Fluorescence measurements were carried out with a Jasco Spectrofluorometer FP-8200, equipped with a Jasco MCB-100 Mini Circulation Bath (20°C, xenon lamp, data interval 0.5 nm, accumulation number 3). The excitation wavelength was 280 nm, fluorescence emission was recorded at 350 nm to exclude self-fluorescence of non-bound peptide. For the measurements, GCAP-2 was mixed with increasing concentrations of ROS-GC peptides in 20 mM HEPES (pH 7.5). As a negative control, separate recordings were performed with N-acetyl-L-tryptophanamide or N-acetyl-L-tyrosinamide, respectively. The composition of the controls correlates with the peptides' composition of tryptophan and tyrosine residues to exclude that a change in fluorescence intensity results from self-fluorescence of the peptides.
Mcb 100 mini circulation bath
Manufactured by Jasco
Sourced in Japan
The MCB-100 Mini Circulation Bath is a compact laboratory equipment designed for temperature-controlled liquid circulation. It features a stainless steel tank with a capacity of 1.5 liters and can maintain temperatures between -20°C to 100°C.
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Lab products found in correlation
5 protocols using mcb 100 mini circulation bath
1
Fluorescence-based Peptide Binding Assay
2
Thermal Stability of Apo-Calmodulin
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Sensitivity of apo-CaM (Ca2+-free) to temperature was assessed by decrease in α-helical content measured by CD. Ellipticity at 222 nm for CaM variants was recorded in a 0.1 cm path length quartz cell using a JASCO J-1100 spectrometer equipped with a JASCO MCB-100 mini circulation bath for temperature control. Purified proteins (10 μM) were measured in 2 mM Hepes (pH 7.5) supplemented with 1 mM EGTA. Temperature ranged from 20 °C to 80 °C in 2 °C increments, with a ramp increase rate of 1 °C/min and a 180s equilibration period between recordings. Data were normalized and fitted to the Boltzmann sigmoid equation (GraphPad Prism) to derive the melting temperature of CaM (Tm).
3
Thermal Melting Profile Characterization
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Thermal melting profiles were performed [20 (link)] with a temperature range from 25–95 °C at the rate of 1 °C/min using a Jasco J-1500 CD Spectrometer (Jasco, Tokyo, Japan) equipped with a MCB-100 Mini circulation Bath, Peltier unit-controlled cell holder, and a xenon lamp.
4
Measuring Rec1-resilin Secondary Structure in Crowding
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The secondary structure of Rec1-resilin in the crowding environment was measured using CD spectroscopy. CD spectra were recorded (at 25°C) in the wavelength range 190 to 260 nm using a JASCO spectropolarimeter (Jasco J-150, Jasco, Japan) connected to a Jasco MCB-100 Mini Circulation Bath. A UV quartz cuvette with a path length of 1 mm was used for all the measurements. The protein concentration used was 0.1 mg/ml, and CD spectra were measured in 1 mM PBS buffer and crowded conditions (at concentrations comparable to those used in the SANS experiment). Before measurements, all systems were stirred (30 min), centrifuged (12,000g for 2 min), and left to equilibrate for 1 hour. The data pitch was set at 0.2 nm, at a scanning speed of 20 nm/min, a bandwidth of 0.2 nm, a digital integration time (DIT) of 1 s, and an accumulation of 10 scans. CD data were analyzed using the CDPro software package. The online fitting program interface Dichroweb was used to extract secondary structure information from CD spectra (53 (link)).
5
Spectroscopic Analysis of Biomolecular Interactions
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Solutions of CT-DNA (Sigma Aldrich) in phosphate buffer (650 μL, 1 mM Na 2 EDTA, 10 mM Na 2 HPO 4 , 20 mM NaCl, pH 7.0) gave a ratio of UV absorbances at 260 and 280 nm, A 260 /A 280 , of 1.80-1.90, indicating that the DNA was sufficiently free from protein. 27 The concentration of CT-DNA stock solution was determined from UV absorption at 260 nm. The circular dichroism spectral titration experiments were performed by keeping the CT-DNA concentration constant (2.7 mM bases) while varying the concentration of metal complexes from 0 to 39 μM. The CD spectra were measured in 1 mm path length quartz cuvettes on a JASCO J-815 circular dichroism spectrometer. Two scans were accumulated at a scan speed of 100 nm min -1 . All CD spectra were recorded at every 0.5 nm from 200 to 350 nm. Sample temperature was maintained at 35 °C using a JASCO MCB-100 mini-circulation bath. Spectra were corrected for buffer signal.
The baker's yeast RNA (Sigma Aldrich) stock solution was also prepared in phosphate buffer in DEPC (Sigma Aldrich) treated water. The A 260 /A 280 value was 2.10, indicating the RNA was pure. 28 The initial RNA concentration was 2.3 mM (bases). The titration of ruthenium complexes and the data collection were the same as indicated for the DNA experiments.
The baker's yeast RNA (Sigma Aldrich) stock solution was also prepared in phosphate buffer in DEPC (Sigma Aldrich) treated water. The A 260 /A 280 value was 2.10, indicating the RNA was pure. 28 The initial RNA concentration was 2.3 mM (bases). The titration of ruthenium complexes and the data collection were the same as indicated for the DNA experiments.
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