Absolute protein quantification using fluorescence measurements with FPCountR.

ABSTRACT: This paper presents a generalisable method for the calibration of fluorescence readings on microplate readers, in order to convert arbitrary fluorescence units into absolute units. FPCountR relies on the generation of bespoke fluorescent protein (FP) calibrants, assays to determine protein concentration and activity, and a corresponding analytical workflow. We systematically characterise the assay protocols for accuracy, sensitivity and simplicity, and describe an 'ECmax' assay that outperforms the others and even enables accurate calibration without requiring the purification of FPs. To obtain cellular protein concentrations, we consider methods for the conversion of optical density to either cell counts or alternatively to cell volumes, as well as examining how cells can interfere with p

SUBMITTER: Csibra E 

PROVIDER: S-EPMC9633595 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature