| Assay Method Information | |
| | NMR Measurements |
| Description: | Uniform 15N isotope labeling was achieved by expression of the protein in the M9 minimal media containing 15NH4Cl as the sole nitrogen source. The final step of purification of Mdm2 (residues 1−118, chosen to enable interactions of N-terminal Mdm2 part [Showalter et al., J. Am. Chem. Soc., 130:6472-6478]) for NMR consisted of gel filtration into the NMR buffer (50 mM phosphate buffer at pH 7.4 containing 150 mM NaCl, 5 mM DTT). Then, 10% (v/v) D2O was added to the samples to provide a lock signal. All the spectra were recorded at 300 K using a Bruker Avance 600 MHz spectrometer. 1H−15N heteronuclear correlations were obtained using the fast HSQC pulse sequence. [Mori et al., J. Magn. Reson., Ser. B, 108:94-98] Assignment of the amide groups of Mdm2 was obtained after Stoll et al. [Stoll et al., Biochemistry, 40:336-344] |
| Affinity data for this assay | |
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