DOI: 10.5061/dryad.9ghx3ffgs
title: Protein quantification in ecological studies: a literature review and empirical comparisons of standard methodologies

description: 1. Protein quantification is a routine procedure in ecological studies
 despite the inherent limitations of well-acknowledged protein
 determination methods which have been largely overlooked by ecologists.
 Thus, we want to bridge this knowledge gap, in hopes of improving the way
 ecologists quantify proteins and interpret findings. 2. We surveyed the
 ecological literature to determine how and why ecologists quantify
 proteins. To determine whether different quantification methods produce
 comparable results across taxa, and between populations of a single
 species, we estimated the protein content of eight phylogenetically
 diverse taxa, and of desert isopods fed different diets, using various
 derived protocols of the ‘crude protein’, Bradford and BCA methods. 3. We
 found that ecologists use many protein quantification procedures, often
 without reporting the crucial information needed to evaluate and repeat
 their methods. Our empirical work demonstrated that the three
 quantification methods examined, and their derived protocols, resulted in
 highly divergent protein estimations that were inconsistent in rank across
 taxa, preventing conversion between methods. We also found that different
 quantification methods yielded different answers to whether isopod protein
 content is affected by diet. 4. We conclude that commonly used
 quantification techniques yield distinct protein estimations with varying
 precision, and no single method is likely to be more accurate than another
 across taxa which may lead to inconsistent results across taxa and between
 conspecifics. Inaccurate protein quantification may explain the observed
 mismatch between organismal N and protein that has plagued some recent
 studies and that contradicts the principles of ecological stoichiometry.
 We recommend using a single BCA protocol to reduce inconsistencies across
 studies, until the promising Amino Acid Analysis becomes more affordable,
 accurate, and accessible to ecologists. Until then, ecologists should
 consider the above-mentioned drawbacks of protein quantification methods
 and interpret their results accordingly.
description: See 'Methods' section in the original paper.
description: Files are separated based
 on the experiment ('Multi-taxa' and 'Conspecifics')
 and the quantification method ('Crude protein, Bradford and BCA), and
 the two are specified in the file name. Crude protein files (for both
 experiments) – Contains columns that specify the sample, a column with the
 measured nitrogen content (% from dry wt.) and a column with the
 calculated Crude protein based on a 6.25 Nitrogen-to-protein
 factor. Bradford
 & BCA files – Multi-taxa experiment: The first
 sheet describes the order in which the 96-well plates were organized. The
 sheet also contains: 1) the concentration of the standard proteins in each
 well, 2) the weight used for extraction for each sample, and 3) the time
 it took us to handle the reagents (i.e., the time since adding the reagent
 until the first wavelength reading) and accordingly what measurement out
 of the 12 was used for calculation (relates only to Bradford). All the
 additional data needed to calculate the protein content is found in the
 'Methods' section of the paper. The following ten sheets contain
 the ten plates' wavelength readings (i.e., ten replicates) throughout
 time. Conspecifics experiment: For each
 of the four plates, a sheet with the plate's order and data about its
 samples is followed by a sheet with the wavelength readings.