TG, AG, DHTG, and the corresponding deuterated stable-labeled internal standards TG-d3, AG-d4, and DHTG-d3 were purchased from Cerilliant corporation (Round Rock, TX). EtioG standard was purchased from Steraloids (Newport, RI). Membrane vesicle overexpressing the human BCRP, MDR1, MRP2, MRP3, and MRP4 were provided by Solvo Biotechnology (Budapest, Hungary). Adenosine 5’-triphosphate (ATP) disodium salt, adenosine 5’-monophosphate (AMP) monohydrate, glutathione, Tris-Base (Tris[hydroxymethyl]aminomethane), MgCl2, NaCl, Sucrose, MOPS (3-[N-Morpholino]propanesulfonic acid) were purchased from Sigma-Aldrich (St. Louis, MO). Bovine serum albumin (BSA), membrane protein extraction kit and trypsin digestion reagents were obtained from Thermo Fisher Scientific (Rockford, IL). The synthetic light peptide and heavy labeled peptides were purchased from New England Peptides (Boston, MA) and Thermo Fisher Scientific (Rockford, IL), respectively. Multiscreen™ HTS Vacuum Manifold and 96-well filter plates with class B glass fiber filters were purchased from EMD Millipore (Billerica, MA). Estradiol-17β-glucuronide (E2-17β-G), N-Methyl Quinidine (NMQ), dehydroepiandrosterone sulfate (DHEAS), and estrone-3-sulfate (E3S) were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals and reagents, unless indicated otherwise, were purchased from Sigma-Aldrich (St. Louis, MO).
3 n morpholino propanesulfonic acid mops
Manufactured by Merck Group
Sourced in United States, Germany, Italy
3-(N-morpholino) propanesulfonic acid (MOPS) is a chemical compound commonly used as a buffer solution in biochemical and cell culture applications. It maintains a stable pH within a specific range, typically between 6.5 and 7.9, making it suitable for various laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (15 mentions)
Rpmi 1640 medium, by Merck Group (8 mentions)
Sodium chloride (nacl), by Merck Group (5 mentions)
Fluconazole, by Merck Group (5 mentions)
Acetonitrile, by Merck Group (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Amphotericin b, by Merck Group (4 mentions)
Itraconazole, by Merck Group (4 mentions)
Sodium hydroxide (naoh), by Thermo Fisher Scientific (4 mentions)
Sabouraud dextrose agar, by Merck Group (4 mentions)
Sucrose, by Merck Group (3 mentions)
Mgcl2, by Merck Group (3 mentions)
Crystal violet, by Merck Group (3 mentions)
Rpmi 1640 broth medium, by Harvard Bioscience (3 mentions)
Dithiothreitol (dtt), by Merck Group (3 mentions)
Formic acid, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Potato dextrose broth (pdb), by Merck Group (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
83 protocols using 3 n morpholino propanesulfonic acid mops
1
Membrane Transport Protein Assay Protocol
2
Broth Microdilution Antifungal Susceptibility Test
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The broth microdilution (BMD) antifungal susceptibility test was performed according to the document M27-S4 (Clinical and Laboratory Standards Institute, 2012) (CLSI) [19] using RPMI-1640 broth (pH 7.0) buffered with 0.165 M MOPS [3-(N-morpholino) propanesulfonic acid] (Sigma Chemical Co. St Louis, MO, USA). Fluconazole (Sigma Chemical) and naphthofuranquinone (compounds 1-3) were dissolved in distilled water and dimethyl sulfoxide (DMSO; Sigma Chemical), respectively. Fluconazole was tested at a range of concentrations from 0.125–64 µg/mL, whereas the naphthofuranquinones were tested at 0.25–128 µg/mL. The 96-well culture plates were incubated at 35°C for 24 h, and the results were examined visually, as recommended by the CLSI [19] . The MIC was considered to be the concentration that inhibited 50% of fungal growth. The in vitro drug interactions were evaluated according to the MIC, and the strains were classified as susceptible (S), or resistant (R). The cutoff points for Fluconazole susceptibility were as follows: MIC≤2 µg/mL (S), MIC≥8 µg/mL (R) [20] (link), [21] (link). The strains C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used as controls [19] .
3
Fungal Pathogen Growth Media Optimization
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The fungal pathogens used in this study are shown in Table 1 . The media used in this study were YPD (1% yeast extract [Bioshop, Canada], 2% peptone [Bioshop], 2% dextrose [Bioshop]), PDB (24 g potato dextrose broth [Himedia, India] in 1 L distilled water), RPMI 1640 medium (10.4 g RPMI 1640 powder [Sigma-Aldrich, USA], 34.5 g MOPS [3-(N-morpholino) propanesulfonic acid, Sigma-Aldrich], 2 g dextrose, in 1 L distilled water, with pH adjusted to 7.0 with NaOH), spider medium (10 g nutrient broth [Himedia], 10 g mannitol [Panreac, Spain], 2 g K2HPO4, in 1 L distilled water, adjusted to pH 7.2 with H3PO4), YNB (0.17% yeast nitrogen base w/o amino acids [Bioshop], 0.5% (NH4)2SO4, 2% dextrose) and modified Dixon medium (36 g malt extract [Merck, Germany], 20 g desiccated oxbile [Sigma-Aldrich], 10 mL Tween 40 [Sigma-Aldrich], 6 g peptone [Bioshop], 2 mL glycerol [Scharlab, Spain], 2 mL oleic acid [Sigma-Aldrich], in 1 L distilled water). All media were solidified by adding 2% agar (Bioshop) if needed, except mDixon medium (1.5% agar).
For synthesis reaction, N, N, N′, N′, N′′-pentamethyldiethylenetriamine [Sigma-Aldrich], alkyl bromides [Sigma-Aldrich], 1, 4-butanesultone [Sigma-Aldrich], and acetonitrile [POCh S. A., Poland] were purchased. All compounds were analytical reagent quality and used without further purification.
For synthesis reaction, N, N, N′, N′, N′′-pentamethyldiethylenetriamine [Sigma-Aldrich], alkyl bromides [Sigma-Aldrich], 1, 4-butanesultone [Sigma-Aldrich], and acetonitrile [POCh S. A., Poland] were purchased. All compounds were analytical reagent quality and used without further purification.
4
Oleuropein and Hydroxytyrosol Isolation Protocol
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Oleuropein (Ole) was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). Hydroxytyrosol (HT, 2-(3,4-dihydroxyphenyl) ethyl alcohol) was purchased from Tokyo Chemical Co. (Tokyo, Japan). Oleuropein aglycone (3,4-DHPEA-EDA, 3,4-dihydroxyphenylethyl elenolate dialdehydic form) was purified using ODS-preparative HPLC according to a previous study [32 (link)]. Homovanillic acid (HVA, 4-hydroxy-3-methoxyphenylacetic acid), homovanillyl alcohol (HVAOH, 4-hydroxy-3-methoxyphenethyl alcohol), MOPS [3-(N-morpholino)propanesulfonic acid], β-glucuronidase Type VII-A, and sulfatase Type H-1 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Trifluoroacetic acid (TFA) was obtained from Wako Pure Chemical Co. (Tokyo, Japan).
5
Antifungal Susceptibility Testing Protocol
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The following chemicals were purchased from different manufacturers: nystatin (Pharma Nostra, Rio de Janeiro, Brazil), fluconazole (Fagron, São Paulo, Brazil), ethanol (AlphaTec®, Santo André, Brazil), hexane, dichloromethane, and ethyl acetate (Cromato Produtos Químicos®, Diadema, Brazil), Sabouraud Dextrose Broth (Acumedia, San Bernardino, CA, USA), and Fetal Bovine Serum (Gibco®, São Paulo, Brazil). Dimethylsulphoxide (DMSO), crystal violet, RPMI 1640 (Roswell Park Memorial Institute Medium, Buffalo, New York), and MOPS (3-[N-Morpholino] propane sulfonic acid) were purchased from Sigma-Aldrich Co. (St. Louis, MI, USA). RPMI1640 medium (without NaHCO3 and with L-glutamine) was buffered with 0.165 M of MOPS to pH 7.4.
6
Broth Microdilution Antifungal Susceptibility
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The broth microdilution (BMD) antifungal susceptibility test was performed according to M27-A3 protocol using RPMI broth (pH 7.0) buffered with 0.165 M MOPS [3-(N-morpholino)propanesulfonic acid (Sigma-Aldrich, St Louis, MO, USA) [57 ]. Compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and tested at concentrations ranging from 1.95 to 1000 μg/mL. The yeasts and compounds were incubated in 96-well culture plates at 35 °C for 24 h, and the results were examined visually. The minimum inhibitory concentration (MIC) of each compound was determined as the concentration that inhibited 50% of fungal growth [58 ].
7
Hydrolytic Activity of LacA β-Galactosidase
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Lactose monohydrate and acetonitrile (HPLC grade) were supplied by VWR (Barcelona, Spain). MOPS (3-(N-morpholino) propanesulfonic acid), xylitol, phenyl-βglucoside, maltulose, maltose, cellobiose, mannitol, sorbitol, sucrose, and pNP-β-Dgalactopyranoside were purchased from Sigma Aldrich (Stenheim, Germany).
Erythritol, isomaltulose, gentiobiose, isomaltose, lactulose and 2'-fucosyl-lactose, were purchased from Carbosynth (Berkshire, UK). KH2PO4 and K2HPO4 were supplied by Merck (Steinheim, Germany). All other chemicals were of analytical grade. The hydrolytic activity of LacA β-galactosidase (1.16 U mL -1 ) was also assayed by incubation at 30⁰C of different carbohydrates such as lactose, lactulose, cellobiose, gentiobiose, maltose, isomaltulose and sucrose at 200 g L -1 with 300 μL of enzyme in 50 mM of MOPS buffer supplemented with 20 mM of NaCl (pH=7) to a final volume of 1 mL. In the case of 2-fucosyl-lactose, isomaltose, nigerose and maltulose the concentration was 0.5 g L -1 . Reactions were stopped at 24 hours by heating at 100ºC for 5 min and samples were stored at -20⁰C for the subsequent analysis. Reactions were carried out in triplicate (n = 3). All these hydrolytic reactions were monitored by GC-FID.
Erythritol, isomaltulose, gentiobiose, isomaltose, lactulose and 2'-fucosyl-lactose, were purchased from Carbosynth (Berkshire, UK). KH2PO4 and K2HPO4 were supplied by Merck (Steinheim, Germany). All other chemicals were of analytical grade. The hydrolytic activity of LacA β-galactosidase (1.16 U mL -1 ) was also assayed by incubation at 30⁰C of different carbohydrates such as lactose, lactulose, cellobiose, gentiobiose, maltose, isomaltulose and sucrose at 200 g L -1 with 300 μL of enzyme in 50 mM of MOPS buffer supplemented with 20 mM of NaCl (pH=7) to a final volume of 1 mL. In the case of 2-fucosyl-lactose, isomaltose, nigerose and maltulose the concentration was 0.5 g L -1 . Reactions were stopped at 24 hours by heating at 100ºC for 5 min and samples were stored at -20⁰C for the subsequent analysis. Reactions were carried out in triplicate (n = 3). All these hydrolytic reactions were monitored by GC-FID.
8
Enzymatic Assay for HRV 3C Protease
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Salts Na2HPO4.2H2O, NaH2PO4.H2O, NaCl, and NaClO4.H2O and buffers citrate phosphate, MOPS (3-(N-morpholino)propanesulfonic acid), HEPES, glycine and PBS (phosphate buffered-saline) were purchased from Sigma Aldrich. NaBr was obtained from Scheme 1. General scheme of HRV 3C protease. The catalytic mechanism of the cysteine protease is analogous to that of the serine protease in which the nucleofilic serine is replaced by the cysteine [29] (link).
Penta, Na2SO4 from Merck. Substrate for the enzyme assay, pentapeptide Glu-Ala-Leu-Phe-Gln conjugated to the chromogenic p-nitroanilide (pNA) through the amide bond (EALFQ-pNA), has been obtained from ThermoFischer with purity higher than 95%.
Penta, Na2SO4 from Merck. Substrate for the enzyme assay, pentapeptide Glu-Ala-Leu-Phe-Gln conjugated to the chromogenic p-nitroanilide (pNA) through the amide bond (EALFQ-pNA), has been obtained from ThermoFischer with purity higher than 95%.
9
Standardized Yeast Suspension Preparation
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Each frozen stock culture was inoculated onto Sabouraud Dextrose Broth (Difco, Milan, Italy) and incubated at 37 °C for 24 h, in an orbital shaker at 60 rpm. Cells were picked up and added to a tube containing RPMI 1640 broth medium with L-glutamine and without bicarbonate buffered to pH 7 with MOPS, 3-(N-morpholino)propanesulfonic acid (165 M, Sigma, Italy). A standardized suspension of 1•10 6 CFU/ml was obtained and immediately used.
10
Membrane Vesicle Transport Assay
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Membrane vesicles from
HEK293 cells overexpressing the human BCRP, P-gp, MRP2, MRP3, and
MRP4 as well as the stably transfected hOATP2B1-MDCK-II cells were
provided by Solvo Biotechnology (Budapest, Hungary). Cell culture
medium, i.e., Dulbecco’s modified Eagle’s medium (DMEM),
fetal bovine serum, 1% GlutaMAX-1, 100 U/mL penicillin, and 100 μg/mL
streptomycin were obtained from Sigma-Aldrich (St. Louis, MO). Furosemide,
diclofenac, estrone-3-sulfate, rosuvastatin, adenosine 5′-triphosphate
(ATP) disodium salt, adenosine 5′-monophosphate (AMP) monohydrate,
glutathione, tris[hydroxymethyl]aminomethane (tris-base), MgCl2, NaCl, sucrose, and 3-[N-morpholino] propane
sulfonic acid (MOPS) were purchased from Sigma-Aldrich (St. Louis,
MO). Bovine serum albumin (BSA), membrane protein extraction kit,
and trypsin digestion reagents were obtained from Thermo Fisher Scientific
(Rockford, IL). Hanks’ balanced salt solution (HBSS), phosphate
buffer saline (PBS), acetonitrile, and formic acid were purchased
from Thermo Fisher Scientific (Rockford, IL). The synthetic unlabeled
and stable labeled peptides were purchased from New England Peptides
(Boston, MA) and Thermo Fisher Scientific (Rockford, IL), respectively.
Multiscreen HTS Vacuum Manifold and 96-well filter plates with class
B glass fiber filters were purchased from EMD Millipore (Billerica,
MA).
HEK293 cells overexpressing the human BCRP, P-gp, MRP2, MRP3, and
MRP4 as well as the stably transfected hOATP2B1-MDCK-II cells were
provided by Solvo Biotechnology (Budapest, Hungary). Cell culture
medium, i.e., Dulbecco’s modified Eagle’s medium (DMEM),
fetal bovine serum, 1% GlutaMAX-1, 100 U/mL penicillin, and 100 μg/mL
streptomycin were obtained from Sigma-Aldrich (St. Louis, MO). Furosemide,
diclofenac, estrone-3-sulfate, rosuvastatin, adenosine 5′-triphosphate
(ATP) disodium salt, adenosine 5′-monophosphate (AMP) monohydrate,
glutathione, tris[hydroxymethyl]aminomethane (tris-base), MgCl2, NaCl, sucrose, and 3-[N-morpholino] propane
sulfonic acid (MOPS) were purchased from Sigma-Aldrich (St. Louis,
MO). Bovine serum albumin (BSA), membrane protein extraction kit,
and trypsin digestion reagents were obtained from Thermo Fisher Scientific
(Rockford, IL). Hanks’ balanced salt solution (HBSS), phosphate
buffer saline (PBS), acetonitrile, and formic acid were purchased
from Thermo Fisher Scientific (Rockford, IL). The synthetic unlabeled
and stable labeled peptides were purchased from New England Peptides
(Boston, MA) and Thermo Fisher Scientific (Rockford, IL), respectively.
Multiscreen HTS Vacuum Manifold and 96-well filter plates with class
B glass fiber filters were purchased from EMD Millipore (Billerica,
MA).
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