NAD +and NADH were then detected using the NAD/NADH-Glo Assay (Promega, G9072) according to the manufacturer’s directions and previous reports (Gui et al., 2016 (link); Sullivan et al., 2015 (link)). To detect NAD+, 50 µL of sample was transferred to a PCR tube and treated with 25 µL of 0.4 N HCl and heated to 60 °C for 15 min where acidic conditions selectively degrade NADH. For NADH detection, 50 µL of sample was transferred to a PCR tube and heated to 75 °C for 30 min for selective basic degradation of NAD+. Following incubation, samples were equilibrated at room temperature, then quenched by neutralizing the NAD +acid-treated samples with 25 µL of 0.5 M Trizma base (Sigma, T6066) and the NADH base-treated samples with 50 µL of 1:1 (v/v) 0.4 N HCl:0.5 M Trizma base. Once neutralized, 50 µL of each sample was then mixed with 50 µL of NAD/NADH-Glo Detection Reagent in an opaque white 96-well flat bottom plate. Selective degradation protocols and detection in the linear range were confirmed using chemical standards for NAD+ (Sigma, N1511) and NADH (Sigma N8129). Enzyme-linked luminescence measurements were then recorded on a Tecan Infinite M Plex microplate reader according to the manufacturer instructions.
Trizma base
Manufactured by Merck Group
Sourced in United States, Germany, Spain, Italy, France, Switzerland, United Kingdom, Sao Tome and Principe, Japan, India, Czechia, China, Ireland, Poland
Trizma base is a buffer compound used in various laboratory applications. It is a stable, water-soluble chemical that can be used to maintain a consistent pH in aqueous solutions. Trizma base is commonly employed in biochemical and molecular biology experiments to create buffer systems with a desired pH range.
Automatically generated - may contain errors
Lab products found in correlation
Sodium chloride (nacl), by Merck Group (58 mentions)
Triton x 100, by Merck Group (55 mentions)
Bovine serum albumin (bsa), by Merck Group (54 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (49 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (45 mentions)
Trizma hydrochloride, by Merck Group (39 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (35 mentions)
Hydrochloric acid (hcl), by Merck Group (34 mentions)
Glycine, by Merck Group (32 mentions)
Tween 20, by Merck Group (31 mentions)
Glycerol, by Merck Group (26 mentions)
Sodium hydroxide (naoh), by Merck Group (24 mentions)
Acetic acid, by Merck Group (23 mentions)
Potassium chloride (kcl), by Merck Group (23 mentions)
Calcium chloride, by Merck Group (20 mentions)
Methanol, by Merck Group (20 mentions)
Trichloroacetic acid, by Merck Group (18 mentions)
Methanol, by Thermo Fisher Scientific (18 mentions)
Formic acid, by Merck Group (18 mentions)
Quercetin, by Merck Group (16 mentions)
469 protocols using trizma base
1
Quantifying NAD+ and NADH Levels
2
Decellularization of Trabecular Bone Plugs
3
Phosphate-Buffered Saline Preparation
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The charcoal and dialysis methods use phosphate-buffered saline with gelatin (PBS; 8.66 g Na2HPO4 [anhydrous], 6.10 g NaH2PO4·2H2O, 1 g gelatin, diluted up to 1 L in ultrapure water and adjusted to pH 7.4 with 1 N NaOH [Sigma-Aldrich, Mississauga, Canada, Cat. Nos. S5136, 711 505, G6144 and 109137, respectively]). We used all the PBS in < 1 week, so we did not a add preservative; for longer storage, we added 0.1 g thimerosal (Sigma-Aldrich, Mississauga, Canada Cat. Nos. T5125 and S2002); sodium azide can be used as an alternative bacteriostatic agent. The harvester method uses a 50-mM Tris acetate buffer (6.05 g Trizma base [Sigma T1503] in 1 L ultrapure water, chilled to 4°C, and pH adjusted to 7.4 with 5 N acetic acid [Sigma 695092 ]) for incubating plasma samples and a 25-mM Tris–HCl rinse buffer (6.05 g Trizma base dissolved in 2 L ultrapure water, chilled to 4°C, and pH adjusted to 7.4 with 6 N hydrochloric acid [Sigma 320331 ]). When we refer to ‘buffer’ throughout this paper, we are referring to the appropriate assay buffer for the selected separation procedure.
4
Measuring Alkaline Phosphatase Activity in Caco-2 Cells
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Alkaline phosphatase (AP) activity was measured as previously described [15 (link),16 (link)] with some modifications. Caco-2 cells were treated with or without 2 mM IIAEK. Micelles were prepared as previously described [13 (link)]. Following treatment, the cells were washed twice with 0.9% NaCl. Subsequently, 691 μL of cold 50 mM Tris buffer [pH 7.5, Trizma base (Sigma-Aldrich, St. Louis, MO, USA, T1503)] was added. Protein was collected on ice and homogenized with an injection needle (26G × 1/2) (TERUMO, Tokyo, Japan, NN-2613S). AP activity was estimated using 1.25 mg/mL disodium p-nitrophenol phosphate as a substrate in Tris-HCl buffer [pH 10.0, 5 mM MgCl2.6H2O (Wako, 135-00165) and 200 mM Trizma base (Sigma-Aldrich, St. Louis, MO, USA, T1503)]. After 30 min of incubation at 37 °C, the absorbance at 405 nm was measured, and AP activity was calculated as μmol/min using a calibration curve for various concentrations of p-nitrophenol. μmol/min was defined as U. U was converted to U/mg protein in order to normalize AP activity to the protein concentration, which was determined using a commercially available kit (BioRad, protein assay; BioRad).
5
Enzymatic Activities in BM-MSCs
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5 × 105 cells from each group of different age were used for the assessment of enzyme activities. The cells were homogenized in 200 μL of 250 mM sucrose(Sigma-Aldrich, St.Louis, MO), 50 mM HEPES (Sigma-Aldrich), 0,5 mM EDTA (Sigma Aldrich) and one tablet protease inhibitor cocktail (Roche, Mannheim, Germany ). Enzymes activities were determined using a SUNRISE spectrophotometer (TECAN, Mannedorf, Switzerland). Reaction rates of enzymes were determined by the increase or decrease in absorbance of NAD(P)H (Sigma-Aldrich, St.Louis, MO) at 340 nm at 37 °C. Lactate dehydrogenise (EC 1.1.1.27) was determined in BM-MSCs using 50 mM Trizma base (pH 7,4), 0,15 mM NADH and 5 mM sodium pyruvate (omitted for control) (all Sigma Aldrich, St.Louis, MO). Glucose-6-phosphate 1-deshydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase, decarboxylating (EC 1.1.1.343) was determined in BM-MSCs using 78 mM Trizma base, 5 mM MgCl2(pH 7,4), 0,1 mM NADP, 0,5 mM D-Glucose 6-phosphate disodium salt hydrate and 6-Phosphogluconic acid trisodium salt (omitted for control) (all Sigma-Aldrich, St.Louis, MO).
6
Comprehensive Protein Extraction Protocol
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DL-Dithiothreitol (DTT, D0632), N-Ethylmaleimide (NEM, E1271), Iodoacetamide (I1149), IGEPAL CA-630 (NP40, I3021), Triton X-100 (T9284), bovine serum albumin (BSA, A7906), Sodium dodecyl sulfate (SDS, L3771), Tween-20 (P9416), Ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA, E4884), and Trizma base (Tris, 93349) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Complete protease inhibitor cocktail (04693116001) and phosphatase inhibitor cocktail (04906837001) were purchased from Roche Diagnostic (Mannheim, Germany). Ciprofloxacin (17850) was purchased from Fluka (Buchs, Switzerland).
7
Optimizing Diclofenac Extraction and Purification
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Methanol (Merck), DMF (Merck), ethanol (Merck), Na diclofenac (Sigma-Aldrich), HCl (smart lab), Bovine serum albumin fraction V (Sigma-Aldrich), NaOH (Merck), Trizma Base (Sigma-Aldrich), NaCl (Merck), TLC plate 60 F254 (Merck).
8
REGN-H098P Antibody Characterization
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The fully human monoclonal antibody REGN-H098P, the mouse anti-REGN-H098P mAb positive control, biotinylated and ruthenium-labeled REGN-H098P, REGN-H098P chimeras (IgG1 CH3 domain or L445P substitution), recombinant human mAbs (mAb1 IgG4, mAb2 IgG1, mAb4 IgG4 G1-CH3 with IgG1 CH3 domain, mAb5 IgG4 CH3-L > P with leucine > proline 445 substitution), and fusion proteins with IgG4 Fc fragment and IgG4 Fab fragment were produced by Regeneron Pharmaceuticals (Tarrytown, NY). Commercial human IgG1 kappa, IgG2 with lambda light chain, and IgG4 kappa (wild-type "CPSC" hinge sequence) were from Sigma (St Louis, MO). Human IgG3 was from Abcam (Cambridge, UK).
All solutions for the REGN-H098P ADA assay, unless otherwise specified, were prepared in 1% BSA, 1X PBS. 10% BSA Diluent/Blocking Solution was from SeraCare (Milford, MA). 1X PBS was from Life Technologies (Grand Island, NY). 1.5 M Trizma base was from Sigma (St Louis, MO). Glacial acetic acid was from Thermo Fisher Scientific (Waltham, MA). Protein G agarose beads were from G Bioscience (St Louis, MO). Rabbit anti-human IgG(H + L) used for immuno-depletion was from Life Technologies. HRP-Rabbit anti-human IgG (H + L) used for western blot was from Abcam. Streptavidin-coated SECTOR® microplates, read buffer, and Sector Imager 2400 were from Meso Scale Discovery (MSD, MD, USA).
All solutions for the REGN-H098P ADA assay, unless otherwise specified, were prepared in 1% BSA, 1X PBS. 10% BSA Diluent/Blocking Solution was from SeraCare (Milford, MA). 1X PBS was from Life Technologies (Grand Island, NY). 1.5 M Trizma base was from Sigma (St Louis, MO). Glacial acetic acid was from Thermo Fisher Scientific (Waltham, MA). Protein G agarose beads were from G Bioscience (St Louis, MO). Rabbit anti-human IgG(H + L) used for immuno-depletion was from Life Technologies. HRP-Rabbit anti-human IgG (H + L) used for western blot was from Abcam. Streptavidin-coated SECTOR® microplates, read buffer, and Sector Imager 2400 were from Meso Scale Discovery (MSD, MD, USA).
9
Peptide A9R Synthesis and Characterization
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Peptide A9R was supplied by Biomatik (Cambridge, Ontario,
Canada). The purity was 98.93% by high-performance liquid chromatography
using an Inertsil ODS-SP column with acetonitrile [0.1% trifluoroacetate
(TFA)]/water (0.1% TFA) gradient. The molar mass by electrospray ionization
mass spectroscopy (ESI-MS) was 814.90 g mol–1 (M
+ H+, 813.92 g mol–1 expected).Scheme S1 shows the chemical structure of A9R. Elastase from porcine pancreas (MW = 25.9 kDa) and Trizma base were purchased from Sigma-Aldrich.
1-bromohexadecane was purchased from Sigma-Aldrich. Lipids DOPC, POPC,
POPG, and POPE were obtained from Sigma-Aldrich.
Canada). The purity was 98.93% by high-performance liquid chromatography
using an Inertsil ODS-SP column with acetonitrile [0.1% trifluoroacetate
(TFA)]/water (0.1% TFA) gradient. The molar mass by electrospray ionization
mass spectroscopy (ESI-MS) was 814.90 g mol–1 (M
+ H+, 813.92 g mol–1 expected).
1-bromohexadecane was purchased from Sigma-Aldrich. Lipids DOPC, POPC,
POPG, and POPE were obtained from Sigma-Aldrich.
10
Characterization of Membrane-Active Compounds
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Histamine, octyl, and R- and S-octan-2-yl isocyanates were purchased from Sigma-Aldrich Co. PC (chicken egg), PS (porcine brain), and Chl were purchased from Avanti Polar Lipids. Trizma hydrochloride, Trizma base, sodium phosphate monobasic, sodium phosphate dibasic, NaCl, Hepes, calcein, EDTA, Triton X-100, SDS, D2O, ethanol, and chloroform were purchased from Sigma-Aldrich Co. Compressed N2, He, and O2 were supplied by Airgas. All buffers were prepared fresh and filtered through a 0.22-μm polyethersulfone membrane (Express PLUS, Millipore). For the SFG experiments, PBS (phosphate-buffered saline) and SDS were purchased from Sigma-Aldrich Co. Sodium chloride was purchased from Mallinckrodt Chemicals. Ultrapure water (Millipore MilliQ, 18.2 megohms·cm, ≤ 5 parts per billion total organic carbon) was used for all solutions. Equilateral CaF2 prisms were obtained from Crystran Ltd.
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