For biophysical validation, Myc910–960 peptides were purchased from Thermo Fisher Scientific with 90% purity. The fluorescence lifetime of Trp in Myc peptide was measured using the DeltaFlex TCSPC system (Horiba Scientific). A quartz cuvette of 1 cm × 1 cm and 1 ml volume were used to record the spectra. The excitation monochromator's wavelength was set up at 284 nm, and the emission monochromator at 345 nm. The measurement range was set up to 200 ns with 32 nm of bandpass and peak preset of 10,000 counts. LUDOX was used to correct the instrument response factor at 284-nm wavelength. 7.5 μM Myc peptide sample with L755507 and 10074-G5 was prepared in 50 mM sodium phosphate buffer (pH 7) and incubated for 10 min at 25 °C before taking the measurement. The decay curve was fitted into a biexponential decay function using Data Analysis Software provided by Horiba Scientific.
Deltaflex tcspc system
Manufactured by Horiba
The DeltaFlex TCSPC system is a time-correlated single-photon counting (TCSPC) instrument designed for time-resolved fluorescence measurements. It provides high-resolution temporal analysis of photon arrival times, enabling the study of various photophysical and photochemical processes. The DeltaFlex system is capable of performing time-resolved fluorescence spectroscopy and lifetime measurements.
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Lab products found in correlation
Data analysis software, by Horiba (2 mentions)
Uv 2501pc spectrophotometer, by Shimadzu (1 mentions)
Uv 3600 plus spectrophotometer, by Shimadzu (1 mentions)
Vnmrs 600 spectrometer, by Agilent Technologies (1 mentions)
Rf 6000 spectrofluorometer, by Shimadzu (1 mentions)
Invia system, by Renishaw (1 mentions)
7 protocols using deltaflex tcspc system
1
Fluorescence Lifetime Analysis of Myc Peptide
2
Tryptophan Fluorescence Lifetime Analysis
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The fluorescence lifetime of tryptophan residue (Trp161) in NSP1-CTR in absence and presence of MTX was measured using the Horiba Scientific DeltaFlex TCSPC system at 25 °C. The samples were excited at 284 nm using the nanoLED as a light source. The wavelength of the emission monochromator was set at 346 nm. The measurement range was adjusted to 400 ns with bandpass of 32 nm and peak preset of 10 000 counts. Ludox was used to correct the instrument response factor (IRF) at 284 nm. The concentration of NSP1-CTR was kept constant at 8 μM and in presence of 50 mM sodium phosphate (pH 7.4) buffer. Finally, the three exponential decay function fitting was used to obtain the tryptophan lifetimes.
3
Time-Resolved Anisotropy Decay Analysis
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To confirm the binding of NSP1-CTR and MTX, time-resolved anisotropy decay was measured using DeltaFlex TCSPC system (Horiba Scientific) at 25 °C. A 1 ml pathlength quartz cuvette was used to record the measurements where the measurement range was set up to 200 ns. The bandpass of 16 nm, peak preset of 1000 counts, and repetition rate of 1 MHz were used during experimentation. The protein concentration was kept constant at 30 μM. At 284 nm wavelength, Ludox was used for adjusting the instrument response factor (IRF). Using the data analysis software DAS6 (Horiba Scientific), the resultant anisotropy decay curves were fitted into a bi-exponential decay function:22 ![]()
where, anisotropy at time t is denoted by r(t) and initial anisotropy is denoted by r0, and A1 and A2 defines the rotational correlation time θ1 and θ2, respectively.
4
Time-Resolved Optical Characterization
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The time-resolved measurements
were obtained using a gated iCCD camera (250-950 nm) system, and for
the temperature-dependent measurements, a helium-closed cycle cryopump,
with optical windows, Si thermodiode, and sample mount, attached directly
to the cold head. TCSPC measurements were recorded with a Horiba DeltaFlex
TCSPC system using a Horiba NanoLED 357 nm and SpectraLED 330 nm as
light sources.
were obtained using a gated iCCD camera (250-950 nm) system, and for
the temperature-dependent measurements, a helium-closed cycle cryopump,
with optical windows, Si thermodiode, and sample mount, attached directly
to the cold head. TCSPC measurements were recorded with a Horiba DeltaFlex
TCSPC system using a Horiba NanoLED 357 nm and SpectraLED 330 nm as
light sources.
5
Optical and Spectroscopic Characterization of Materials
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UV-visible spectroscopy measurements were performed using a Shimadzu UV-2501PC spectrophotometer and Shimadzu UV-3600 Plus spectrophotometer using quartz cuvettes. Photoluminescence spectroscopy was performed using Cary Eclipse spectrophotometer and a Shimadzu RF-6000 spectrofluorometer. Raman spectra were acquired using an NT-MDT NTEGRA Spectra system with 473 nm laser excitation and Renishaw inVia system with 532 nm laser excitation. The photoluminescence background was subtracted using spine interpolation and the spectra were then normalized to the Raman mode at ~2900 cm−1. Time-resolved photoluminescence measurements were performed with a Horiba DeltaFlex TCSPC system with excitation at 336 nm, 349 nm and 409 nm using a 6 nm bandpass. 1H NMR spectroscopy was performed on a Varian VNMRS 600 spectrometer operating at a 1H frequency of 599.7 MHz.
6
Anisotropy Decay Measurement of Protein-Compound Interactions
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The anisotropy decay was measured using the DeltaFlex TCSPC system (Horiba Scientific). A quartz cuvette of 1 cm × 1 cm and 1 ml volume were used to record the spectra. The measurement range was set up to 200 ns with 16 nm of bandpass, peak preset of 1000 counts, and repetition rate of 1 MHz. The protein sample (20 μM Myc) with compound L755507 was incubated for half an hour at 25 °C before taking the measurement. LUDOX was used for correcting the instrument response factor at 284-nm wavelength. The anisotropy decay curve was fitted into two exponential decay function using Data Analysis Software provided by Horiba Scientific. The anisotropy decay curve can be expressed by the following equation (33 ). Where, r(t) is the anisotropy at time t, r0 is the intrinsic or initial anisotropy, A1 and A2 are the amplitude associated with the rotational correlation time and , respectively.
7
Tyr Fluorescence Lifetime Measurement
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The fluorescence lifetime of Tyr in the E-CTD was measured using the DeltaFlex TCSPC system (Horiba Scientific). The wavelengths for excitation monochromator was set up at 280 nm and emission monochromator at 310 nm. The measurement range was set up to 200 ns with 32 nm of bandpass and a peak preset of 10000 counts. Ludox was used to correct the instrument response factor (IRF), and the wavelength for prompt measurement was set up at 280 nm. 20 µM protein sample was prepared in 50 mM sodium phosphate buffer at pH 7.4.
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