The milled harvested matured barley grains were used for hordein determination as well. Barley alcohol-soluble proteins (hordeins) were extracted from 50 mg of flour and the isolated proteins were separated on SDS-PAGE according to Uddin et al. [40] . Maltose binding protein (MBP5) (6 mg/mL stock) (New EnglandBioLabs) as a negative control and yeast alcohol dehydrogenase (1 µg/µL stock) (Sigma-Aldrich Inc) as a positive control was used with following dilutions in the sample buffer: maltose binding protein (MBP5) in 1∶20; and alcohol dehydrogenase in 1∶1. Pre-stained high molecular weight protein standard (HiMark) were purchased from Invitrogen (Life Technologies).
Yeast alcohol dehydrogenase
Manufactured by Merck Group
Sourced in United Kingdom
Yeast alcohol dehydrogenase is an enzyme that catalyzes the interconversion of alcohols and aldehydes or ketones. It is commonly used in biochemistry and molecular biology laboratories for various applications.
Automatically generated - may contain errors
Lab products found in correlation
Avidin, by Merck Group (2 mentions)
Synapt hdms mass spectrometer, by Waters Corporation (2 mentions)
β lactoglobulin a, by Merck Group (2 mentions)
Masslynx v4, by Waters Corporation (2 mentions)
Driftscope 2, by Waters Corporation (2 mentions)
Massign, by Waters Corporation (2 mentions)
Concanavilin a, by Merck Group (2 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (2 mentions)
Glycogen phosphorylase b from rabbit muscle, by Merck Group (2 mentions)
Glutamate dehydrogenase from bovine liver, by Merck Group (2 mentions)
Rabbit aldolase, by Merck Group (2 mentions)
Horse liver alcohol dehydrogenase, by Merck Group (2 mentions)
Pyruvate kinase, by Merck Group (2 mentions)
Beta galactosidase from escherichia coli, by Merck Group (2 mentions)
Groes, by Merck Group (2 mentions)
Alcohol dehydrogenase, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Glucose 6 phosphate dehydrogenase, by Merck Group (1 mentions)
Bradford protein reagent, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Bio-Rad (1 mentions)
14 protocols using yeast alcohol dehydrogenase
1
Barley Hordein Protein Extraction
2
Continuous Spectrophotometric Assay for NMNAT Activity
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Enzyme activity of NMNAT was measured in a continuous spectrophotometric coupled assay by monitoring the increase in absorbance of NADH at 340 nm, The reaction process is as follows Balducci et al. (1995) (link):
The reaction solution contains 28 mM HEPES buffer (pH 7.4), 11.2 mM MgCl2, 16 nM semicarbazide-HCl, 0.046 mM ethanol, 1.5 mM ATP, and 0.03 mg/ml yeast alcohol dehydrogenase (Sigma, A7011), and NMNAT or variants. The reaction was initiated by adding NMN to a final concentration of 0.625 mM. All measurements were performed at 37 °C. The activity was calculated using the equation below.
Where C0β-NADH, the extinction coefficient of β-NADH at 340 nm, is 6.22 (Zhai et al., 2006 (link)).
The reaction solution contains 28 mM HEPES buffer (pH 7.4), 11.2 mM MgCl2, 16 nM semicarbazide-HCl, 0.046 mM ethanol, 1.5 mM ATP, and 0.03 mg/ml yeast alcohol dehydrogenase (Sigma, A7011), and NMNAT or variants. The reaction was initiated by adding NMN to a final concentration of 0.625 mM. All measurements were performed at 37 °C. The activity was calculated using the equation below.
Where C0β-NADH, the extinction coefficient of β-NADH at 340 nm, is 6.22 (Zhai et al., 2006 (link)).
3
Quantitative NAD Assay in Tissues
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Ten volumes (µl/mg) of buffered 75% ethanol and 25% HEPES (10 mM, pH 7.1) were used to extract 20 mg of kidney tissues. Extracts were diluted 1:20 in distilled water for a total minimum volume of 50 µl per dilution to reach a concentration within the standard curve. Afterward, 25 µl of NAD samples were loaded in a 96-well microtiter-plate in duplicate. Ten µl/well of reaction buffer (600 mM ethanol, 0.5 mM 3-(4.5dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide [MTT]), 2 mM phenazine ethosulfate (PES), 120 mM bicine (pH 7.8) was added. Yeast alcohol dehydrogenase (Sigma-Aldrich A3263 > 300 units/mg) was then added to the NAD samples. The kinetics of the reaction (OD at 550 nm, every 30 s for 20 min) was examined on an LB 942 multimode reader and NAD was quantified using a linear regression curve equation method between NAD standard concentration and the slope of the regression line.
4
E. coli Cross-Linking and Proteomics
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E. coli DH3α cells were grown
in lysogeny broth (LB) media for 16 h at 37 °C. Cells were pelleted
at 800xg for 10 min at 4 °C. The cell pellet, chicken ovotransferrin,
bovine serum albumin, and yeast alcohol dehydrogenase (Sigma-Aldrich)
were resuspended in 20 mM HEPES pH 7.4 to a concentration of 10 mg/mL.
2.5–5 mM tBu-PhoX (Thermo Fisher Scientific) in DMSO was added
to each solution and incubated for 30–60 min at room temperature.
The cross-linking reaction was quenched by adding 20 mM Tris pH 8
and incubated for 15 min at room temperature. Cross-linked E. coli were incubated for 10 min at 95 °C after
the addition of 4% wt/v SDS and lysed by sonication for 10 min using
a Bioruptor (30 s cycles). E. coli proteins
were precipitated by chloroform–methanol precipitation. Cross-linked
proteins were supplemented with 8 M urea, reduced, alkylated, and
proteolyzed with Lys-C endopeptidase (1:200 wt/wt) and trypsin (1:100
wt/wt). The digestion was stopped after 16 h with 1% formic acid (FA).
Cross-linked peptides were desalted using Sep-Pak C8 cartridges (Waters)
and dried. Cross-linked and mono-linked peptides were enriched as
described before.19 (link)
in lysogeny broth (LB) media for 16 h at 37 °C. Cells were pelleted
at 800xg for 10 min at 4 °C. The cell pellet, chicken ovotransferrin,
bovine serum albumin, and yeast alcohol dehydrogenase (Sigma-Aldrich)
were resuspended in 20 mM HEPES pH 7.4 to a concentration of 10 mg/mL.
2.5–5 mM tBu-PhoX (Thermo Fisher Scientific) in DMSO was added
to each solution and incubated for 30–60 min at room temperature.
The cross-linking reaction was quenched by adding 20 mM Tris pH 8
and incubated for 15 min at room temperature. Cross-linked E. coli were incubated for 10 min at 95 °C after
the addition of 4% wt/v SDS and lysed by sonication for 10 min using
a Bioruptor (30 s cycles). E. coli proteins
were precipitated by chloroform–methanol precipitation. Cross-linked
proteins were supplemented with 8 M urea, reduced, alkylated, and
proteolyzed with Lys-C endopeptidase (1:200 wt/wt) and trypsin (1:100
wt/wt). The digestion was stopped after 16 h with 1% formic acid (FA).
Cross-linked peptides were desalted using Sep-Pak C8 cartridges (Waters)
and dried. Cross-linked and mono-linked peptides were enriched as
described before.19 (link)
5
Quantifying Isotope Labeling in Yeast ADH
6
Pyruvate Decarboxylase Activity Assay
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The pyruvate decarboxylase activity was measured using a coupled assay with yeast alcohol dehydrogenase [26 (link)]. The reaction mix contained: 50 mM pH 7.0 Tris–HCl buffer (The pH was adjusted at 25 °C), 2 mM MgCl2, 0.2 mM thiamine pyrophosphate, 0.3 mM NADH, 10 mM pyruvate and 1 U/mL of yeast alcohol dehydrogenase from Sigma (A3263). Pyruvate was used to start the reaction and the consumption of NADH was followed spectrophotometrically at 340 nm (molar extinction coefficient ε of NADH = 6.22 mM−1 cm−1). The protein concentration was determined using the Bradford protein reagent with bovine serum albumin as the standard (BioRad, Hercules, CA).
For the thermostability assay, the Pdc protein was mixed with the reaction mix without NADH and Adh and then incubated at various temperatures for 30 min. The pH of reaction mix was set to 7.0 at 25 °C. Then the incubated mix was chilled on ice for 15 min before the assay. Both NADH and Adh protein could be used to start the reaction. In cases where Adh protein was used to start the reaction, the rate of the reaction was determined after the reaction had reached steady state (i.e. the brief, transient initial rate was ignored).
For the thermostability assay, the Pdc protein was mixed with the reaction mix without NADH and Adh and then incubated at various temperatures for 30 min. The pH of reaction mix was set to 7.0 at 25 °C. Then the incubated mix was chilled on ice for 15 min before the assay. Both NADH and Adh protein could be used to start the reaction. In cases where Adh protein was used to start the reaction, the rate of the reaction was determined after the reaction had reached steady state (i.e. the brief, transient initial rate was ignored).
7
Synthesis of Organometallic Rhodium Electron Mediator
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Formate dehydrogenase (FateDH), formaldehyde dehydrogenase (FaldDH), yeast alcohol dehydrogenase (ADH), β-nicotinamide adenine dinucleotide (β-NAD+), reduced nicotinamide adenine dinucleotide (NADH), dicyandiamide, triethanolamine (TEOA), dichloro (pentamethylcyclopentadienyl) rhodium(iii ) dimer, 1,10-phenanthroline were purchased from Sigma-Aldrich. Tungsten disulfide (WS2) was purchased from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China).
The organometallic electron mediator (M), [Cp*Rh(phen)H2O]2+, (Cp* = 5-C5Me5, phen = 1,10-phenanthroline) was synthesized as follows. Briefly, 103.01 mg of dichloro (pentamethylcyclopentadienyl) rhodium(iii ) dimer was added to 10 mL of methylene chloride, where the solid was insoluble. Then, 60.07 mg of 1,10-phenanthroline was added to the mixture. After stirring at room temperature for 3 h, the color of the solution changed from dark orange to orange. After removing the solvent by evaporation under reduced pressure, M was obtained.
The organometallic electron mediator (M), [Cp*Rh(phen)H2O]2+, (Cp* = 5-C5Me5, phen = 1,10-phenanthroline) was synthesized as follows. Briefly, 103.01 mg of dichloro (pentamethylcyclopentadienyl) rhodium(
8
Characterization of Skp-OMP Complexes by IMS-MS
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Skp:OMP complexes were prepared by rapid dilution of the denatured OMP (400 μM in 8 M urea, 50 mM glycine-NaOH, pH 9.5) to a final concentration of 5 μM into a solution of Skp (5 μM in 50 mM glycine-NaOH, pH 9.5). The samples were then buffer exchanged into 200 mM ammonium acetate, pH 10 using Zeba spin desalting columns (Thermo Scientific, UK) immediately prior to MS analysis. nanoESI-IMS-MS spectra were acquired using a Synapt HDMS mass spectrometer (Waters Corporation, UK) using platinum/gold-plated borosilicate capillaries prepared in-house. Typical instrument parameters include: capillary voltage 1.2-1.6 kV, cone voltage 40 V, trap collision voltage 6 V, transfer collision voltage 10 V, trap DC bias 20 V, backing pressure 4.5 mBar, IMS gas pressure 0.5 mBar, travelling wave height 7 V, travelling wave velocity 250 ms-1. Data were processed using MassLynx v4.1, Driftscope 2.5 (Waters Corporation, UK) and Massign55 (link). CCSs were estimated by a calibration approach28 (link),33 (link),56 using arrival time data for ions with known CCSs (β-lactoglobulin A, avidin, concanavilin A and yeast alcohol dehydrogenase, all Sigma Aldrich, UK). Estimated modal CCSs are shown as mean ± standard deviation of three independent experiments. Theoretical CCSs for globular proteins with a given effective gas phase density were calculated according to published methods57 (link).
9
NMNAT Enzymatic Activity Assay
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The enzymatic activity of NMNAT was measured in a continuous coupled enzyme assay by monitoring the increase in absorbance at 340 nm caused by the reduction of NAD to NADH48 (link). The reaction was performed at 37 °C in 16 nM semicarbazide-HCl, 0.625% (v/v) ethanol, 30 nM HEPES buffer (pH=7.4), 1.17 mM ATP, 15 U yeast alcohol dehydrogenase (Sigma), purified recombinant NMNAT protein variants and was initiated by adding nicotinamide mononucleotidase (NMN) to a final concentration of 0.625 nM. The activity is determined from the linear progression curve using the following formula:
where C0β-NADH, the extinction coefficient of β-NADH at 340 nm, is 6.22.
where C0β-NADH, the extinction coefficient of β-NADH at 340 nm, is 6.22.
10
Holdase Activity of HSPB5 Mutants
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All holdase assays were performed in duplicate using a 96-well plate reader (BioTek) with PBS solutions at pH 7.5 and 250 μL well volumes. DTT-denatured bovine αLactalbumin (Sigma L6010) was used as a model substrate and light scattering at 360 nm was used to monitor protein aggregation in the presence and absence of WT- and mutant HSPB5 at 42°C. 40 µM sHSP (subunit concentration) was added to 600 µM αLac. Aggregation of αLac was induced by the addition of DTT to final concentrations of 50 mM. Aggregation of the model substrate Yeast Alcohol Dehydrogenase (Sigma A8656) was achieved with the addition of EDTA and DTT to final concentrations of 5 mM at 37°C. Light scattering by aggregates was monitored in the presence and absence of WT- and mutant HSPB5. 20 µM sHSP (subunit concentration) was added to 100 µM Alcohol Dehydrogenase.
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