Sodium hyaluronate (72 kDa, Lifecore Biomedical), methacrylic anhydride (Sigma-Aldrich, 94%), copper(II) bromide (CuBr2, Sigma-Aldrich, 99%), tris(2-pyridylmethyl)amine (TPMA, Sigma-Aldrich, 98%), 2-hydroxyethyl 2-bromoisobutyrate (HEBIB, Sigma-Aldrich, 95%), poly(ethylene glycol) bis(2-bromoisobutyrate) (PEGBBIB, Mn,avg = 700 g/mol, Sigma-Aldrich, PDI ≤ 1.1) sodium dl -lactate (NaC3H5O3, TCI, 60% in water), sodium fumarate (Na2C4H2O4, VWR, 98%), HEPES buffer solution (C8H18N2O4S, VWR, 1 M in water, pH = 7.3), potassium phosphate dibasic (K2HPO4, Sigma-Aldrich), potassium phosphate monobasic (KH2PO4, Sigma-Aldrich), sodium chloride (NaCl, VWR), ammonium sulfate ((NH4)2SO4, Fisher Scientific), magnesium(II) sulfate heptahydrate (MgSO4·7H2O, VWR), trace mineral supplement (ATCC), casamino acids (VWR), silicone oil (Alfa Aesar), isopropyl ß-d -1-thiogalactopyranoside (IPTG, Teknova), kanamycin sulfate (C18H38N4O15S, Growcells), nail polish (Electron Microscopy Sciences), BacLight Live/Dead Stain (Invitrogen), deuterium oxide (D2O, Sigma-Aldrich, 99.9%), hyaluronidase from bovine testes (Sigma-Aldrich, Type I–S, 400–1000 units/mg), hydrogen peroxide solution (H2O2, Sigma-Aldrich, 30% in H2O), and 3,3′,5,5′-tetramethylbenzidine (TMBZ, Alfa Aesar, 98%) were used as received. All media components were autoclaved or sterilized using 0.2 μm PES filters.
Nh4 2so4
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Ammonium sulfate, (NH4)2SO4, is a chemical compound commonly used in laboratory settings. It is a white, crystalline salt that is soluble in water. Ammonium sulfate is a source of ammonium ions and sulfate ions, which are important for various chemical and biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Ktaprime plus chromatography system, by GE Healthcare (2 mentions)
Hitrap protein g hp column, by GE Healthcare (2 mentions)
Agarose, by Thermo Fisher Scientific (2 mentions)
Fm4 64, by Thermo Fisher Scientific (2 mentions)
Softworx v5, by Cytiva (2 mentions)
Kh2po4, by Thermo Fisher Scientific (2 mentions)
K2hpo4 3h2o, by Thermo Fisher Scientific (2 mentions)
C6h5o7na3 2h2o, by Thermo Fisher Scientific (2 mentions)
Sytox green, by Thermo Fisher Scientific (2 mentions)
Lb broth, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
K2hpo4, by Merck Group (1 mentions)
Cubr2, by Merck Group (1 mentions)
Mgso4 7h2o, by Avantor (1 mentions)
Live dead baclight stain, by Thermo Fisher Scientific (1 mentions)
Kh2po4, by Merck Group (1 mentions)
Tryptophan, by Merck Group (1 mentions)
Methionine, by Merck Group (1 mentions)
Threonine, by Merck Group (1 mentions)
Proline, by Merck Group (1 mentions)
10 protocols using nh4 2so4
1
Hyaluronic Acid Hydrogel Synthesis and Characterization
2
Amino Acid Spectrophotometric Analysis
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All
reagents were of analytical
grade and used without further purification. All of the reagents were
prepared using Millipore water (18.2 MΩ·cm). Proline (99%),l -leucine (98%), phenylalanine (98%), threonine (98%), arginine
(98%), asparagine (98%), glycine (99%), valine (98%), alanine (98%),
methionine (98%), tryptophan (98%), histidine (98%), acrylamide (99%),
NaOH, CH3COONa·3H2O (99.5%), Na2HPO4·H2O (99%), NaH2PO4·2H2O (99%), and KCl (99%) were purchased
from Merck. Methylene blue monohydrate (96%) was purchased from Acros;
HCl (33–37%) and (NH4)2SO4 (99%) were purchased from Fisher Scientific.
reagents were of analytical
grade and used without further purification. All of the reagents were
prepared using Millipore water (18.2 MΩ·cm). Proline (99%),
(98%), asparagine (98%), glycine (99%), valine (98%), alanine (98%),
methionine (98%), tryptophan (98%), histidine (98%), acrylamide (99%),
NaOH, CH3COONa·3H2O (99.5%), Na2HPO4·H2O (99%), NaH2PO4·2H2O (99%), and KCl (99%) were purchased
from Merck. Methylene blue monohydrate (96%) was purchased from Acros;
HCl (33–37%) and (NH4)2SO4 (99%) were purchased from Fisher Scientific.
3
Affinity Chromatography for mAb Purification
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The purification of mAbs was performed by affinity chromatography. Briefly, novel mouse mAbs were purified by salt fractionation (solid ammonium sulphate ([NH4]2SO4; 45% of saturation - 270 g/L; Fisher Scientific) followed by affinity chromatography using a 5 ml HiTrap Protein G HP column in an ÄKTAprime plus chromatography system (both from GE Healthcare, UK). The antibody sample was loaded into the HiTrap Protein G HP column pre-equilibrated with 25 ml binding buffer (0.1 M phosphate buffer, 0.15 M NaCl, pH 7.4) at a flow rate of 1 ml/min. Unbound proteins were removed by washing the column with 50 ml binding buffer and bound antibodies were eluted with 25 ml elution buffer (0.1 M glycine, pH 2.5). Selected fractions were pooled together and subjected to buffer exchange with PBS using the HiPrep 26/10 Desalting column (GE Healthcare Life Sciences). Following buffer exchange, the purified antibodies were filtered through a 0.2 μM syringe filter (Merck Millipore, UK), aliquoted and stored at -20°C for further studies.
4
GAS Cell Morphology Visualization
5
Purification of Novel Mouse Monoclonal Antibodies
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Isotyping of novel mAbs was determined using a mouse mAb isotyping kit (AbD Serotec, UK) according to the manufacturer’s protocol and the antibodies were purified by affinity chromatography as described previously9 (link). Briefly, novel mouse mAbs were purified by salt fractionation (solid ammonium sulphate ([NH4]2SO4; 45% of saturation − 270 g/L; Fisher Scientific) followed by affinity chromatography using a 5 ml HiTrap Protein G HP column in an ÄKTAprime plus chromatography system (GE Healthcare, UK), as described previously9 (link). The purified antibodies were filtered through a 0.2 µm syringe filter (Merck Millipore, UK), aliquoted and stored at −20 °C for further studies.
6
GAS Cell Morphology Visualization
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To visualize the morphology of GAS cells, bacteria from late stationary (overnight cultures) or exponential (4 hours of growth post 1:20 back-dilution of overnight culture in fresh medium) phase of growth were mixed by vortexing. 6 μl of each cell suspension was added to 1.5 μl dye mix composed of 60 μg/mL FM4–64 (Life Technologies Corporation), 10 μg/mL DAPI (Sigma Aldrich), and 2.5 μM SYTOX Green (Life Technologies Corporation) in 1X T-base (2 g (NH4)2SO4 [Fisher Scientific], 18.3 g K2HPO4 ⋅ 3H2O [Fisher Scientific], 6 g KH2PO4 [Fisher Scientific], 1 g C6H5O7Na3 ⋅ 2H2O [Fisher Scientific] per 1 L of ddH2O) and transferred onto an agarose pad (20% LB broth, 1% agarose [Fisher Scientific]). Samples were air-dried under a fume hood (care was taken to prevent over-drying). Cells were visualized on an Applied Precision DV Elite optical sectioning microscope equipped with a Photometrics CoolSNAP-HQ2 camera. Pictures were deconvolved using SoftWoRx v5.5.1 (Applied Precision). Images for figures were prepared using FIJI. Bacterial cell diameters from multiple pictures were quantified with CellProfiler (Kamentsky et al., 2011 (link)) on two separate occasions.
7
Capsule and LOS Outer Core Typing
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For the determination of capsule and lipooligosaccaharide (LOS) outer core (OC) locus types, a conventional polymerase chain reaction (PCR) was undertaken using primers listed in Supplementary Table S2 . Conventional PCR volume was 20 μl and consisted of 2 μl of 10x Taq DNA polymerase reaction buffer with (NH4)2SO4 (Thermo Fisher Scientific), 2 mM MgCl2, 0.4 mM of each dNTPs, 400 nM of primers, 0.2 U Taq DNA polymerase (Thermo Fisher Scientific) and 1 μl of DNA template. Tm was calculated based on the primers sequences.
8
Pilot Study of Ammonia Removal from Wastewater
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The pilot study was carried out on site in a meat processing plant located in ABP Food Group, Kilcommon, Co. Tipperary, Ireland. Figure 9 shows the conventional method of treatment which is being used in the plant using biological nitrification in an aerobic tank. (NH3) rich WW was obtained from RCWW produced by a rendering plant processing animal products. The (NH3) stripping unit and membrane materials (PP and PTFE) were designed and sourced from BLUE-tec bv, Industrieweg 16, 6871 KA, Renkum, The Netherlands. Bulk NaOH (30%) (w/w) and H2SO4 (96%) (w/w) which were of analytical grade were procured from Brenntag Chemicals Distribution LTD, Ireland. (NH4)2SO4 (99%) (w/w%) was procured from Fischer, Ireland and is of an analytical grade.
9
Second Strand cDNA Synthesis
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49 μL of the second strand mix, containing 33.5 μL water, 12 μL 5x second strand buffer [100 mM Tris-HCl (pH 6.9), 23 mM MgCl2, 450 mM KCl, 0.75 mM β-NAD, 50 mM (NH4)2 SO4, Invitrogen, Cat. # 10812–014], 1.2 μL 10 mM dNTPs, 0.4 μL E. coli ligase (Invitrogen, Cat. # 18052–019), 1.5 μL DNA polymerase I (Invitrogen, Cat. # 18010–025), and 0.4 μL RNase H (Invitrogen, Cat. # 18021–071), was added to the product from the previous step. The mixture was incubated at 16 °C for 2 h. cDNA was purified with 1x AMPure XP DNA beads (Beckman Coulter, Cat. # A63881) and eluted in 24 μL nuclease-free water that was subsequently concentrated to 6.4 μL.
10
Genotyping 5-HTTLPR Polymorphism
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Genomic DNA was extracted using a whole blood genomic DNA extraction kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instructions, and then analyzed with 0.8% agarose gel electrophoresis. Amplification primers for the promoter region of the 5-HTT gene were as follows: Forward primer, 5′-GCGCTCCTGCATCCCCCATTA-3′; and reverse primer, 5′-GGGATGCGGGGGAATACTGGT-3′. 5-HTTLPR polymorphism in the 5-HTT promoter region was assessed using PCR amplification. A total of 25 μl PCR reaction solution was prepared by mixing 10X PCR reaction buffer [containing 2.0 mmol/l (NH4)2SO4 and MgCl2; Invitrogen Life Technologies, Carlsbad, CA, USA], 0.16 μmol/l each primer, 1.5 units Taq enzymes and 160 μmol/l deoxyribonucleotide triphosphates. The PCR conditions were as follows: Denaturation at 95°C for 2 min, followed by 35 cycles of 95°C for 60 sec, 62°C for 60 sec and 72°C for 60 sec, and then extension at 72°C for 10 min. The PCR products were separated by 2% agarose gel electrophoresis, and the gel was visualized and analyzed by a Bio-Rad image analysis system (Bio-Rad, Hercules, CA, USA). Two different allelic fragments with the lengths of 297 bp (L) and 253 bp (S) were detected among the PCR products, and the genotype was determined accordingly (Fig. 1 ) (14 (link)).
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