Journal: Science Advances

Article Title: Multiplexed mapping of the interactome of GPCRs with receptor activity–modifying proteins

doi: 10.1126/sciadv.ado9959

Figure Lengend Snippet: ( A ) An SBA assay experimental workflow was developed ( , ). Abs were grouped by the phylogenetic subfamily of their GPCR target and were coupled to unique color-coded beads and pooled to generate six subfamily-specific SBAs . A library of DuET GPCRs and RAMPs was expressed pairwise. Cells were solubilized to create heterogeneous mixtures of proteins, and concentrations were normalized across samples before incubation of aliquots with the SBAs . PE-conjugated anti-1D4 or anti-OLLAS mAbs were used to detect the GPCR-RAMP complexes captured by the Ab-coupled beads . The data were collected on a Luminex FlexMap three-dimensional (3D) instrument and processed to identify GPCR-RAMP complexes. Results were integrated into an interactive web interface . ( B ) The GPCR-RAMP complex capture and detection schemes are shown schematically. In all cases, the reporter fluorescence produced by the PE-conjugated detection Ab was associated with the bar code of each bead. From a single well, GPCR-RAMP complexes could be detected simultaneously using anti-epitope tag Abs, anti-GPCR Abs, or anti-RAMP Abs. ( C ) Data analysis workflow. Data generated as described in (A) were first tested for GPCR or RAMP expression, then the fluorescence intensity data were normalized, and threshold values were calculated. Adapted from . Created in Biorender.com .

Article Snippet: The fluorescence associated with each bead was measured using a Luminex FlexMap 3D instrument (Luminex Corp., xPONENT Software, build 4.3.309.1).

Techniques: Incubation, Luminex, Fluorescence, Produced, Generated, Expressing