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

Biosensors ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 180
Author(s):  
Roberto F. Delgadillo ◽  
Katie A. Carnes ◽  
Nestor Valles-Villarreal ◽  
Omar Olmos ◽  
Kathia Zaleta-Rivera ◽  
...  

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.

1979 ◽  
Vol 50 (9) ◽  
pp. 1104-1107 ◽  
Author(s):  
Muneo Sasaki ◽  
Fujitsugu Amita ◽  
Jiro Osugi

2006 ◽  
Vol 91 (12) ◽  
pp. 4605-4610 ◽  
Author(s):  
Bálint Kintses ◽  
Zoltán Simon ◽  
Máté Gyimesi ◽  
Júlia Tóth ◽  
Balázs Jelinek ◽  
...  

2006 ◽  
Vol 60 (12) ◽  
pp. 1477-1481 ◽  
Author(s):  
Jia Tang ◽  
Feng Gai
Keyword(s):  

1989 ◽  
Vol 60 (7) ◽  
pp. 2356-2358 ◽  
Author(s):  
Hirotsugu Tsuruta ◽  
Toshihiko Nagamura ◽  
Kazumoto Kimura ◽  
Yoshihiko Igarashi ◽  
Akihiko Kajita ◽  
...  

1974 ◽  
Vol 5 (28) ◽  
pp. no-no
Author(s):  
PAOLO PAPOFF ◽  
BASILIO MORELLI ◽  
LEONARDO LAMPUGNANI
Keyword(s):  

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