Purified protein was diluted to 1:3 times with 5 mM phosphate buffer, pH 7.0, making a final volume of 800 μL. The protein was analyzed on FP-8000 (Jasco, Easton, MD, USA). 10 µM protein was used for analysis using a path length of 10 mm.
Fp 8000
Manufactured by Jasco
Sourced in United States
The FP-8000 is a fluorescence spectrometer designed for analytical applications. It features a wide wavelength range, high sensitivity, and advanced optics for accurate and reliable measurements. The FP-8000 is suitable for a variety of sample types and is a versatile tool for researchers and laboratories.
Automatically generated - may contain errors
4 protocols using fp 8000
1
Protein Dilution and Analysis
2
Recombinant Indicator Protein Purification
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Recombinant indicators, N-terminally tagged with polyhistidine, were expressed in E. coli (JM109(DE3)) for 4-5 days at 22 °C with gentle shaking at 130 rpm. The E. coli cells were lysed using B-PER. Subsequently, the recombinant indicator was purified using a Ni-NTA column (Wako) and underwent buffer exchange employing a Microsep Advance Centrifugal Device with a 30 K MWCO (PALL). The buffers used for the purified indicators were exchanged with the appropriate ones for GECIs 6 , iGluSnFR 31 , and GINKO 32 . Protein concentrations were determined as previously reported 13 . Fluorescence spectra were recorded using a fluorescence spectrophotometer (F-7000, Hitachi), and absorption spectra were recorded using a microplate reader (SpectraMax iD3, Molecular Devices). For kinetic analysis, the dissociation dynamics were measured using a stopped-flow device (SFS-853 and FP-8000, JASCO), as previously reported 9 . Briefly, fluorescence signal changes by depletion of Ca 2+ from the indicators through rapid mixing with 10 mM EGTA were recorded every 5 ms up to 30 s, then were fitted to a single exponential curve to determine the k off value using OriginPro 9 software (LightStone). The k on value was calculated using the independently determined K d through the following equation:
3
Characterization of Quantum Dots
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QDs were characterized using a JEM-2100 (JEOL, Japan) transmission electron microscope (TEM), and a D8 Advance (Bruker, Germany) X-ray diffractometer (Cu Kα). Samples were prepared by drying sample droplets from water dispersion onto a 100-mesh Cu grid coated with a lacey carbon film, which was dried prior to imaging by TEM. Absorption and emission spectra were collected using fluorescence spectroscopy (JASCO FP-8000).
4
Tryptophan Fluorescence Quenching Assay
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Fluorescence measurements were obtained by monitoring the changes in intrinsic fluorescence emission of the protein samples in a 10 mm path length quartz cell using a Jasco FP-8000 spectrofluorimeter equipped with thermostatically controlled cell holder (25 C). The MtgA mutants (concentration ~0.26 mM) in the buffer 20 mM Tris-HCl pH 8, 500 mM NaCl, CHAPS 0.7% were titrated with increasing concentration of ligands (MoeA, or compounds 44 or 62) and the quenching of tryptophan fluorescence emission spectra were recorded between 305 et 450 nm, following excitation at 295 nm. Each spectrum was the average of 3 acquisitions and was corrected for the contribution of the experimental solution without protein. The data from the titration experiment of the mutant Y181W by MoeA or substrate analogs were analyzed using nonlinear regression in Graphpad Prism 6 software.
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