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A new ELISA for quantifying Na,K-ATPase proteins across a wide range of taxa and sample sources
  • Jana Löptien,
  • Susanne Dobler,
  • Shabnam Mohammadi
Jana Löptien
Universität Hamburg
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Susanne Dobler
Universität Hamburg
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Shabnam Mohammadi
University of Nebraska-Lincoln

Corresponding Author:[email protected]

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Abstract

Large transmembrane proteins are difficult to quantify with traditional methods due to their low binding affinity to traditional assay dyes (e.g., Bradford and Lowry assays). Enzyme-linked immunosorbent assays (ELISAs) provide higher quantification specificity for such proteins. An ELISA can detect a specific protein in a heterogenous sample. However, commercially available ELISAs are limited to a few model organisms and are highly dependent on the source of the protein and the isolation method used. Here we provide a solution to these problems with a versatile and readily reproducible indirect ELISA that relatively quantifies the transmembrane protein Na,K-ATPase (NKA). The NKA has become a model for studying the evolutionary ecology of plant-herbivore and predator-prey interactions, but functional studies of the proteins have been hindered by the lack of robust quantification methods. In our ELISA, the antigen is directly coated to the surface of a well in a 96-well plate, the primary antibody is used for detection, and an enzyme-linked secondary antibody is used for signal amplification and colorimetric detection. Two factors make this ELISA highly applicable across a wide range of species and protein sources: (1) the use of a commercially available antibody that binds universally across the animal kingdom and (2) the production of a malleable relative standard that can be adapted to various sample types (species and protein source). Comparisons of in vitro enzyme assays performed based on traditional protein quantification methods vs. our ELISA reveal that the ELISA consistently provides more robust NKA quantification necessary for subsequent functional data.