mtq2  (Addgene inc)

Journal: eLife

Article Title: Differential interaction patterns of opioid analgesics with µ opioid receptors correlate with ligand-specific voltage sensitivity

doi: 10.7554/eLife.91291

Figure Lengend Snippet: ( A ) Representative Förster resonance energy transfer (FRET)-based single-cell recording of µ opioid receptor (MOR)-induced Gα i activation under voltage clamp conditions used for fit in and , the voltage protocol indicated below (mean ± SEM, n=4). ( B ) Representative FRET-based single-cell recording of MOR-induced Gα o activation under voltage clamp conditions used for fit in C, the voltage protocol indicated below (mean ± SEM, n=7). ( C ) Voltage dependence of naloxone-induced Gα i activation (blue) compared to Gα o activation (magenta). Activation was determined by clamping the membrane from –90 mV to different potentials and plotted relative to 0 mV. Data was fitted to Boltzmann function resulting in a z-factor of 1.17 for Gα i and 1.2 for Gα o and a V 50 -value of 31 mV for Gα i and 27 mV for Gα o . ( D ) Average FRET-based single-cell recording of arrestin-mTur2 interaction with MOR-sYFP2 under voltage clamp conditions, the voltage protocol indicated below (mean ± SEM, n=7). ( E ) Average FRET-based single-cell recording of arrestin-mTur2 interaction with MOR-sYFP2 induced by the weak partial agonist tramadol (mean ± SEM, n=5).

Article Snippet: Gβ 1 -2A-cpV-Gy 2 -IRES-Gα i2 -mTur2 was purchased from Addgene (Watertown, MA, USA, plasmid #69624; ).

Techniques: Förster Resonance Energy Transfer, Activation Assay, Membrane

Journal: STAR Protocols

Article Title: Protocol for deriving proximity, affinity, and stoichiometry of protein interactions using image-based quantitative two-hybrid FRET

doi: 10.1016/j.xpro.2023.102459

Figure Lengend Snippet: Expected outcomes: Spectral correction factors and FRET calibration (A) Determination of spectral correction factors: R A1 : Plot of fluorescence in the FRET channel (y-axis) vs. fluorescence in the YFP channel (x-axis). R A1 describes cross excitation of YFP by CFP excitation (FRET/YFP). R D1 : Plot of fluorescence in the FRET channel (y-axis) vs. fluorescence in the CFP channel (x-axis). R D1 describes the bleed-through of CFP emission into the FRET emission detection range (FRET/CFP). R D2 : Plot of fluorescence in the YFP channel vs. the fluorescence in the CFP channel. R D2 describes cross excitation of CFP from the YFP excitation (YFP/CFP). These spectral correction factors are determined by linear regressions between the specified channels. (B) Correlating sensitized emission versus donor quenching displays an affine relationship between these quantities when using dimer constructs. The G ratio corresponds to the slope of a linear regression line, while the F ratio is calculated from the y-axis offset. (C) Left: cartoon of the dimer constructs. Right: Plot of FRET efficiencies determined from donor quenching ( E D ) vs. A free for the shown dimers. FRET calibration results in stable FRET efficiencies independent of the fluorophore concentration ( A free or D free ). The plot for sensitized emission ( E A ) should look similar. All calibration images were processed with the “FRET_PIX” macro instead of the “FRET_ROI” macro.

Article Snippet: Spectral correction R A1 , R D1 , R D2 , mTq2 (donor only) mVen (acceptor only) , mTq2 mVen , AddGene#198196 AddGene#198192.

Techniques: Fluorescence, Construct, Concentration Assay

Journal: STAR Protocols

Article Title: Protocol for deriving proximity, affinity, and stoichiometry of protein interactions using image-based quantitative two-hybrid FRET

doi: 10.1016/j.xpro.2023.102459

Figure Lengend Snippet: Expected outcomes: Spectral correction factors and FRET calibration (A) Determination of spectral correction factors: R A1 : Plot of fluorescence in the FRET channel (y-axis) vs. fluorescence in the YFP channel (x-axis). R A1 describes cross excitation of YFP by CFP excitation (FRET/YFP). R D1 : Plot of fluorescence in the FRET channel (y-axis) vs. fluorescence in the CFP channel (x-axis). R D1 describes the bleed-through of CFP emission into the FRET emission detection range (FRET/CFP). R D2 : Plot of fluorescence in the YFP channel vs. the fluorescence in the CFP channel. R D2 describes cross excitation of CFP from the YFP excitation (YFP/CFP). These spectral correction factors are determined by linear regressions between the specified channels. (B) Correlating sensitized emission versus donor quenching displays an affine relationship between these quantities when using dimer constructs. The G ratio corresponds to the slope of a linear regression line, while the F ratio is calculated from the y-axis offset. (C) Left: cartoon of the dimer constructs. Right: Plot of FRET efficiencies determined from donor quenching ( E D ) vs. A free for the shown dimers. FRET calibration results in stable FRET efficiencies independent of the fluorophore concentration ( A free or D free ). The plot for sensitized emission ( E A ) should look similar. All calibration images were processed with the “FRET_PIX” macro instead of the “FRET_ROI” macro.

Article Snippet: Spectral correction R A1 , R D1 , R D2 , mTq2 (donor only) mVen (acceptor only) , mTq2 mVen , AddGene#198196 AddGene#198192.

Techniques: Fluorescence, Construct, Concentration Assay