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…
開示
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. |
