Frequently Asked Questions

Nuclear Magnetic Resonance (NMR) spectroscopy takes advantage of the natural magnetism present in certain atomic nuclei. The nuclei most often studied in biological applications of NMR are: ¹H, ¹³C, ¹⁵N, and ³¹P. All of these nuclei are stable (non-radioactive) isotopes. When a molecule is placed in a magnetic field, these nuclei act like tiny little bar magnets and align like iron filings. The nuclear bar magnets can align either in the same direction (up) or in the opposite direction (down) as the external magnetic field; these two alignments have different energies. An analogy of this energy difference can be physically experienced by holding the north poles of two magnets together versus holding the north and south poles together. In the first case, they repel and in the latter they attract. It is the difference in energy between these two alignments of the nuclei in the magnetic field that is the fundamental quantity measured by NMR. The great strength of the technique comes from the fact that the energy difference between the up and down states (a.k.a. the chemical shift) is exquisitely sensitive to the molecular environment of the nucleus. Virtually every nucleus in a molecule experiences a different environment and thus has a chemical shift different from that of every other nucleus. For example, the chemical shift of a C atom bonded to another C and three H atoms will be different from that of a C bonded to two C and two H atoms. This means the structure of a molecule is encoded in the chemical shifts of the nuclei. By using rather sophisticated experiments, the chemical shift of every nucleus in the molecule and its physical location in the molecule can be determined.

Just as the nuclear bar magnets in a molecule interact with the external magnetic field, they also interact with each other. The strength of this interaction depends on the distance between the nuclei. This feature makes it possible to use NMR to determine distances between nuclei that are closer than about 5Å. Thus NMR can be used to determine the folded conformation of a protein or nucleic acid or to discover how a drug binds to its receptor molecule. Three-dimensional structures of molecules are determined by combining information on chemical shifts assigned to particular atoms with internuclear distances estimated from NMR experiments. Specialized NMR experiments can be used to determine how rapidly the molecule or parts of the molecule are moving in solution. The time scale of motions over which NMR is sensitive covers an enormous range: 10^-11s to >1s. In the picosecond to nanosecond time range, effects of librations, vibrations, and overall molecular tumbling can be quantified. The microsecond to the second time frame includes motions involved in conformational changes, ligand binding, and catalysis.

Diffusion measurements, kinetics, metabolism, oil well logging, imaging, fluid flow, analysis of foodstuffs, analysis of zeolite catalysts, clinical quantification of serum cholesterol, combinatorial chemistry, and quantum computing are among the far-ranging applications of NMR. All of this versatility comes at a cost. NMR spectroscopy is an insensitive spectroscopic technique. The problem arises from the very small energy differences between the up and down states of alignment. The experimental problem is the detection of the weak absorption of photons that excite the excess population in the ground state. Due to the lack of sensitivity, liquid state NMR requires micromoles of compound at relatively high concentration (1 mM) and detection by very expensive equipment. NMRFAM supplies the equipment and expertise necessary to do these types of experiments.

No. The theory behind NMR is very complicated, but you do not need to have an in-depth understanding of it in order to collect data to answer a particular biochemical question or obtain a structure. This is analogous to an MRI scan in a hospital the doctor does not need to understand the theory of the instrument in order to do the scan and interpret the data. The NMRFAM staff includes experts in multiple areas of NMR spectroscopy who can train you to run the samples and analyze the data. The staff is also available to answer questions. Eight hours of training are provided for new users free of charge. If additional training is needed, the fee is $50/hour. The training occurs on-site, although questions can be answered via phone or e-mail. If you need training or have questions, please contact us at nmrfam-head@biochem.wisc.edu.

If you have a small molecule and do not want to perform the experiment yourself, you can submit a sample to NMRFAM, and we will collect standard spectra for you.

Yes, several questions that should be addressed first (below). Our staff can help you determine the best experimental approach for your particular question:

1 – What problem do you want to study? NMR is very good at some and poor at others.
2 – What is the molecular mass of your biomacromolecule and does it dimerize or oligimerize? If it is ~20 kDa or more, there may be limits on what can be studied.
3 – How much sample can you produce? Is it soluble at the concentration needed by NMR, and can you incorporate stable isotopes.

It generally depends on the type of information that is desired. Structural studies are the most limited case. The molecular mass of most biomacromolecules under investigation by NMR are less than ~20-25 kDa with the majority < 20 kDa. If you want to investigate the binding of ligands or determine the binding surface in a protein-protein interaction, molecules with much higher masses can be studied.

New techniques and stable isotope labeling methods have greatly increased the maximum molecular mass of biomacromolecules available for study by NMR. The structures of several b-barrel membrane spanning proteins inserted in a micelle have been determined. These systems had an aggregate molecular mass of ~50-60 kDa. Generally, structure determinations of proteins have been limited to molecules with less than ~400 amino acid residues, however, the global fold for a 82 kDa single chain protein in solution has been reported in Tugarinov et al., PNAS 102(3)622-627 (2005), and there are some reported studies of protein complexes with molecular masses between 200 and 800 kDa. For reviews, see Tugarinov, et al., Ann Rev Biochem 73:107-146 (2004) and Riek et al., TIBS 25(10):462-468 (2000).

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 300μM (e.g., ~1.5 mg of a 15,000 Da protein) in 300 µl solvent. For other experiments, such as ligand binding or pK_a 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.

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.

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.

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 Lai Bergeman or 608-262-3173 to arrange training. See our User Access page 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).

Yes, NMRFAM offers routine service spectroscopy of submitted samples! An abstract describing the proposed project must be submitted with the time request, if it’s not already on file with NMRFAM and then time is scheduled via iLab.

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, a collaborator, or an NMRFAM staff member.

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.

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 0^oC and 50^oC 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.

NMRFAM provides a wet lab directly neighboring the spectrometers for users and staff to perform any final sample preparation. Users may ship solution NMR samples already in NMR tubes or in microfuge tubes for transfer to an NMR tube by an NMRFAM staff member.

NMRFAM has test spinning stations and all solid-state NMR samples will be tested for spinning stability prior to scheduled NMR time, whether packed by the user or packed by NMRFAM staff.

Any biological tissues, fluids, cells, toxins or other potential biohazards require prior notification and discussion with NMRFAM staff to ensure appropriate safety protocols are in place.

Any questions regarding the optimal isotopic labeling scheme for macromolecular NMR experiments, sample preparation or handling protocols for metabolomics workflows, or sample quantity or concentration should be directed to NMRFAM staff for consultation and advice.

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.

iLab is an online scheduling and billing system that allows researchers to submit work requests and to schedule equipment with UW shared resources. Visit our core directory for more information on each core.

Group/lab administration includes the principal investigator, the manager(s) and the primary contact. These roles have the following capabilities:

• Manage memberships
• Manage fund assignments
• Manage budgets
• Approve service requests
• Manage payment selections on services
• Review, approve and manage all invoices

As a PI, you are responsible for accepting your group members into your group/lab, adding in funding strings and assigning them to the appropriate researchers. These responsibilities may be delegated to a manager once the PI account and group is created in iLab.

Existing users log into iLab at https://uwmadison.ilabsolutions.com/account/login

New PIs and new members should follow the instructions in the “Registering for an account” tabs below.

REGISTERING FOR AN ACCOUNT in iLab

1. Navigate to the iLab login page and click the ‘Register’ button in the top right of the screen then click the ‘Registering using UW credentials’ link.
2. Log in with your NetID and password.
3. Search the ‘PI/Group’ dropdown for your name. If your name is not there, select the ‘My lab is not listed’ option. Fill in your contact phone number. Click the ‘register’ button.
4. Once your request has been submitted, the iLab team will work with you to get your group/lab created. When this is complete, you will be able to access iLab.
   1. Ensure that your time zone is set to Central Standard Time
5. Before you are able to request services and schedule equipment with NMRFAM, the following steps must be complete for your lab:

ADDING YOUR GROUP/LAB MEMBERS

If your group/lab members are already registered to another group within iLab, you may add them to your group:

1. In your group/lab, click the “Members” tab
2. At the bottom of the page, click “Link existing user”
3. Enter a name to search, click on the correct name in the results, and click “Add”
4. The group/lab member will be able to select the group when ordering services or reserving resources using iLab

Most group/lab members will need to request access to your group. Group/lab members should follow the steps in the tab “Getting Started: Group/Lab Members,” below. Once they’ve requested access, complete the following steps:

1. Log into iLab and select “My Groups” on the left navigation bar
2. Select the name of the group/lab to which you want to add lab members
3. Select the “Membership Requests & UW fundings” tab. New membership requests will show at the top of this page
4. Click “Approve” to accept a member into your lab. Click “Reject” if they are not a member of your group/lab.

ADDING FUNDING ACCOUNTS (FUNDING STRINGS)

1. Log into iLab and select “My Groups” on the left navigation bar
2. Select the name of the group/lab to which you want to add funding account
3. On the lab page, open the “Membership Requests & UW fundings” tab
4. Scroll to the bottom and select “Manually add a new UW funding”
5. Fill out the required information and select “Add”
6. The funding account will now be available in the Manage UW fundings table.
7. Select the corresponding staff check boxes in the column for the funding account to give them access to that funding string. Group/lab members will now be able to use that funding string to request services or make appointments

OPTIONAL: DESIGNATE A LAB MANAGER/PROXY

1. Log into iLab and select “My Groups” on the left navigation bar
2. Select the name of the group/lab for which you want to designate a manager
3. On the lab page, open the “Members” tab
4. Find the member you want to designate as a manager and select the yellow pencil icon to the right of their name
5. In the “Lvl:” dropdown, change their level to “Manager”
6. If the member you have designated as a manager should also receive invoices for core services, select the “Core Financial Contact” checkbox.
7. Select “Save” to the right of the member’s name.

REGISTERING FOR AN ACCOUNT

1. Request access to UW—Madison’s service centers: https://uwmadison.ilabsolutions.com/account/signup/1802

ADDING YOUR LAB MEMBERS

If your group/lab members are already registered to another group within iLab, you may add them to using the following workflow:

1. In your group/lab, click the “Members” tab
2. At the bottom of the page, click “Link existing user”
3. Enter a name to search, click on the correct name in the results, and click “Add”
4. The member will be able to select the group/lab name when ordering services or reserving resources using iLab

Most members will need to request access to your lab. Once they’ve requested access, complete the following steps:

1. Log into iLab and select “My Groups” on the left navigation bar
2. Select the name of the group/lab to which you want to add members
3. Select the “Membership Requests & UW fundings” tab. New membership requests will show at the top of this page
4. Click “Approve” to accept a member into your group/lab. Click “Reject” if they are not a member of your group.

OPTIONAL: DESIGNATE A GROUP MANAGER/PROXY

1. Log into iLab and select “My Groups” on the left navigation bar
2. Select the name of the group/lab for which you want to designate a manager
3. On the group/lab page, open the “Members” tab
4. Find the member you want to designate as a manager and select the yellow pencil icon to the right of their name
5. In the “Lvl:” dropdown, change their level to “Manager”
6. If the member you have designated as a manager should also receive invoices for core services, select the “Financial Contact” checkbox.
7. Select “Save” to the right of the member’s name.

REGISTERING FOR AN ACCOUNT

1. Navigate to the core page: https://uwmadison.ilabsolutions.com/account/login
2. Click the ‘Register’ link in the top right of the page
3. If you have a NetID, click the ‘Register using UW Credentials’ button then follow the onscreen instructions to log in with your UW NetID
4. If you do not have a NetID, click the ‘Register for an iLab account’ button and follow the onscreen instructions to create an account.
5. You will be directed to an iLab Registration page where you will need to select your PI/Group, and verify your contact information
   1. If your PI/Group is not available in the drop down, refer your PI to the instructions “Registering for an account as a UW PI” above
6. Once your registration has been submitted, your PI will receive a notification from iLab that you have requested membership to their group/lab. They will need to approve your membership and assign one or more funding accounts for your use.

Notes: Ensure that your time zone is set to Central Standard Time to make reservations

If you are in one group/lab and need access to another, you may either request to join the group directly, or the group management must add you.

METHOD ONE

The first method to join a new group is to request access directly. To do so, follow these steps:

1. Click on the navigation menu
2. Click on “Manage Groups”

Next:

1. Click “Request Group Access”

You will now see the option to choose your institution (University of Wisconsin – Madison) and the group/lab you would like access to. Simply start typing the name of the PI and the group will appear. Click on the desired group/lab and click “Request Access”. The PI or manager will now be able to approve your request.

METHOD TWO

The second method to join a group/lab is for the PI to add you. They will complete the following steps:

1. In the group/lab, click on the “Members” tab
2. At the bottom of the page, click “Link existing user”
3. Enter a name to search, click on the correct name in the results, and click “Add”
4. The member will be able to select the group/lab name when ordering services or reserving resources using iLab