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

Published: June 29, 2023

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 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. 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…

Offenlegungen

The authors have nothing to disclose.

Materials

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.

Tags

Play Video

Diesen Artikel zitieren
Quantitative In-Cell Hydrogen NMR Spectroscopy to Monitor Protein-Ligand Interactions. J. Vis. Exp. (Pending Publication), e21446, doi: (2023).

View Video