This video describes the nuclear magnetic resonance spectroscopy technique to study protein-protein interactions between 15N-labeled wild-type and mutant envoplakin proteins and the unlabeled vimentin protein. The successful interaction between wild-type envoplakin and vimentin leads to extensive line broadening and peak disappearance in the NMR spectra, whereas the absence of an interaction between the mutated envoplakin and vimentin results in well-resolved peaks in the NMR spectra.
Protocol
1. NMR Methods NMR Sample Preparation Purify 15N-labeled wild-type or R1914E E-PRD protein using 20 mM Tris-HCl, 1 mM DTT, pH 7. Protein stock solutions usually range from 0.3 to 1 mM with volumes of about 1 mL. NOTE: Protein can be concentrated to >100 µM using an MWCO 3 kDa, 5 mL centrifugal ultrafiltration device to bring the concentration into a suitable range for sample preparation. Purify a sample of unlabeled VimRod pr…
Representative Results
Figure 1: Screen Capture of the Setup of the NMR Experiment. The window shown is used to set up a standard experiment to collect an HSQC dataset. Experiment parameters are read adjacent to Experiment. The ZGPR experiment shown is chosen as an initial experiment to load the standard and solvent-dependent proton parameters. The Title window is used to input experimental details for record-keeping p…