NADH oxidoreductase activity was deliberated in isolated mitochondria by the method of King and Howard, 1967. The reaction mixture contained 0.2 M glycylglycine buffer pH 8.5, 6 mM NADH in 2 mM glycylglycine buffer and 10.5 mM cytochrome c. Reaction was started with the addition of isolated mitochondrial sample in required quantity. Absorbance change was taken after 30 sec interval at 550 nm for 2 min using Perkin Elmer Lambda 20 spectrophotometer. NADH oxidoreductase activity was expressed as μmole of NADH oxidized per minute per milligram of protein. Absorbance index used cytochrome c (reduced minus oxidised) which is 19.2 mM−1 × cm−1 at 550 nm:
Lambda 20 spectrophotometer
Manufactured by PerkinElmer
Sourced in United States
The Lambda 20 spectrophotometer is a compact and versatile instrument designed for a wide range of UV-Vis spectroscopy applications. It features a high-performance double-beam optical system, a wavelength range of 190-1100 nm, and a photometric range of -4 to 4 Abs. The instrument provides accurate and reliable measurements with a spectral bandwidth of 1 nm.
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Lab products found in correlation
J 800 series spectropolarimeter, by Jasco (1 mentions)
6520 accurate mass q tof lc ms, by Agilent Technologies (1 mentions)
Cary eclipse fluorescence spectrophotometer, by Agilent Technologies (1 mentions)
Dihydrofolic acid, by Merck Group (1 mentions)
Genelute hp plasmid miniprep kit, by Merck Group (1 mentions)
T4 rna ligase, by Promega (1 mentions)
Bis acrylamide, by Merck Group (1 mentions)
35s methionine, by Cytiva (1 mentions)
Pyruvate kinase, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Ammonium persulfate, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Acrylamide, by Merck Group (1 mentions)
Erythromycin, by Merck Group (1 mentions)
810 spectropolarimeter, by Jasco (1 mentions)
Aβ42 peptide, by Bachem (1 mentions)
Ls 50b luminescence spectrophotometer, by PerkinElmer (1 mentions)
38 protocols using lambda 20 spectrophotometer
1
NADH Oxidoreductase Activity in Mitochondria
2
Recombinant alpha-synuclein purification
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Expression and purification of recombinant aS and aS variant were obtained as previously described (29 (link), 35 (link)). Protein concentrations were determined by absorption measurements at 280 nm using a double-beam Lambda-20 spectrophotometer from PerkinElmer Life Sciences. The extinction coefficient for aS and H50Q at 280 nm was 5960 m −1 cm−1 (61 (link)). DHA and AA were purchased from Sigma. All other chemicals were of analytical reagent grade and were obtained from Sigma or Fluka (Buchs, Switzerland). Aliquots of DHA and AA were stored at a concentration of 76 mm in 100% ethanol at −80 °C under a helium atmosphere to prevent oxidation. Fibrils were obtained after 2 weeks of incubation of aS in PBS buffer, pH 7.4, using a protein concentration of 70 μm , at 37 °C under shaking (500 rpm). To isolate fibrils from monomers, the protein sample was ultracentrifuged at 380,000 × g for 1 h at 4 °C.
3
Quantitative Measurement of Lipid Peroxidation in Brain
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The quantitative measurement of LPO in brain was performed according to the method of Wills.[12 (link)] The amount of malondialdehyde (MDA), a measure of LPO was measured by reaction with thiobarbituric acid at 532 nm using Perkin Elmer lambda 20 spectrophotometer (Norwalk, CT, USA). The values were calculated using molar extinction coefficient of chromophore (1.56 × 105/M/cm) and expressed as nanomoles of MDA per milligram of protein.
4
Spectroscopic Analysis of Protein Structure
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Protein concentrations were determined by absorption measurements at 280 nm using a double‐beam Lambda‐20 spectrophotometer (Perkin Elmer Life Sciences). The molar absorptivity at 280 nm for Syn and E46K samples was 5,960 cm−1 M−1, as evaluated from their amino acid composition by the method of Gill and von Hippel.53 Secondary structure conformation of E46K and wild‐type protein was assessed by far‐UV circular dichroism (CD). The measurements were performed on a J‐800 Series spectropolarimeter (JASCO, Japan) in a 1‐mm quartz cuvette at room temperature. Data were collected in the wavelength range of 250–190 nm. All protein samples were measured at the same settings averaged in five and the buffer data subtracted. Only data with high‐tension voltage <600 V were collected and shown to avoid too noisy signals. The mean residue ellipticity [θ] (degree cm2 dmol−1) was calculated from the formula [θ] (θobs/10) (MRW/lc), where θobs is the observed ellipticity in degrees; MRW is the mean residue molecular weight of the protein; l is the optical path length in cm; and c is the protein concentration in g/ml. A protein concentration of 7 μM was used. The spectra were recorded in 25 mM sodium phosphate buffer pH 7.4.
5
Analytical Techniques for Chemical Compound Characterization
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Column chromatography was performed
on a Biotage Isolera Spektra One with Biotage SNAP KP-sil (silica
gel) or KP-C18-HS (C18, reverse phase) columns. NMR spectra, 1H NMR and 13C NMR, were recorded on an Agilent
400 MHz (101 MHz for 13C NMR). The chemical shifts for 1H and 13C NMR spectra are reported in parts per
million (ppm) using the residual solvent peak for reference. The following
abbreviations are used for reporting NMR peaks: singlet (s), doublet
(d), triplet (t), quartet (q), heptet (hept), multiplet (m), broad
(br), and apparent (app). All coupling constants (J) are reported in hertz (Hz). For diastereomeric mixtures, peaks
that can be attributed to single diastereomers are labeled d1/d2. ATR-FTIR spectra
were recorded on a PerkinElmer Spectrum Frontier infrared spectrometer
with a pike-GladiATR module and reported in wavenumber (cm–1). Melting points were recorded on a Büchi Melting Point B-545.
High-resolution mass spectrometry (HRMS) was performed on an Agilent
1290 infinity LC system equipped with an autosampler tandem to an
Agilent 6520 Accurate Mass Q-TOF LC/MS.29a Fluorescence spectra were acquired on an Agilent Technologies Cary
Eclipse fluorescence spectrophotometer. UV–vis spectra were
acquired on a PerkinElmer Lambda20 Spectrophotometer, using a Starna
Silica (quartz) cuvette with 10 mm path length, two faces polished.
on a Biotage Isolera Spektra One with Biotage SNAP KP-sil (silica
gel) or KP-C18-HS (C18, reverse phase) columns. NMR spectra, 1H NMR and 13C NMR, were recorded on an Agilent
400 MHz (101 MHz for 13C NMR). The chemical shifts for 1H and 13C NMR spectra are reported in parts per
million (ppm) using the residual solvent peak for reference. The following
abbreviations are used for reporting NMR peaks: singlet (s), doublet
(d), triplet (t), quartet (q), heptet (hept), multiplet (m), broad
(br), and apparent (app). All coupling constants (J) are reported in hertz (Hz). For diastereomeric mixtures, peaks
that can be attributed to single diastereomers are labeled d1/d2. ATR-FTIR spectra
were recorded on a PerkinElmer Spectrum Frontier infrared spectrometer
with a pike-GladiATR module and reported in wavenumber (cm–1). Melting points were recorded on a Büchi Melting Point B-545.
High-resolution mass spectrometry (HRMS) was performed on an Agilent
1290 infinity LC system equipped with an autosampler tandem to an
Agilent 6520 Accurate Mass Q-TOF LC/MS.29a Fluorescence spectra were acquired on an Agilent Technologies Cary
Eclipse fluorescence spectrophotometer. UV–vis spectra were
acquired on a PerkinElmer Lambda20 Spectrophotometer, using a Starna
Silica (quartz) cuvette with 10 mm path length, two faces polished.
6
Quantifying Reduced Glutathione Levels
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Reduced glutathione was estimated according to the method described by Ellman, 1959. Reduced Glutathione levels were measured at 412 nm using a Perkin Elmer Lambda 20 spectrophotometer were calculated using molar extinction co-efficient of the chromophore (1.36 × 104 (mol/L) −1 cm−1) [21 (link)].
7
Catalase Activity Assay Protocol
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Catalase activity was assessed by the method of Luck (1965) . The change in absorbance was recorded for 2 min at 30 s interval at 240 nm using Perkin Elmer Lambda 20 spectrophotometer. The results were expressed as micromoles of hydrogen peroxide decomposed/min/mg of protein.
8
Purification and Analysis of Proteins
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Tris, acrylamide,
bis-acrylamide, urea, ammonium persulfate, N,N,N′,N′-tetramethylenediamine
(TEMED), dihydrofolic acid,
glycerol, ampicillin, pyruvate kinase, lysozyme, erythromycin, isopropyl
β-d -thiogalactopyranoside (IPTG), dithiothreitol (DTT),
and 2-mercaptoethanol were purchased from Sigma Chemicals (St. Louis,
MO). [35S]Methionine (10 μCi/μL) was obtained
from Amersham (Pitscataway, NJ). BL-21(DE-3) competent cells and T4
RNA ligase were from Promega (Madison, WI). The Plasmid MaxiKit (Life
Science Products, Inc., Frederick, CO) and the GenEluteHP plasmid
miniprep kit (Sigma) were used for plasmid purification.
Phosphorimager
analysis was performed using a Molecular Dynamics
400E PhosphorImager equipped with ImageQuant version 3.2. Ultraviolet
and visible spectral measurements were taken using a PerkinElmer lambda
20 spectrophotometer. Circular dichroism spectra were recorded using
a Jasco-810 spectropolarimeter.
bis-acrylamide, urea, ammonium persulfate, N,N,N′,N′-tetramethylenediamine
(TEMED), dihydrofolic acid,
glycerol, ampicillin, pyruvate kinase, lysozyme, erythromycin, isopropyl
β-
and 2-mercaptoethanol were purchased from Sigma Chemicals (St. Louis,
MO). [35S]Methionine (10 μCi/μL) was obtained
from Amersham (Pitscataway, NJ). BL-21(DE-3) competent cells and T4
RNA ligase were from Promega (Madison, WI). The Plasmid MaxiKit (Life
Science Products, Inc., Frederick, CO) and the GenEluteHP plasmid
miniprep kit (Sigma) were used for plasmid purification.
Phosphorimager
analysis was performed using a Molecular Dynamics
400E PhosphorImager equipped with ImageQuant version 3.2. Ultraviolet
and visible spectral measurements were taken using a PerkinElmer lambda
20 spectrophotometer. Circular dichroism spectra were recorded using
a Jasco-810 spectropolarimeter.
9
Absorbance Spectroscopy Protocol
10
Preparation of Amyloid Beta-42 Fibrils
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Aβ-42 peptide (Bachem, Bubendorf, Switzerland) was dissolved in 100% hexafluoroisopropanol (HFIP) to a final concentration of 1 mM. The solution was aliquoted, HFIP was evaporated and the resulting pellets were stored at −20 °C until needed. These aliquots were stable for at least three months. To generate amyloid aggregates, Aβ-42 peptide was dissolved to a 30 μM concentration in 20 mM sodium phosphate buffer (PBS), pH 7.4, at 25 °C. The samples were sonicated for 15 min and then centrifuged at 14,000× g for 15 min at 4 °C. The clear supernatant was collected, and the peptide concentration was checked by evaluating the absorbance of the resulting solution by means of a double-beam Lambda-20 spectrophotometer (Perkin Elmer Life Sciences, Norwalk, CT, USA) (ε280 = 1490 mol−1 cm−1) and adjusted to a final concentration of 25 μM. Enriched solutions of fibrillar Aβ-42 (Fib) were collected after 72 h of incubation of the supernatant at room temperature (RT).
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