A step by step protocol for absolute quantification of protein using Arabidopsis thaliana transgenic lines carrying NanoLUC-tagged genes

Quantification of protein levels in absolute units will allow testing quantitative models of gene
regulatory networks. Better quantitative models have application in fundamental plant biology
but also in synthetic biology. Models in absolute units can, for example, help guide the design
of promoter modifications using CRISPR genome editing technologies, such as base editing.
Absolute protein quantification can also be applied to assessing the quality and biological
meaning of affinity-constant measurements of transcription factors for DNA- or Protein-Protein
interactions. Some absolute quantification methods using targeted proteomics have been
described for mammalian cells and plant cells. However, gathering data using such methods is
expensive, making it prohibitive for experimental set ups in which dynamic data is required
(e.g.: inferring model parameters from time series in chronobiology applications). To overcome
these shortcomings, we developed a Nano Luciferase based toolkit and methodology for
performing absolute quantifications using recombinant NanoLUC and tagged lines. The steps
presented in this chapter are extensively exemplified in the Doctoral thesis “A Mathematical
Model in Absolute Units For the Arabidopsis Circadian Clock” (Urquiza Garcia 2018) and
preprint (Urquiza Garcia et al. 2024; to be published in Molecular Systems Biology, 2025). Here
we described steps to follow and tips for using NanoLUC for absolute quantification of a
protein of interest.