Dual-Channel Stopped-Flow Apparatus for Simultaneous Fluorescence, Anisotropy, and FRET Kinetic Data Acquisition for Binary and Ternary Biological Complexes

Abstract

The Stopped-Flow apparatus (SF) tracks molecular events by mixing the reactants in sub-millisecond regimes. The reaction of intrinsically or extrinsically labeled biomolecules can be monitored by recording the fluorescence, F(t), anisotropy, r(t), polarization, p(t), or FRET, F(t)FRET, traces at nanomolar concentrations. These kinetic measurements are critical to elucidate reaction mechanisms, structural information, and even thermodynamics. In a single detector SF, or L-configuration, the r(t), p(t), and F(t) traces are acquired by switching the orientation of the emission polarizer to collect the IVV and IVH signals however it requires two-shot experiments. In a two-detector SF, or T-configuration, these traces are collected in a single-shot experiment, but it increases the apparatus’ complexity and price. Herein, we present a single-detector dual-channel SF to obtain the F(t) and r(t) traces simultaneously, in which a photo-elastic modulator oscillates by 90° the excitation light plane at a 50 kHz frequency, and the emission signal is processed by a set of electronic filters that split it into the r(t) and F(t) analog signals that are digitized and stored into separated spreadsheets by a custom-tailored instrument control software. We evaluated the association kinetics of binary and ternary biological complexes acquired with our dual-channel SF and the traditional methods; such as a single polarizer at the magic angle to acquire F(t), a set of polarizers to track F(t), and r(t), and by energy transfer quenching, F(t)FRET. Our dual-channel SF economized labeled material and yielded rate constants in excellent agreement with the traditional methods.

A FLIM Microscopy Based on Acceptor-Detected Förster Resonance Energy Transfer

“…In this work, we present the equations and experimental strategies to observe trFRET-sensitized acceptor (A), creating the basis of a new type of FLIM, herein called FLIM-trADFRET, that increases the FRET resolution up to 300 Å (Figure A–D, Database S1). For trDDFRET and trADFRET, the rate of transfer ( k t ) and the FRET efficiency is the same in both cases (Figure E–J), − but the latter has unlimited photon accumulation and background noise reduction (red line, Figure G,H) allowing better signal–noise ratio (S/N) (Figure K,L). Our new approach is based on the photon accumulation of the trADFRE ⟨τ trADFRET Obs ⟩ over a reference lifetime ⟨τ Std ⟩ that has been acquired with a standard solution (Figure J,K).…”