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.

See 'Methods' section in the original paper.

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.