A novel and robust method for counting components within bio-molecular complexes using fluorescence microscopy and statistical modelling

Abstract

Cellular biology occurs through myriad interactions between diverse molecular components, many of which assemble in to specific complexes. Various techniques can provide a qualitative survey of which components are found in a given complex. However, quantitative analysis of the absolute number of molecules within a complex (known as stoichiometry) remains challenging. Here we provide a novel method that combines fluorescence microscopy and statistical modelling to derive accurate molecular counts. We have devised a system in which batches of a given biomolecule are differentially labelled with spectrally distinct fluorescent dyes (label A or B), and mixed such that B-labelled molecules are vastly outnumbered by those with label A. Complexes, containing this component, are then simply scored as either being positive or negative for label B. The frequency of positive complexes is directly related to the stoichiometry of interaction and molecular counts can be inferred by statistical modelling. We demonstrate this method using complexes of Adenovirus particles and monoclonal antibodies, achieving counts that are in excellent agreement with previous estimates. Beyond virology, this approach is readily transferable to other experimental systems and, therefore, provides a powerful tool for quantitative molecular biology.

Trim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates

“…We used two model substrates to assay for TRIM21 function; Adenovirus 5 (Adv5) targeted with an anti-Hexon antibody (Adv5 neutralization) and GFP-tagged Caveolin-1 (CAV1-mEGFP) targeted with an anti-GFP antibody (Trim-Away). The multi-epitope (Adv5 59 , 60 ) and oligomeric (CAV1 61 ) nature of these substrates induce TRIM21 clustering and its activation 15 . For the Trim-Away assay, either R-R-PS or Ac-R-R-PS were electroporated together with anti-GFP antibody into cells expressing CAV1-mEGFP and the kinetics of degradation monitored over time (Fig.…”