aceti at email@example.com or 608-262-3173 to arrange training. See our User Access pages for more information about becoming an NMRFAM user.
Before independent use of any of the facility instruments, all users must be trained. Facility staff will oversee this training, and solo use on a particular class of experiment will not be permitted until the staff is satisfied that a minimum competency level is reached. New users receive up to 8 hours of free training. The cost for training is $50/hr (minimum 1 hr).
Can I have someone run a sample for me?
Yes, NMRFAM offers routine service spectroscopy of submitted samples twice weekly on a first-come-first-serve basis on the DMX 400. The rate for the DMX 400 is $53/hr and most samples can be run in less than 30 minutes. See the schedule via Sundial. This service is generally only for obtaining 1H or 13C spectra of small molecules (< 1000 Da). See our Service page for application forms and detailed instructions for submitting service spectroscopy samples. An abstract describing the proposed project must be submitted with the time request, if it's not already on file with NMRFAM.
How much does it cost?
NMRFAM charges hourly fees for data collection. The rate depends on the field strength and whether the instrument has a cryogenic probe. See our Instrumentation page for a complete list of our instrumentation and the per hour rate for each. Spectrometer time is billed in 0.5 hr increments.
If you choose to send us your sample or need to have our staff set up your experiment and run it, we apply a surcharge for the time it takes to set up your experiment (typically 0.5-1 hr at $50/hr) and then the charge drops to the per hour rate for the remainder of the experimental time. This does not include data analysis. Data analysis is usually done by the user or a collaborator, who may be a NMRFAM staff member. If the project is a collaboration with a staff member, there is no surcharge, but the cost of the spectrometer time will be charged as in the table below.
Fees will be waived if problems develop as the result of equipment failure, but not for problems resulting from sample quality, quantity, or outside operator error.
IMPORTANT: If you sign up for time and then do not use it or do not arrange for someone else to use it, you will be billed for that time. If you know that you will not use time you have signed up for then you can cancel it but you must do so at least 24 hours in advance to avoid being charged for the requested time.
There is no charge for the use of the computers and software at NMRFAM.