In vitro activity was assayed for 1 h at 30°C in 50 mM Tris pH 8, 150 mM ammonium acetate, 1 mM dithiothreitol (DTT), 2 mM MgCl2, 10% v/v glycerol. Bulk tRNAs (100 μM) were incubated with 5 μM of protein in a total volume of 50 μl and reaction was started with addition of 2 mM NADPH. Quenching was performed by adding 50 μl of acidic phenol (Sigma Aldrich) followed by centrifugation at 13 000 rpm for 10 min. tRNA in the aqueous phase were ethanol precipitated and further purified using a MicroSpin G-25 column (GE-healthcare). Dihydrouridine quantification was carried out by means of a colorimetric method as described previously (18 (link)). Briefly, samples were incubated at 40°C for 30 min after addition of 5 μl of 1 M KOH. The solutions were neutralized with 25 μl of 96% H2SO4 followed by 25 μl of a 3% solution 2,3-butanedione monoxime (Sigma Aldrich) and 25 μl of a saturated solution in N-Phenyl-p-phenylenediamine (Sigma-Aldrich). Samples were then heated at 95°C for 10 min and cooled to 55°C. Following addition of 50 μl of 1 mM FeCl3, a violet-red coloration appeared allowing quantification via absorption at 550 nm. Concentration of D in tRNA was determined by using a standard curve obtained with variable amounts of dihydrouracil.
N phenyl p phenylenediamine
Manufactured by Merck Group
Sourced in United States
N-phenyl-p-phenylenediamine is a chemical compound used in the manufacturing of various products. It is a pale yellow crystalline solid. The core function of this product is as a chemical intermediate in the production of dyes, rubber accelerators, and other industrial chemicals.
Automatically generated - may contain errors
Lab products found in correlation
Sodium alginate, by Merck Group (2 mentions)
P phenylenediamine, by Merck Group (2 mentions)
Microspin g 25 column, by GE Healthcare (1 mentions)
2 3 butanedione monoxime, by Merck Group (1 mentions)
Acidic phenol, by Merck Group (1 mentions)
Syringe filter, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine tetrahydrochloride hydrate, by Merck Group (1 mentions)
Dopamine hydrochloride, by Merck Group (1 mentions)
Recombinant mmp 2, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
N n dicyclohexylcarbodiimide, by Merck Group (1 mentions)
1 6 diphenyl 1 3 5 hexatriene dph, by Merck Group (1 mentions)
Anhydrous tetrahydrofuran thf, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Ethanol, by Avantor (1 mentions)
Chloroform, by Thermo Fisher Scientific (1 mentions)
Ammonium hydroxide, by Merck Group (1 mentions)
Dichloromethane, by Avantor (1 mentions)
Diethyl ether, by Merck Group (1 mentions)
Succinic anhydride, by Merck Group (1 mentions)
7 protocols using n phenyl p phenylenediamine
1
Quantification of Dihydrouridine in tRNA
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In vitro activity was assayed for 1 h at 30°C in 50 mM Tris pH 8, 150 mM ammonium acetate, 1 mM dithiothreitol (DTT), 2 mM MgCl2, 10% v/v glycerol. Bulk tRNAs (100 μM) were incubated with 5 μM of protein in a total volume of 50 μl and reaction was started with addition of 2 mM NADPH. Quenching was performed by adding 50 μl of acidic phenol (Sigma Aldrich) followed by centrifugation at 13 000 rpm for 10 min. tRNA in the aqueous phase were ethanol precipitated and further purified using a MicroSpin G-25 column (GE-healthcare). Dihydrouridine quantification was carried out by means of a colorimetric method as described previously (18 (link)). Briefly, samples were incubated at 40°C for 30 min after addition of 5 μl of 1 M KOH. The solutions were neutralized with 25 μl of 96% H2SO4 followed by 25 μl of a 3% solution 2,3-butanedione monoxime (Sigma Aldrich) and 25 μl of a saturated solution in N-Phenyl-p-phenylenediamine (Sigma-Aldrich). Samples were then heated at 95°C for 10 min and cooled to 55°C. Following addition of 50 μl of 1 mM FeCl3, a violet-red coloration appeared allowing quantification via absorption at 550 nm. Concentration of D in tRNA was determined by using a standard curve obtained with variable amounts of dihydrouracil.
+ Expand
In vitro activity was assayed for 1 h at 30°C in 50 mM Tris pH 8, 150 mM ammonium acetate, 1 mM dithiothreitol (DTT), 2 mM MgCl2, 10% v/v glycerol. Bulk tRNAs (100 μM) were incubated with 5 μM of protein in a total volume of 50 μl and reaction was started with addition of 2 mM NADPH. Quenching was performed by adding 50 μl of acidic phenol (Sigma Aldrich) followed by centrifugation at 13 000 rpm for 10 min. tRNA in the aqueous phase were ethanol precipitated and further purified using a MicroSpin G-25 column (GE-healthcare). Dihydrouridine quantification was carried out by means of a colorimetric method as described previously (18 (link)). Briefly, samples were incubated at 40°C for 30 min after addition of 5 μl of 1 M KOH. The solutions were neutralized with 25 μl of 96% H2SO4 followed by 25 μl of a 3% solution 2,3-butanedione monoxime (Sigma Aldrich) and 25 μl of a saturated solution in N-Phenyl-p-phenylenediamine (Sigma-Aldrich). Samples were then heated at 95°C for 10 min and cooled to 55°C. Following addition of 50 μl of 1 mM FeCl3, a violet-red coloration appeared allowing quantification via absorption at 550 nm. Concentration of D in tRNA was determined by using a standard curve obtained with variable amounts of dihydrouracil.
2
Polymerization Reactions with Aniline Dimer and DAB
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For polymerization reactions, cells were incubated in a mixture of polymer precursor solutions and hydrogen peroxide solution for 30 min unless specified and washed three times in Tyrode’s solution. All solutions were prepared fresh each time. Aniline dimer solution (3 mM) was prepared by dissolving 5.5 mg of N-phenyl-p-phenylenediamine (Sigma-Aldrich, 241393) in 10 ml of Tyrode’s solution ~20 hours at room temperature using a magnetic stir bar. DAB solution (10 mM) was prepared by dissolving 36 mg of 3,3′-diaminobenzidine tetrahydrochloride hydrate (Sigma-Aldrich, D5637) in 10 ml of Tyrode’s solution. The pH of the solution was adjusted to 7.35 by 1 M NaOH solution. When ready to perform the reactions, the above solutions were filtered with 0.45 μm syringe filters (Thermo Fisher Scientific), and H2O2 (EMD Millipore, 386790) was added to the solutions for a final concentration of 0.05 mM unless specified.
3
Multifunctional Hydrogel for Tissue Repair
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Sodium alginate (ALG, low viscosity, 4-12 cP), sodium periodate (NaIO4), dopamine hydrochloride (DOPA), N-phenyl-p-phenylenediamine, and recombinant MMP-2 were purchased from Sigma-Aldrich. Thiol-modified hyaluronic acid (HA-SH, Mw = 300 kDa, degree of thiol substitution = 33%) was purchased from ESI BIO, USA. MMP-SP (Seq: CGPLGGGRMSMPV RDGSC where C is a thiol-containing cysteine and GGRMSMPV is the MMP-cleavable sequence.) was purchased from Bankpeptide Ltd., Hefei, China. DPCA was purchased from Santa Cruz Biotechnology Ltd. L929 Mouse fibroblast cell line (L929) and rat cardioblasts (H9C2) were purchased from National Biomedical Experimental Cell Bank, Beijing, China.
4
Synthesis and Characterization of Functionalized Polymers
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Agarose low electrophoreses, Dimethyl sulfoxide (DMSO), epichlorohydrin and dimethyl formamide (DMF), N-hydroxysuccinimide (NHS) and N, N-di cyclohexyl carbodiimide (DCC) were purchased from Merck. N-Phenyl-p-Phenylenediamine, p-Phenylenediamine was received from Sigma-Aldrich.
5
Synthesis and Characterization of Polymeric Materials
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l -Lactide (LA, Serva Feinbiochemica) was recrystallized from toluene twice, polyethylene glycol monomethyl ether (mPEG2k, Sigma-Aldrich) was dried under reduced pressure, and stannous 2-ethylhexanoate (Sn(Oct)2, ∼95%, Sigma-Aldrich) was dried over molecular sieves. Dicyclohexylcarbodiimide (DCC), 4-(dimethylamino)pyridine (DMAP), ammonium hydroxide, hydrochloric acid (37%), succinic anhydride, p-phenylenediamine, N-phenyl-p-phenylenediamine, and phenyl hydrazine (97%) were all obtained from Sigma-Aldrich and used without further purification; ammonium persulfate, ethanol, and dichloromethane were obtained from VWR. Dimethylformamide (DMF, HPLC, VWR) was dried over CaH2 and distilled under reduced pressure before use. Methanol (MeOH, HPLC, Fisher Scientific), trichloroacetyl isocyanate (96%, Sigma-Aldrich), 1,6-diphenyl-1,3,5-hexatriene (DPH, 98%, Sigma-Aldrich), chloroform (99%, Fisher Scientific), diethyl ether (99.8%, Sigma-Aldrich), anhydrous tetrahydrofuran (THF, Sigma-Aldrich), and hexamethylene diisocyanate (HMDI, 99%, Sigma-Aldrich) were all used without further purification.
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6
Detailed Preparation of Biomolecule Samples
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Human insulin, myoglobin
from equine skeletal
muscle, 1,5-diaminonaphtalene (1,5-DAN; handle with care, check material
safety data sheet), N-phenyl-p-phenylenediamine
(PPDA; handle with care, check material safety data sheet), sinapinic
acid (SA), and acetonitrile (ACN; MS grade) were purchased from Sigma-Aldrich.
Trifluoroacetic acid (TFA) was purchased from Thermo Fisher Scientific.
Formic acid (FA) was purchased from Honeywell. Ultrapure water (Milli-Q;
Merck) was used throughout this study. 2-Propanol was purchased from
Biosolve BV. NIST mAb standard (HzIgG1-kappa, NS0) was provided by
the Consortium for Top-Down Proteomics (CTDP).39 (link),59 A 5 mg/mL solution of human insulin was prepared in water/TFA,
99.95%:0.05%. A 2 mg/mL solution of myoglobin was prepared in water.
NIST mAb standard was diluted from an initial concentration of 10
mg/mL to a final concentration of 2 mg/mL using water. A saturated
solution of 1,5-DAN was prepared in water/ACN/FA, 50%:49.95%:0.05%.
A saturated solution of PPDA was prepared in water/2-propanol/TFA,
50%:49.95%:0.05%. A 10 mg/mL solution of SA was prepared in water/ACN,
50%:50%. MALDI matrices were freshly prepared prior to the analysis.
from equine skeletal
muscle, 1,5-diaminonaphtalene (1,5-DAN; handle with care, check material
safety data sheet), N-phenyl-p-phenylenediamine
(PPDA; handle with care, check material safety data sheet), sinapinic
acid (SA), and acetonitrile (ACN; MS grade) were purchased from Sigma-Aldrich.
Trifluoroacetic acid (TFA) was purchased from Thermo Fisher Scientific.
Formic acid (FA) was purchased from Honeywell. Ultrapure water (Milli-Q;
Merck) was used throughout this study. 2-Propanol was purchased from
Biosolve BV. NIST mAb standard (HzIgG1-kappa, NS0) was provided by
the Consortium for Top-Down Proteomics (CTDP).39 (link),59 A 5 mg/mL solution of human insulin was prepared in water/TFA,
99.95%:0.05%. A 2 mg/mL solution of myoglobin was prepared in water.
NIST mAb standard was diluted from an initial concentration of 10
mg/mL to a final concentration of 2 mg/mL using water. A saturated
solution of 1,5-DAN was prepared in water/ACN/FA, 50%:49.95%:0.05%.
A saturated solution of PPDA was prepared in water/2-propanol/TFA,
50%:49.95%:0.05%. A 10 mg/mL solution of SA was prepared in water/ACN,
50%:50%. MALDI matrices were freshly prepared prior to the analysis.
7
Oxidized Sodium Alginate-Gelatin Hydrogel
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Gelatin (type A, Bloom 300), sodium alginate (medium viscosity grade) and N-phenyl-p-phenylenediamine were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). OA, with an oxidation degree (OD, the percentage of oxidized structure unit in sodium alginate) of 58.10% and weight-average Mw of ~2.64×104 g/mol (data from gel permeation chromatograph), was prepared through periodate oxidation of sodium alginate in a water–ethanol mixture following the published method.19 (link),20 (link) The fertilized chicken eggs were obtained from the Institute of Livestock Science, South China Agricultural University (Guangzhou, People’s Republic of China). Sprague Dawley rats were purchased from the laboratory animal center of Sun Yat-sen University. MSCs were obtained from neonatal Sprague Dawley rats (1–3 days old), their isolation, purification, culture, and passage were performed according to the standard method.21 (link) All animal care and experimental procedures were conducted according to institutional animal care and use guidelines, and approved by the Animal Ethics Committee of Sun Yat-sen University. The other reagents were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, People’s Republic of China). All chemicals were used as received unless specified.
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