N-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxy sulfosuccinimide (sulfo-NHS), glucose oxidase (GOx), and horseradish peroxidase (HRP) were purchased from Sigma (Selangor, Malaysia). The solution of PBS buffer, pH 7, were prepared in house by the addition of disodium hydrogen diphosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4) until it reaches pH 7. Both Na2HPO4 and NaH2PO4 were purchased from Scharlau (Selangor, Malaysia). All the starting materials for enzyme conjugation and glucose detection ordered were of highest grade and were used directly without any further purification.
Na2hpo4
Manufactured by Scharlab
Sourced in Spain
Sodium phosphate dibasic (Na2HPO4) is an inorganic chemical compound used as a lab reagent. It is a white, crystalline powder that is soluble in water. Na2HPO4 is commonly used as a buffer solution, pH adjusting agent, and as a source of sodium and phosphate ions in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Nah2po4, by Scharlab (6 mentions)
Kh2po4, by Scharlab (4 mentions)
Sodium chloride (nacl), by Scharlab (4 mentions)
Potassium chloride (kcl), by Scharlab (2 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
Gallic acid, by ITW Reagents (1 mentions)
Ascorbic acid, by Avantor (1 mentions)
Fluoresceine, by Merck Group (1 mentions)
Acetonitrile, by Avantor (1 mentions)
Hesperidin, by Merck Group (1 mentions)
Apigenin, by Merck Group (1 mentions)
Ethanol, by ITW Reagents (1 mentions)
Hesperetin, by Merck Group (1 mentions)
Trans cinnamic acid, by Merck Group (1 mentions)
Na2co3, by Avantor (1 mentions)
Chlorogenic acid, by Merck Group (1 mentions)
Trolox, by Merck Group (1 mentions)
Catechin, by Merck Group (1 mentions)
P coumaric acid, by Merck Group (1 mentions)
Caffeic acid, by Merck Group (1 mentions)
13 protocols using na2hpo4
1
Enzyme Conjugation and Glucose Detection
2
Quantification of Polyphenol Compounds
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HCl (hydrochloric acid), 2,6-DCFI (2,6-dichloroindophenol), FCR (Folin–Ciocalteu reagent), K2S2O8 (potassium persulfate), ethanol, and gallic acid were purchased from Panreac (Barcelona, Spain). Na2HPO4 (sodium hydrogen phosphate), KH2PO4 (potassium dihydrogen phosphate), and NaHCO3 (sodium hydrogen carbonate) were obtained from Scharlab (Barcelona, Spain). Na2CO3 (sodium carbonate), acetone, acetonitrile, and ascorbic acid were provided by VWR International (Lovaina, Belgium). Formic acid was purchased from Merck (Darmstadt, Germany). Acetic acid, hexane, and methanol were supplied by J.T. Baker (Deventer, The Netherlands). HPO3 (metaphosphoric acid), Trolox ((+/−)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)), AAPH (2,2′-azobis (2-methyl)propionamidine), fluoresceine, protocatechuic acid, (+)-catechin, caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, trans-cinnamic acid, naringin, hesperetin, hesperidin, and apigenin were obtained from Sigma-Aldrich (St. Louis, USA). Rutin trihydrate and quercetin were obtained from HWI Analytik GmbH (Ruelzheim, Germany). Analytical grade chemicals and distilled water were used.
3
Synthesis and Characterization of Oligonucleotide Probes
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Highest available
grade reagents were used. NaH2PO4, Na2HPO4, NaCl, and KCl were purchased from Scharlab. Tris-(hydroxymethyl)aminomethane
hydrochloride (Tris–HCl), mercaptohexanol (MCH), hydroquinone,
H2O2 (30%, w/v), ethylenediaminetetraacetic
acid (EDTA), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich
(Germany).
ProtA-polyHRP40, a native ProtA labeled with a homopolymer
containing 40 HRP molecules (antibodies-online), and an anti-DNA–RNA
hybrid antibody (AbS9.6) from Kerafast (Boston, MA) were commercially
available. A commercial blocker solution, consisting of a phosphate-buffered
saline (PBS) containing 1% w/v purified casein, from Thermo Scientific
(Waltham, MA) was also used.
The buffer solutions were prepared
with Milli-Q water (18 MΩ
cm at 25 °C) and sterilized after their preparation to avoid
RNase degradation. Phosphate-buffered saline (PBS) consisted of 0.01
M phosphate-buffered solution containing 137 mM NaCl and 2.7 mM KCl
(pH 7.5), Tris–EDTA buffer (pH 8.0) formed by mixing 10 mM
Tris–HCl, 1 mM EDTA, and 0.3 M NaCl (pH 8.0), and phosphate
buffer (0.05 M, pH 6.0) were used.
The synthetic oligonucleotides
used (sequences shown inTable 3 ) were purchased from
Sigma-Aldrich (Germany). Once received, they were reconstituted in
nuclease-free water to give a final concentration of 100 μM
and stored at −80 °C divided into small aliquots.
grade reagents were used. NaH2PO4, Na2HPO4, NaCl, and KCl were purchased from Scharlab. Tris-(hydroxymethyl)aminomethane
hydrochloride (Tris–HCl), mercaptohexanol (MCH), hydroquinone,
H2O2 (30%, w/v), ethylenediaminetetraacetic
acid (EDTA), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich
(Germany).
ProtA-polyHRP40, a native ProtA labeled with a homopolymer
containing 40 HRP molecules (antibodies-online), and an anti-DNA–RNA
hybrid antibody (AbS9.6) from Kerafast (Boston, MA) were commercially
available. A commercial blocker solution, consisting of a phosphate-buffered
saline (PBS) containing 1% w/v purified casein, from Thermo Scientific
(Waltham, MA) was also used.
The buffer solutions were prepared
with Milli-Q water (18 MΩ
cm at 25 °C) and sterilized after their preparation to avoid
RNase degradation. Phosphate-buffered saline (PBS) consisted of 0.01
M phosphate-buffered solution containing 137 mM NaCl and 2.7 mM KCl
(pH 7.5), Tris–EDTA buffer (pH 8.0) formed by mixing 10 mM
Tris–HCl, 1 mM EDTA, and 0.3 M NaCl (pH 8.0), and phosphate
buffer (0.05 M, pH 6.0) were used.
The synthetic oligonucleotides
used (sequences shown in
Sigma-Aldrich (Germany). Once received, they were reconstituted in
nuclease-free water to give a final concentration of 100 μM
and stored at −80 °C divided into small aliquots.
4
Preparation of Fungal and Bacterial Inocula
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The mold inocula were prepared by growing on potato dextrose agar (PDA; Scharlab, S.L.; Barcelona, Spain) at 25 °C for 7 days. Conidia were harvested by washing the surface of the plates with 3 mL of phosphate-buffered saline (PBS) containing 0.32 g/L NaH2PO4 (Scharlab, S.L.), 1.09 g/L Na2HPO4 (Scharlab, S.L.), and 9 g/L of NaCl (Fisher Scientific S.L.; Waltham, MA, USA). Each conidia suspension was quantified using a Thoma counting chamber (Blaubrand®; Wertheim, Germany) and adjusted to 105 spores/mL to be used as inoculum.
D. hansenii strains were incubated in yeast extract-sucrose broth (YES; 20 g/L yeast extract (Scharlab, S.L.)) and 125 g/L sucrose (Scharlab, S.L.) at 25 °C for 24 h under stirring conditions (150 rpm). After centrifuging, the pellet was resuspended in PBS and quantified using the Thoma counting chamber before adjusting to 106 cfu/mL.
E. faecium and S. vitulinus were cultured in brain heart infusion broth (BHI, Scharlab, S.L.) and incubated for 48 h at 30 °C under stirring (150 rpm). After their centrifugation, the pellets were resuspended in PBS and turbidimetrically adjusted to 106 cfu/mL.
The protein PgAFP was extracted by fast protein liquid chromatography (FPLC) using the method previously described by Delgado et al. [16 (link)].
D. hansenii strains were incubated in yeast extract-sucrose broth (YES; 20 g/L yeast extract (Scharlab, S.L.)) and 125 g/L sucrose (Scharlab, S.L.) at 25 °C for 24 h under stirring conditions (150 rpm). After centrifuging, the pellet was resuspended in PBS and quantified using the Thoma counting chamber before adjusting to 106 cfu/mL.
E. faecium and S. vitulinus were cultured in brain heart infusion broth (BHI, Scharlab, S.L.) and incubated for 48 h at 30 °C under stirring (150 rpm). After their centrifugation, the pellets were resuspended in PBS and turbidimetrically adjusted to 106 cfu/mL.
The protein PgAFP was extracted by fast protein liquid chromatography (FPLC) using the method previously described by Delgado et al. [16 (link)].
5
Acid Digestion and Phosphorus Determination
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The reagents used to carry out the acid digestions were purchased from SCP Science (Montreal, Quebec, Canada): HCl PlasmaPURE (34–37%), HNO3 PlasmaPURE (67–69%), and from Sigma-Aldrich (St. Louis, MO, USA): H2O2 (≥30%). On the other hand, to determine the concentration of P, the reagents were purchased from Labbox (Barcelona, Spain): H2SO4 (98%), from Panreac Química S.A (Castellar del Vallès, Barcelona, Spain): Na2HPO4 (100%), N2H6SO4 (99%), and from Scharlau (Sant Feliu de Llobregat, Barcelona, Spain): (NH4)6Mo7O24·4H2O (99%). A buffer was used to eliminate interferences associated with the measurement of magnesium, which was purchased from Merck KGaA (Darmstadt, Germany): CsCl, LaCl3 (100%). All the solutions were prepared using ultrapure water obtained by passing twice-distilled water through a Milli-Q system (18 MΩ/cm, Millipore, Bedford, MA, USA).
6
Luteolin-Loaded Phospholipid Nanoformulation
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Luteolin (LUT) was purchased from Beijing Mesochem Technology Co. Pvt. Ltd. (Beijing, China). Phospholipon® 90G (P-90G) (GmbH, Nattermannallee 1, Koln, Germany) is chemically phosphatidylcholine (PC) containing ascorbyl palmitate (0.1%). Span 60, Span 80 and Brij 35 were procured from Thermo-Fisher Scientific (Waltam, MA, USA). DMSO (VWR Chemicals, France), MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (Invitrogen, Thermo Fisher, USA), Advanced DMEM (Dulbecco’s Modified Eagle Medium) (Gibco, Life Technologies Ltd., London, UK), NaCl (sodium chloride), KCl (potassium chloride), Na2HPO4 (disodium hydrogen phosphate) and KH2PO4 (potassium dihydrogen phosphate) were procured from Scharlab S.L., Barcelona, Spain. Millipore water was used as an aqueous medium.
7
Porcine Eye-Based In Vitro Model for Ophthalmic Drugs
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Dexamethasone sodium phosphate (CAS 2392-39-4) and bromfenac sodium (CAS 91714-93-1) were purchased from Carbosynth (Compton, UK). Phosphate buffer saline (PBS pH 7.4) was prepared with the following composition: KCl 2.7 mM (Scharlau, Barcelona, Spain), NaCl 137 mM (Labkem, Barcelona, Spain), KH2PO4 1.8 mM (ITW Reagents—Barcelona, Spain) and Na2HPO4 10 mM (Scharlau, Barcelona, Spain). Phosphate buffer (pH 6) was prepared with the following composition: NaOH 1.15 mM (VWR, Paris, France) and KH2PO4 10 mM. Porcine eyes were provided by a local slaughterhouse (Compostelana de Carnes S.L.—Santiago de Compostela, Spain), transported immersed in PBS solution in an ice bath and used within three hours from eye collection.
8
Mould Strain Preservation and Inoculum Preparation
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Mould strains were maintained as stock cultures at −80 °C in PBS with 10% (v/v) glycerol as cryoprotectant. For this study, the inocula of each mould were prepared by growing on potato dextrose agar (PDA, Conda Pronadisa, Madrid, Spain) for 7 days at 25 °C. Conidia were harvested by rubbing the surface using saline phosphate buffer PBS; 0.32 g/L of NaH2PO4 (Scharlab S.L, Barcelona, Spain), 1.09 g/L of Na2HPO4 (Scharlab S.L.) and 0.9 of NaCl (Scharlab S.L.) with a glass rod. Spores were quantified by using a Thoma counting chamber BLAUBRAND® (Brand, Germany) and adjusting to 107 spores/mL to be used as inoculum.
9
Antifungal Protein Assay on Penicillium expansum
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Penicillium allii-sativi CECT 20922 (formerly Penicillium chrysogenum RP42C) isolated from drycured ham (Acosta et al., 2009) (link) was used as a source of the antifungal protein PgAFP. This protein was assayed upon five Penicillium expansum strains (Table 1), which previously showed susceptibility against PgAFP (Delgado et al., 2019a) . Each strain was 3-point inoculated on potato dextrose agar (PDA) plates and incubated for 10 days at 25 °C. Then, spores were harvested by addition of 4 mL of phosphate buffer saline (PBS) [0.32 g of NaH2PO4 (Scharlab, S.
L.), 1.09 g of Na2HPO4 (Scharlab, S.L.), 9 g of NaCl (Scharlab, S.L.), 1 L of distilled water],
scraping the plate surface with a glass spatula. Conidia concentrations were determined using a Thoma counting chamber Blaubrand® (Brand, Germany), visualising them in a microscope (NIKON, Japan) and adjusted to 10 5 spores/mL.
L.), 1.09 g of Na2HPO4 (Scharlab, S.L.), 9 g of NaCl (Scharlab, S.L.), 1 L of distilled water],
scraping the plate surface with a glass spatula. Conidia concentrations were determined using a Thoma counting chamber Blaubrand® (Brand, Germany), visualising them in a microscope (NIKON, Japan) and adjusted to 10 5 spores/mL.
10
Essential Oil Compound Effects on Nematodes
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Carvacrol (≥ 98% w/w), eugenol (≥ 98%), thymol (98.5% w/w), vanillin (≥ 98% w/w), sodium azide (NaN3) and 30% hydrogen peroxide (H2O2) solution (w/w) were obtained from Sigma-Aldrich (Spain). Dimethyl sulfoxide (DMSO), NaOH, sodium hypochlorite (NaClO), and all the other reagents used to prepare Nematode Growth Medium (NGM) agar (3 g/L NaCl, 2.5 g/L peptone, 17 g/L agar, 1 M potassium phosphate buffer [25 mL],
and [1 mL] 5% cholesterol in ethanol), K-medium (32 mM KCl, 51 mM NaCl) and M9 buffer (3 g/L KH2PO4, 6 g/L Na2HPO4, 5 g/L NaCl, 1 ml 1 M MgSO4) were purchased from Scharlab (Spain), except for cholesterol (95% w/w), which was supplied by Acros Organics (Spain). All the chemicals used in this study were of standard reagent grade.
Stock solutions of Carvacrol, eugenol, thymol and vanillin (2.5 M) were completely dissolved in DMSO and remained frozen until used. The final tested EOCs concentrations were prepared in K-medium (final DMSO concentration ≤ 0.6% (v/v)).
and [1 mL] 5% cholesterol in ethanol), K-medium (32 mM KCl, 51 mM NaCl) and M9 buffer (3 g/L KH2PO4, 6 g/L Na2HPO4, 5 g/L NaCl, 1 ml 1 M MgSO4) were purchased from Scharlab (Spain), except for cholesterol (95% w/w), which was supplied by Acros Organics (Spain). All the chemicals used in this study were of standard reagent grade.
Stock solutions of Carvacrol, eugenol, thymol and vanillin (2.5 M) were completely dissolved in DMSO and remained frozen until used. The final tested EOCs concentrations were prepared in K-medium (final DMSO concentration ≤ 0.6% (v/v)).
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