Quantitative In-Cell Hydrogen NMR Spectroscopy to Monitor Protein-Ligand Interactions

Abstract

Source: Barbieri, L. et al., Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor. J. Vis. Exp. (2021)

In this video, we demonstrate the in-cell nuclear magnetic resonance spectroscopy technique to study protein-ligand interactions between unlabeled overexpressed proteins and small molecules. The successful ligand-protein interaction is confirmed visually by the appearance of an additional set of peaks in the spectral region of interest that gradually replaces the original peaks.

Protocol

1. Reagent and solution setup

1. To prepare complete DMEM, add 5 mL L-glutamine 200 mM, 5 mL penicillin-streptomycin 100x, and 50 mL fetal bovine serum (FBS, 10% vol/vol final concentration) to 440 mL DMEM.     
   NOTE: This solution can be stored at 4 °C for 1 month.
2. Prepare agarose solution by dissolving 150 mg of low-gelling agarose in 10 mL of phosphate-buffered saline (PBS) at 85 °C to obtain a 1.5% (w/v) solution. Sterilize by filtration with a 0.22 µm filter. Prepare 1 mL aliquots of agarose solution in 1.5 N
3. Prepare the bioreactor medium.
   1. Dis....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials
Citric acid	Sigma-Aldrich	251275
D2O	Sigma-Aldrich	453366
DMEM, high glucose	Life Technologies	10313-021
DMEM, high glucose, powder	Sigma-Aldrich	D5648
FBS	Life Technologies	10270
HCl	Sigma-Aldrich	30721
L-glutamine (200 mM)	Life Technologies	25030
Low-gelling agarose, powder	Sigma-Aldrich	A4018
NaHCO3, powder	Carlo Erba	478537
PBS	Life Technologies	10010
Penicillin-streptomycin (10,000 U/ml)	Life Technologies	15140-122
NaOH pellets	Sigma-Aldrich	30620
Trypsin-EDTA (0.05% (wt/vol))	Life Technologies	25300-054
Equipment
Avance III Spectrometer equipped with a 5 mm CryoProbe	Bruker	n/a	All modern spectrometers and narrow-bore magnets equipped with 5 mm probes are compatible.
InsightMR flow unit	Bruker	n/a
P-920 pump module from ÄKTA FPLC	GE Healthcare	n/a	Any FPLC, HPLC peristaltic or syringe pump should be compatible with the flow unit.