Nuclear Magnetic Resonance Spectroscopy to Identify Multiple Phosphorylations in Proteins

Abstract

Source: Danis, C. et. al., Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins. J. Vis. Exp.  (2016).

This video demonstrates the use of nuclear magnetic resonance spectroscopy (NMR) techniques to identify multiple phosphorylations in a protein. The phosphorylation of a protein at specific amino acid causes the deshielding of the neighboring amide hydrogen, which generates the spectral difference.

Protocol

1. Production of 15N, 13C-Tau (Figure 1) ATransform pET15b-Tau recombinant T7 expression plasmid into BL21(DE3) competent Escherichia coli bacterial cells. NOTE: the cDNA coding for the longest (441 amino acid residues) Tau isoform is cloned between NcoI and XhoI restriction sites in the pET15b plasmid. Mix gently 50 µl of competent BL21(DE3) cells, forming 1-5 x 107 colonies per µg of pl…

Representative Results

Figure 1: Scheme of the main steps of recombinant protein production and isotopic labeling. Steps from bacteria transformation to recombinant protein production are outlined as described in paragraph 1 of the protocol. Figure 2: Scheme of the main …