Samples of P26 (0.2 mg/ mL), Type I collagen (1 mg/mL) and P26-collagen mix were dissolved in HEPES buffer (10mM) t pH 7.15 and incubated for 30 min at RT. Circular Dichroism (CD) spectra were collected in high-transparency quartz cuvettes (250 μL) with a path length of 1 mm and band width of 2 nm at 25 °C in the far UV spectral range (190–260 nm) using a JASCO J-815 circular dichroic spectrometer. Conformational changes in the secondary structure of the peptide induced by collagen were investigated at physiological pH by subtracting the spectra of collagen alone.45 (link) All the CD spectra collected were normalized to mean residual ellipticity (MRE) values.
J 815 circular dichroic spectrometer
Manufactured by Jasco
Sourced in United States
The J-815 is a circular dichroic spectrometer, a laboratory instrument used to measure the circular dichroism of a sample. Circular dichroism is the difference in absorption of left-handed and right-handed circularly polarized light, which can provide information about the structure and conformation of molecules. The J-815 is designed to accurately measure circular dichroism across a wide range of wavelengths.
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Lab products found in correlation
3 protocols using j 815 circular dichroic spectrometer
1
Circular Dichroism Analysis of P26-Collagen Interactions
2
Probing Protein Secondary Structure
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Changes in the secondary structure of FDH as a function of pH and addition of divalent cations to the solution were investigated through CD measurements using a CD spectrometer (J-815 Circular Dichroic Spectrometer, JASCO, Easton, MD, USA) [66 (link)]. These measurements were carried out in a 0.1 cm cuvette at 25 °C using a final protein concentration of 0.15 mg mL-1, diluted in 50 mM NaH2PO4 at pH 4.5 (unfortunately we could not use NaAc because the –COOH groups adsorb the light so we needed to use a non-adsorbing medium to obtain reliable data [67 (link)]). Then, the concentration of CaCl2 or MgCl2 was increased in the range of 0–100 mM to receive information about any changes in the secondary structure. The obtained spectra were further fitted with a method called β-structure selection (BeStSel) that takes into account the twist of β-structures for estimation of the secondary structure. This method can reliably distinguish parallel and anti-parallel β-sheets and accurately estimate the secondary structure for a broad range of proteins [68 (link), 69 (link)]. Moreover, the secondary structure components applied by the method are characteristic to the protein fold, which in turn can be predicted to the level of topology in the CATH classification from a single CD spectrum.
3
Conformational Changes of Peptides
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Samples of P26 and P32 (0.2 mg/mL) were dissolved in 5
mM HEPES buffer at pH 7.4 for 5 min at RT. CD spectra were collected
in high-transparency quartz cuvettes with a path length of 1 mm and
band width of 2 nm at 25 °C in the far UV spectral range (190–250
nm) using a JASCO J-815 circular dichroic spectrometer. The experiments
were conducted using the peptides in the absence and presence of calcium
ions (3 and 10 mM CaCl2). Conformational changes in the
secondary structure of the peptides in the presence of bivalent calcium
ions were investigated at pH 7.4.
mM HEPES buffer at pH 7.4 for 5 min at RT. CD spectra were collected
in high-transparency quartz cuvettes with a path length of 1 mm and
band width of 2 nm at 25 °C in the far UV spectral range (190–250
nm) using a JASCO J-815 circular dichroic spectrometer. The experiments
were conducted using the peptides in the absence and presence of calcium
ions (3 and 10 mM CaCl2). Conformational changes in the
secondary structure of the peptides in the presence of bivalent calcium
ions were investigated at pH 7.4.
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