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.
The authors were actually asked by the journal to consider submitting this method after publishing the PLOS Pathology paper. Like all JoVE papers, it was submitted as a normal manuscript and peer reviewed. When accepted, JoVE writers came up with a script. Professional videographers filmed all the UK-based authors, on location in their labs, describing the research and doing the protocol. Senior author Vardis Ntoukakis said the experience was interesting and very worthwhile – he’ll certainly consider JOVE for publication of methods in future.
Immunoprecipitation is a laboratory assignment in a Master’s degree course at the University of Warwick. Ntoukakis hopes the paper will be useful to the students on the course, as well as the wider scientific community. I followed a JoVE protocol during my PhD so I can personally vouch for the fact that the clear step-by-step instructions and visual tutorial make it much easier to attempt a new method with confidence.
Unfortunately not all articles are open access, but JoVE has a good search tool and a section on general techniques, so next time you’re trying new plant science protocols, have a look for a useful video!
Highlighted paper (Open access): Piquerez, S. J. M., Balmuth, A. L., Sklenář, J., Jones, A. M. E., Rathjen, J. P., Ntoukakis, V. Identification of Post-translational Modifications of Plant Protein Complexes. J. Vis. Exp. (84), e51095, doi:10.3791/51095 (2014).