Researchers from the University of Warwick published a methodology paper with a twist this week. The paper, published online by the Journal of Visualized Experiments (JoVE), gives step-by-step instructions and video demonstration of a method for purifying a protein and identifying proteins that perform its post-translational modifications.
Authors Sophie Piquerez, Alexi Balmuth, Jan Skenář, Alex Jones, John Rathjen and Vardis Ntoukakis developed the method in order to characterize the interactions between nucleotide-binding leucine-rich-repeat proteins and the Prf/Pto complex in effector-triggered immunity. In principle the method could be applied to any protein – the protein of interest is epitope-tagged, immunoprecipitated and analysed by MS.
A video journal lends itself to new or improved methodology rather than high impact conclusions. As with a lot of JoVE articles, the scientifically significant results obtained using the protocol have already been published; in this case in Ntoukakis et al. 2013 (PLOS Pathogens, 10.1371/journal.ppat.1003123).
The authors conclude the abstract by saying the paper demonstrates:
- Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry;
- It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate;
- The immunoprecipitation step is essential to get enough protein to do the MS. (more…)
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