Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells.

ABSTRACT: Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application

SUBMITTER: Nederveen-Schippers LM 

PROVIDER: S-EPMC8307594 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature