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Related Concept Videos

NMR Spectroscopy: Chemical Shift Overview01:15

NMR Spectroscopy: Chemical Shift Overview

The position of the absorption signal of a sample is reported relative to the position of the signal of tetramethylsilane (TMS), which is added as an internal reference while recording spectra. The difference between the absorption frequencies of the sample and TMS (in Hz) is divided by the spectrometer operating frequency (in MHz) to obtain a dimensionless quantity called the chemical shift. It is reported on the δ (delta) scale and expressed in parts per million.
For instance, the proton...
¹H NMR: Complex Splitting01:13

¹H NMR: Complex Splitting

A proton M that is coupled to a proton X results in doublet signals for M. However, NMR-active nuclei can be simultaneously coupled to more than one nonequivalent nucleus. When M is coupled to a second proton A, such as in styrene oxide, each peak in the doublet is split into another doublet.
Splitting diagrams or splitting tree diagrams are routinely used to depict such complex couplings. While drawing splitting diagrams, the splitting with the larger coupling constant is usually applied first.
Proton (¹H) NMR: Chemical Shift01:07

Proton (¹H) NMR: Chemical Shift

Organic molecules primarily contain carbon and hydrogen atoms. While all the hydrogen isotopes are NMR-active, protium or hydrogen-1 is the most abundant. It has a significant energy separation between its nuclear spin states due to its large gyromagnetic ratio. As per Boltzmann's distribution, an increase in the energy separation implies a greater excess population of nuclei available for excitation, resulting in a strong NMR absorption signal.
Absorption signals of all the protium nuclei in a...
¹H NMR: Interpreting Distorted and Overlapping Signals01:02

¹H NMR: Interpreting Distorted and Overlapping Signals

Spin systems where the difference in chemical shifts of the coupled nuclei is greater than ten times J are called first-order spin systems. These nuclei are weakly coupled, and their chemical shifts and coupling constant can generally be estimated from the well-separated signals in the spectrum.
As Δν decreases and the signals move closer, the doublets appear increasingly distorted. The intensities of the inner lines increase at the cost of those of the outer lines as the signals are slanted or...
¹³C NMR: Distortionless Enhancement by Polarization Transfer (DEPT)01:20

¹³C NMR: Distortionless Enhancement by Polarization Transfer (DEPT)

When proton-coupled carbon-13 spectra are simplified by a broadband proton decoupling technique, structural information about the coupled protons is lost. Distortionless enhancement by polarization transfer (DEPT) is a technique that provides information on the number of hydrogens attached to each carbon in a molecule. While the DEPT experiment utilizes complex pulse sequences, the pulse delay and flip angle are specifically manipulated. The resulting signals have different phases depending on...
Chemical Shift: Internal References and Solvent Effects01:17

Chemical Shift: Internal References and Solvent Effects

In an NMR sample, precise measurement of the absolute absorption frequencies of nuclei is difficult. A standard internal reference compound is added, and the frequency difference between the reference signal and sample signals is measured.
The internal reference compound generally used in NMR spectroscopy is tetramethylsilane (TMS). TMS is preferred because it is chemically inert, soluble in NMR solvents, and easily removable. Also, the highly shielded methyl protons in TMS yield an intense...

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Expression and Purification of Recombinant Macrophage Migration Inhibitory Factor (MIF).

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Related Experiment Video

Updated: May 15, 2026

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy
14:55

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Published on: September 17, 2017

MIF NMR Chemical Shift Perturbation Mapping.

Emmanuel K Yeboah1, Natalie A Borg1, Stephen J Headey2

  • 1Immunity and Immune Evasion Laboratory, School of Health and Biomedical Sciences, RMIT University, Bundoora, VIC, 3083, Australia.

Methods in Molecular Biology (Clifton, N.J.)
|May 13, 2026
PubMed
Summary
This summary is machine-generated.

Macrophage migration inhibitory factor (MIF) is a therapeutic target. This study presents a 15N-labeling technique and Heteronuclear Single Quantum Coherence (HSQC) NMR methods to determine MIF

Keywords:
15N-MIFChemical shift perturbation (CSP) mappingHSQC NMRMIF

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Biophysics

Background:

  • Macrophage migration inhibitory factor (MIF) is a crucial cytokine involved in cancer, autoimmunity, and viral infections.
  • MIF is an attractive therapeutic target due to its role in various diseases.
  • Biophysical techniques are essential for compound screening and validating biological assays by estimating binding affinities.

Purpose of the Study:

  • To present an isotopic 15N labeling technique for MIF.
  • To establish conditions for high-quality 15N-HSQC NMR spectra acquisition.
  • To demonstrate the use of chemical shift mapping for determining MIF binding sites and affinities.

Main Methods:

  • Isotopic 15N labeling of MIF.
  • Heteronuclear Single Quantum Coherence (HSQC) NMR spectroscopy.
  • Chemical shift mapping of NMR resonances perturbed by compound binding.

Main Results:

  • Successful implementation of a 15N labeling technique for MIF.
  • Optimized conditions for obtaining high-quality 15N-HSQC NMR spectra.
  • Demonstrated ability to map compound binding sites and determine binding affinities using chemical shift perturbation.

Conclusions:

  • The developed 15N labeling and HSQC NMR methods provide a robust approach for MIF-ligand interaction studies.
  • This technique enables precise determination of binding sites and affinities at micromolar to millimolar concentrations.
  • These findings facilitate the therapeutic development targeting MIF.