Fp 6500 spectrofluorimeter

PL measurements were performed by incubating 5 μM NS samples (with or without EMB incubation) on a Jasco FP-6500 spectrofluorimeter using a quartz cuvette with a 1.5 mm optical path. The temperature was controlled within 0.05 °C with a temperature-controlled recirculating bath. The normalised emission bands PL(λ) were measured with a 275 nm excitation wavelength, buffer subtracted and normalised by the total PL area. The first momentum λ₁ of the emission band was calculated as λ₁ = ∫ λ PL(λ)dλ.

Fluorescence analysis was performed using a JASCO FP-6500 spectrofluorimeter at 25°C with 1-cm quartz cells.

Fluorescence spectroscopy was performed on a FP-6500 spectrofluorimeter (Jasco). Spectra were recorded by exciting the protoporphyrin IX soret band at 410 nm for the in vitro conversion of protoporphyrin IX into heme b by Fra/HemH. The emission peak analysed was 630 nm. The bandwidth for the excitation and emission was set to 5 nm, the sensitivity to medium and the response to 0.5 s. The heme emission band at 450 nm was analysed upon soret band excitations at 380 nm for the estimation of the heme concentration in crude cell extract. In this case, the bandwidth for the excitation and emission was set to 3 nm, the sensitivity to high and the response to 0.5 s. All spectra were recorded at 20°C.

The Brookhaven LS measurements were complemented by measuring the area underneath the elastic peak obtained in the fluorescence spectrum of the vesicle dispersions. In fact, when particles are dissolved in a solution and hit by radiation, they produce elastic scattering radiation in all directions. Like in a traditional light scattering setup, the intensity of the elastically diffused light will be proportional to the mass of the particles and to their concentration. The elastically diffused light can be measured by using a spectrofluorimeter, which is a quite common spectroscopy lab facility, by simply setting equal values for the excitation and the emission wavelength [26 ]. Therefore, the same samples and dilutions used in the Brookhaven setup were used in a JASCO FP-6500 spectrofluorimeter, and the area underneath the elastic peak obtained with a 532 nm excitation wavelength, 1 nm excitation and emission slits and 500 V PMT voltage was taken as a measure of the light scattered by vesicle dispersions. Each experiment was performed in triplicate.

Emission spectra of COS-7 cell lysates or YFP purified from E. coli cultures were performed at 25 °C on a Jasco FP-6500 spectrofluorimeter equipped with a thermostated cell. A 3 mm path cuvette sealed with Teflon caps was used. The spectral slit-widths were set to 3 nm for each monochromator. The excitation wavelength was set to 480 nm and emission spectra were collected in the range 500–560 nm.

Time-dependent bulk fluorescence intensities of Hsp90 were measured in a quartz glass cuvette using a FP-6500 spectrofluorimeter (Jasco, Spectra Manager version 2). Wavelengths of fluorescence excitation/emission were set to 542/562 nm or 646/668 nm, for Hsp90 samples modified with Atto542 or JF646, respectively. A Peltier thermocouple was used to adjust the sample temperature to 25 °C. Hsp90 samples were prepared in assay buffer supplemented with 0.3 mg/ml BSA and 0.05% Tween 20 to suppress glass surface interactions. 150 nM fluorescently modified Hsp90 was reacted with 5 µM non-labelled wild-type or mutant Hsp90 for 30 min at room temperature, prior to measurement, in order to ensure that only one subunit in dimeric Hsp90 constructs carried a fluorophore. Closure of the Hsp90 molecular clamp was initiated by adding 2 mM AMP-PNP. Fluorescence intensity time traces were fitted using a bi-exponential function.
Fluorescence quenching experiments were carried out using 150 nM fluorophore in 50 mM phosphate, pH 7.5, with the ionic strength adjusted to 200 mM using potassium chloride, and at 25 °C. The concentration of tested compounds was 25 mM each.