HCT enzyme assays towards 4-coumaroyl CoA (“forward reaction”) were perform with 10 or 20 ng of recombinant proteins or 4 to 7 µg of crude plant protein extracts in a reaction solution of 100 mM sodium phosphate buffer pH 7.5, 500 µM shikimic acid and 500 µM dithiothreitol (Roche, Madison, WI) in a final volume of 100 µL. The 4-coumaroyl CoA concentration was 50 µM for assays with crude extracts, and varied from 5 to 100 µM to determine kinetics of recombinant enzymes. HCT enzyme assays towards caffeoyl shikimate (“reverse reaction”) were carried out with 50 ng of recombinant proteins or 13 to 17 µg of plant crude protein extracts in a reaction solution of 100 mM sodium phosphate buffer pH 7.5, 500 µM Coenzyme A and 500 µM dithiothreitol (Roche, Madison, WI) in a total volume of 100 µL. The caffeoyl shikimate concentration was 50 µM for assays with crude extracts, and varied from 20 to 400 µM to determine kinetics of recombinant enzymes. Reactions were terminated by addition of 10 µL of glacial acetic acid and products were analyzed by HPLC as previously described (Escamilla-Trevino et al. [27 ]) Products were quantified by measuring peak areas and converting to units of quantity using calibration curves that were constructed with authentic standards of each product.
Dithiothreitol (dtt)
Manufactured by Roche
Sourced in Germany, United States, Switzerland, United Kingdom
DTT (Dithiothreitol) is a common reducing agent used in biochemical applications. It acts by reducing disulfide bonds in proteins, which can be useful for maintaining protein structure and activity. DTT is often used in buffer solutions to help preserve the native state of proteins during experimental procedures.
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Lab products found in correlation
Iodoacetamide, by Merck Group (7 mentions)
Trypsin, by Promega (4 mentions)
Sodium chloride (nacl), by Merck Group (4 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Roche (4 mentions)
Ms grade trypsin, by Promega (3 mentions)
Iodoacetic acid, by Merck Group (3 mentions)
Urea, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Mgcl2, by Thermo Fisher Scientific (3 mentions)
Sequencing grade modified trypsin, by Promega (2 mentions)
Lc ms grade formic acid, by Thermo Fisher Scientific (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Iodoacetomide, by Roche (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (2 mentions)
Ammonium bicarbonate, by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Phosstop, by Roche (2 mentions)
73 protocols using dithiothreitol (dtt)
1
HCT Enzyme Assays for Kinetic Analysis
2
Induction of ER Biogenesis System
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Precultures were grown in liquid SCD medium during the day, diluted into fresh medium and grown overnight for 16 h so that they reached mid log phase (OD600 = 0.5 -1). For induction of the ER biogenesis system, overnight cultures were diluted to OD600 = 0.05 in fresh medium and treated with the indicated concentrations of ßestradiol (Sigma) for up to 6 h. For DTT treatment, overnight cultures were diluted to OD600 = 0.1 and treated with 8 mM DTT (Roche, Mannheim, Germany) for up to 2 h.
Immediately before imaging, cells were harvested by centrifugation, mounted on coverslips and covered with a 1% (w/v) agarose pad. Images were acquired with a DMi8 inverted microscope (Leica, Wetzlar, Germany) equipped with a CSU-X1 spinning-disk confocal scanning unit (Yokogawa, Musashino, Japan) and an ORCA-Flash 4.0 LT camera (Hamamatsu, Hamamatsu, Japan). A HC PL APO 63x/1.40-0.60 or a HC PL APO 100x/1.4 CS2 oil objective lens (Leica) was used.
Immediately before imaging, cells were harvested by centrifugation, mounted on coverslips and covered with a 1% (w/v) agarose pad. Images were acquired with a DMi8 inverted microscope (Leica, Wetzlar, Germany) equipped with a CSU-X1 spinning-disk confocal scanning unit (Yokogawa, Musashino, Japan) and an ORCA-Flash 4.0 LT camera (Hamamatsu, Hamamatsu, Japan). A HC PL APO 63x/1.40-0.60 or a HC PL APO 100x/1.4 CS2 oil objective lens (Leica) was used.
3
Disulfide Bond Reduction and Alkylation
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Reduction and alkylation of disulfide bonds on proteins were carried out using 1 M dithiothreitol (Roche, Switzerland; final sample concentration 10 mM) for 45 min and 1 M Iodoacetamide (Sigma-Aldrich; final sample concentration 30 mM) for 30 min in a dark, respectively. Following alkylation and reduction, the samples were diluted with ammonium bicarbonate buffer (pH 8.0) until the urea concentration was 1 M (Sigma-Aldrich). The proteins were digested with trypsin (MS grade; Promega, Madison, WI, United States) overnight at 37°C at an enzyme to protein ratio of 1:20. Finally, the peptides were acidified with 100% Trifluoroacetic acid (TFA; Sigma-Aldrich) to a final concentration of 1% TFA and then desalted using macro spin columns (Harvard apparatus, Holliston, MA, United States).
4
Quantification of Bile Salt Hydrolase Activity
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Faecal water was prepared and total faecal protein quantified similarly to a method previously-described41 (link), but with the addition of bacterial and mammalian protease inhibitor cocktails (G Biosciences, Uttar Pradesh, India), as well as Dithiothreitol to 1 mM final concentration (Roche, Basel, Switzerland) (to minimise enzyme oxidation42 (link)).
The BSH assay has been described previously37 (link). In brief, BSH activity was determined by measuring insoluble DCA precipitated (determined by absorbance at 600 nm (A600)) following incubation of 500 µg of faecal protein with taurodeoxycholic acid (Sigma-Aldrich, St Louis, US). Samples were compared with a standard curve of known DCA concentrations and measured in triplicate.
The BSH assay has been described previously37 (link). In brief, BSH activity was determined by measuring insoluble DCA precipitated (determined by absorbance at 600 nm (A600)) following incubation of 500 µg of faecal protein with taurodeoxycholic acid (Sigma-Aldrich, St Louis, US). Samples were compared with a standard curve of known DCA concentrations and measured in triplicate.
5
Evaluating Cell Viability and Redox State
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Pa03C cells were seeded at 2500 cells/well in 96-well plates and cell viability was measured with Alamar blue (Invitrogen, Eugene, USA) 48 h after treatment with various concentrations of methyl methanesulfonate (MMS) (Sigma, Cat#129925). Cellular response to MMS was normalized to a non-treated (media only or vehicle) control [30 (link)]. At least three replicates were performed.
For examining alteration of Ref-1 redox state, Pa03C cells were treated with different concentrations of H2O2 (Thermo Fisher, Cat#H325-500) for 30min. Dithiothreitol (DTT; reducing agent, 5 mM (Roche, Cat#38225822)) and diamide (oxidizing agent, 4 mM (Sigma, Cat#D3648)) were used as controls.
For examining alteration of Ref-1 redox state, Pa03C cells were treated with different concentrations of H2O2 (Thermo Fisher, Cat#H325-500) for 30min. Dithiothreitol (DTT; reducing agent, 5 mM (Roche, Cat#38225822)) and diamide (oxidizing agent, 4 mM (Sigma, Cat#D3648)) were used as controls.
6
Glycoprotein Purification and Identification
7
Mass Spectrometry Sample Preparation
8
Proteomic analysis of hiPSC-CMs
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Monolayer-cultured hiPSC-CMs were rinsed twice with 1× PBS and lysed with Pierce RIPA lysis buffer (Thermo Fisher, 89901, Waltham, MA, USA) supplemented with Halt protease and phosphatase inhibitor (Invitrogen, 78443, Waltham, MA, USA) and dithiothreitol (Roche, 3483-12-3, Basel, Switzerland). Lysates were rocked at 4 °C for 20 min and centrifuged 10 min at 15,000× g, with supernatant collected. Total protein concentration of supernatant was assessed through a Bradford assay (Bio Rad, 5000006, Hercules, CA, USA) with 560 nm absorbance per manufacturer’s protocol using BSA standards (Thermo Fisher, 23208, Waltham, MA, USA). Lysates were diluted with 4× Laemmli SDS Sample Buffer (Bio Rad, 1610747, Hercules, CA, USA) to 1 μg/μL total protein concentration.
9
CSF Proteome Sample Preparation
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CSF samples were diluted with 8 M urea. Each resulting denatured protein sample was chemically reduced with 100 mM dithiothreitol (Roche) at 56°C for 30 min and alkylated using 100 mM iodoacetamide for 1 h. Proteins were digested with trypsin using the Filter-Aided Sample Preparation technique48 (link). Specifically, 10 μl of each protein mixture was loaded onto a 10 kDa molecular-weight cutoff filter unit that was then centrifuged (14,500 g, 15 min). Proteins in the recovered solution were then reduced with dithiothreitol and alkylated with iodoacetamide as noted above. The filter membrane was subsequently washed three times with 50 mM NH4HCO3 to eliminate any remaining urea, and proteins were digested with 1 μg trypsin per membrane overnight at 37°C. The resulting tryptic peptides were eluted from the membrane by centrifugation (14,500 g, 15 min). An additional 50 μl of 50 mM NH4HCO3 was added to the filter membrane, and the membrane was centrifuged again (14,500 g, 15 min). Finally, the two eluates were combined for subsequent analysis.
10
In-Gel Tryptic Digestion of Proteins
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Proteins were separated by 10-20% tris-tricine gel and in-gel digested according to a previous published protocol with modifications (9 ).
Briefly, gel pieces containing proteins were cut, transferred, and mashed into small pieces. The pieces were incubated in 500 μL of acetonitrile for 10 minutes until they shrunk. The shrunken pieces were incubated in 50 mM dithiothreitol (Roche) in 50 mM ammonium bicarbonate at 56°C for 30 minutes, washed with 500 μL acetonitrile, incubated in 100 mM iodoacetamide in dark at room temperature for 20 minutes, and washed with 500 μL acetonitrile.
The gel pieces were then incubated with 60 μL of a 50 mM ammonium bicarbonate solution of trypsin (MS grade, Promega) at 4°C for 90 minutes and incubated at 37°C overnight. The protease to protein ratio is roughly 1:50. The liquid in the tubes were collected before gel pieces were eluted by 100 μL of 66.6% acetonitrile in 0.1% formic acid. The solutions were combined and concentrated with a SpeedVac (Thermo Fisher Scientific, US).
Briefly, gel pieces containing proteins were cut, transferred, and mashed into small pieces. The pieces were incubated in 500 μL of acetonitrile for 10 minutes until they shrunk. The shrunken pieces were incubated in 50 mM dithiothreitol (Roche) in 50 mM ammonium bicarbonate at 56°C for 30 minutes, washed with 500 μL acetonitrile, incubated in 100 mM iodoacetamide in dark at room temperature for 20 minutes, and washed with 500 μL acetonitrile.
The gel pieces were then incubated with 60 μL of a 50 mM ammonium bicarbonate solution of trypsin (MS grade, Promega) at 4°C for 90 minutes and incubated at 37°C overnight. The protease to protein ratio is roughly 1:50. The liquid in the tubes were collected before gel pieces were eluted by 100 μL of 66.6% acetonitrile in 0.1% formic acid. The solutions were combined and concentrated with a SpeedVac (Thermo Fisher Scientific, US).
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