All activity assays were performed in duplicate or triplicate, on 96-well microtiter plates, and analyzed with a microplate reader. Peroxidase activity, with 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma, Oakville, ON, Canada), was measured as described previously [26 (link)]. Briefly, 10 μL of sample was combined with 80 μL 0.75 mM H2O2 (Sigma) and 110 μL TMB solution (2.9 mM TMB in 14.5% DMSO (Sigma), and 150 mM sodium phosphate buffer at pH 5.4), and the plate was incubated at 37 °C for 5 min. The reaction was stopped by adding 50 μL of 2 M H2SO4 (Sigma), and absorption was measured at 450 nm, to estimate MPO activity [41 (link),93 (link)].
3 3 5 5 tetramethylbenzidine (tmb)
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, China, Italy, Sao Tome and Principe, Spain, Macao, Canada, India, Australia, Brazil, Japan, France, Netherlands, Poland
TMB is a chromogenic substrate used in enzyme-linked immunosorbent assays (ELISAs) to detect and quantify the presence of specific analytes. It undergoes a color change upon reaction with the enzyme-labeled detection antibody, allowing for the visualization and measurement of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (61 mentions)
Tween 20, by Merck Group (26 mentions)
Sodium hydroxide (naoh), by Merck Group (19 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (16 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (16 mentions)
N hydroxysuccinimide, by Merck Group (13 mentions)
Phosphate buffered saline (pbs), by Merck Group (13 mentions)
Microplate reader, by Bio-Rad (12 mentions)
Horseradish peroxidase, by Merck Group (12 mentions)
Ethanol, by Merck Group (11 mentions)
Hydrochloric acid (hcl), by Merck Group (11 mentions)
Maxisorp plate, by Thermo Fisher Scientific (11 mentions)
Maxisorp, by Thermo Fisher Scientific (10 mentions)
Hexadecyltrimethylammonium bromide, by Merck Group (10 mentions)
Hydrogen peroxide h2o2, by Merck Group (9 mentions)
Sodium acetate, by Merck Group (9 mentions)
Acetic acid, by Merck Group (9 mentions)
Nunc maxisorp, by Thermo Fisher Scientific (9 mentions)
Sucrose, by Merck Group (8 mentions)
Poly l lysine (pll), by Merck Group (8 mentions)
781 protocols using 3 3 5 5 tetramethylbenzidine (tmb)
1
Quantitative Peroxidase Activity Assay
2
Myeloperoxidase Activity Assay for Cornea Samples
3
Colorimetric Enzyme Assay using TMB Substrate
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All used reagents are analytical grade from commercial sources. FeCl3·6H2O, TMB, H2O2 (30%), NaOH, LOx from Aeroccoccus viridans, Zn(O2CH3)2·2H2O, anhydrous sodium acetate, acetic acid, 3,3,5,5-tetramethylbenzidine TMB, and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich. Phosphate buffer solution (PBS) was prepared using Na2HPO4 and KH2PO4 (pH 7.4) from J.T. Baker. Acetate buffer solution (HAc-NaAc) was prepared from acetic acid and sodium acetate (pH 5) obtained from J. T. Baker. All aqueous solutions were prepared using deionized water DI (σ ≥ 18 MΩ cm).
4
Bismuth-Copper Nanoparticle Synthesis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
Materials. Bismuth nitrate pentahydrate (Bi(NO3)3·5H2O, 99%), copper nitrate trihydrate (Cu(NO3)2·3H2O, 99%), TAA, dimethyl sulfoxide (DMSO), nitric acid (HNO3), methylene blue (MB, ≥82%) were purchased from Sinopharm Chemical Reagent Co., Ltd (China). DATS (≥98%) was bought from Beijing Jin Ming Biotechnology Co., Ltd. BSA, GSH, 3,3′,5,5′-tetramethylbenzidine (TMB), terephthalic acid (TPA) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) were obtained from Sigma-Aldrich. No further purification for all used reagents were operated. The catalog numbers of reagents: Bi(NO3)3·5H2O (Sinopharm Chemical Reagent Co., Ltd, catalog number: 80018318), Cu(NO3)2·3H2O (Sinopharm Chemical Reagent Co., Ltd, catalog number: 10007916), TAA (Sinopharm Chemical Reagent Co., Ltd, catalog number: 30177714), DMSO (Sinopharm Chemical Reagent Co., Ltd, catalog number: 30072428), DATS (Beijing Jin Ming Biotechnology Co., Ltd, catalog number: D60006), nitric acid (Sinopharm Chemical Reagent Co., Ltd, catalog number: 10014528), BSA (Sigma-Aldrich, B265993-25g), MB (Sinopharm Chemical Reagent Co., Ltd, catalog numbers: 71024544), GSH (Sigma-Aldrich, G105427-5g), 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich, T100417), TPA (Sigma-Aldrich, P108506-100g), 5,5′-dithiobis-(2-nitrobenzoic acid) (Sigma-Aldrich, D105559).
+ Expand
Materials. Bismuth nitrate pentahydrate (Bi(NO3)3·5H2O, 99%), copper nitrate trihydrate (Cu(NO3)2·3H2O, 99%), TAA, dimethyl sulfoxide (DMSO), nitric acid (HNO3), methylene blue (MB, ≥82%) were purchased from Sinopharm Chemical Reagent Co., Ltd (China). DATS (≥98%) was bought from Beijing Jin Ming Biotechnology Co., Ltd. BSA, GSH, 3,3′,5,5′-tetramethylbenzidine (TMB), terephthalic acid (TPA) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) were obtained from Sigma-Aldrich. No further purification for all used reagents were operated. The catalog numbers of reagents: Bi(NO3)3·5H2O (Sinopharm Chemical Reagent Co., Ltd, catalog number: 80018318), Cu(NO3)2·3H2O (Sinopharm Chemical Reagent Co., Ltd, catalog number: 10007916), TAA (Sinopharm Chemical Reagent Co., Ltd, catalog number: 30177714), DMSO (Sinopharm Chemical Reagent Co., Ltd, catalog number: 30072428), DATS (Beijing Jin Ming Biotechnology Co., Ltd, catalog number: D60006), nitric acid (Sinopharm Chemical Reagent Co., Ltd, catalog number: 10014528), BSA (Sigma-Aldrich, B265993-25g), MB (Sinopharm Chemical Reagent Co., Ltd, catalog numbers: 71024544), GSH (Sigma-Aldrich, G105427-5g), 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich, T100417), TPA (Sigma-Aldrich, P108506-100g), 5,5′-dithiobis-(2-nitrobenzoic acid) (Sigma-Aldrich, D105559).
5
Electrochemical Detection of Heavy Metals
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemicals used in this work were of analytical grade and used without further purification. Sodium acetate buffer solution (0.1 M, pH 5) was prepared from sodium acetate and acetic acid. Phosphate buffer solution (0.1 M, pH 7) was prepared from sodium hydrogen phosphate and sodium dihydrogen phosphate. 3,3′,5,5′-Tetramethylbenzidine (TMB) ready to use supersensitive liquid chromogenic substrate, containing 1.25 mM TMB and 2.21 mM H2O2, was purchased from Sigma-Merck, Germany. Horseradish peroxidase (EC 1.11.1.7 type VI-A from horseradish 1280 units mg−1, type XII from horseradish 1000 units mg−1), trehalose, and EDTA were also purchased form Sigma-Merck, Darmstadt, Germany.
The standard solution of Cd2+ was purchased as an atomic absorption standard solution (1000 µg/mL) from Alfa Aesar, Kandel, Germany, and Cu2+ was purchased as an atomic absorption standard solution (1000 mg/L) from Sigma-Aldrich, Darmstadt, Germany.
The pre-activated immunodyne ABC nylon membrane (⌀ = 3 µm) was supplied by Pall Europe Limited, Portsmouth, UK.
Screen-printed electrodes (SPEs) were obtained as a gift from professor F. Arduini of Tor-Vergata University (Italy), they consisted of a graphite working electrode, a silver/ silver chloride reference electrode (Ag/AgCl), and a counter electrode; 3 mm in diameter and with an active surface of about 0.07 cm2.
The standard solution of Cd2+ was purchased as an atomic absorption standard solution (1000 µg/mL) from Alfa Aesar, Kandel, Germany, and Cu2+ was purchased as an atomic absorption standard solution (1000 mg/L) from Sigma-Aldrich, Darmstadt, Germany.
The pre-activated immunodyne ABC nylon membrane (⌀ = 3 µm) was supplied by Pall Europe Limited, Portsmouth, UK.
Screen-printed electrodes (SPEs) were obtained as a gift from professor F. Arduini of Tor-Vergata University (Italy), they consisted of a graphite working electrode, a silver/ silver chloride reference electrode (Ag/AgCl), and a counter electrode; 3 mm in diameter and with an active surface of about 0.07 cm2.
6
Quantification of PAD Enzyme Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected in lysis buffer [20mM Tris-HCl, pH 7.4; 100mM NaCl; 10mM β-mercaptoethanol; 10% glycerol; protease inhibitor complete (Roche)] and sonicated for 5 minutes at high power with 30 s on/off cycles at 4°C. Then the samples were centrifuged at 13000 x g at 4°C for 30 minutes and the supernatant was immediately used for further application. PAD activity was determined with the Antibody Based Assay for PAD activity (ABAP) kit (Modiquest Research, MQ17.101-96) and performed according to manufacturer’s protocol. HRP-conjugated secondary antibody was visualized with a TMB substrate solution [1 mg/ml TMB (Sigma Aldrich); 0.1M sodium acetate, pH 5.2; 0.01% hydrogen peroxide] and the reaction was stopped with sulfuric acid [2M H2SO4]. The absorbance at 450 nm was determined with a FLUOstar Omega plate reader (BMG Labtech). Human PAD4 enzyme was used as control enzyme activity and was diluted in deimination buffer [40 mM TrisHCl, pH 7.5; 5 mM CaCl2; 1mM DTT] with concentrations between 0.002 mU (minimum deimination) and 2.0 mU (maximum deimination) to create a standard curve to correlate activity to the optical density measured at 450 nm.
7
Blinded Selenium Supplementation Trial
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All patients, interviewers, and staff involved in outcome assessment were blinded to treatment. The blinding was conducted using the described strategy of allocation concealment by numbered boxes. In brief, numbers were assigned to volunteers’ treatments, but only one pharmacist, who was not involved in the tasks, was aware of the content of each numbered box.
Compliance of patients for using selenium and placebo was measured by telephone every week and all subjects who stopped using their selenium or placebo were excluded from the study.
Determination of POX1 and MPO activity: POX1 activity was measured with phenyl acetate as the substrate [14 (link)]. The assay tube contained 1000 µl of 0.1 mol/l Tris-HCl (pH 8.5), 2 mmol/l CaCl2, 2 mmol/l phenyl acetate, and 3.5 µl of serum. The absorbance was continuously monitored at 270 nm for 2 min. POX1 activity was expressed as millimoles of phenyl acetate hydrolyzed per minute [15 (link)].
MPO activity was measured with 3,3’,5,5’-tetramethylbenzidine (TMB, Sigma) as the substrate. Briefly, 50 µl samples were combined with 240 ml of 0.75 mM H2O2 (Sigma) and 50 ml of TMB solution (2.9 mM TMB in 14.5% DMSO and 150 mM sodium phosphate buffer) at pH 5.4. The absorbance was continuously monitored at 450 nm for 1 min. MPO activity was expressed as millimoles of TMB oxidized per minute [16 (link)].
Compliance of patients for using selenium and placebo was measured by telephone every week and all subjects who stopped using their selenium or placebo were excluded from the study.
Determination of POX1 and MPO activity: POX1 activity was measured with phenyl acetate as the substrate [14 (link)]. The assay tube contained 1000 µl of 0.1 mol/l Tris-HCl (pH 8.5), 2 mmol/l CaCl2, 2 mmol/l phenyl acetate, and 3.5 µl of serum. The absorbance was continuously monitored at 270 nm for 2 min. POX1 activity was expressed as millimoles of phenyl acetate hydrolyzed per minute [15 (link)].
MPO activity was measured with 3,3’,5,5’-tetramethylbenzidine (TMB, Sigma) as the substrate. Briefly, 50 µl samples were combined with 240 ml of 0.75 mM H2O2 (Sigma) and 50 ml of TMB solution (2.9 mM TMB in 14.5% DMSO and 150 mM sodium phosphate buffer) at pH 5.4. The absorbance was continuously monitored at 450 nm for 1 min. MPO activity was expressed as millimoles of TMB oxidized per minute [16 (link)].
8
Hydrogen Peroxide Production in LAB
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hydrogen peroxide production from each LAB was analysed according to a previously described method 29 . Shortly, LAB were initially cultured for 3 days in supplemented MRS. 3,3′,5,5′‐Tetramethyl‐benzidine (TMB) plates were prepared by adding solution A [25 mg of TMB (Sigma‐Aldrich), dissolved in 6 ml methanol] and solution B [2 mg horseradish peroxidase, type 1, approximately 100 purpurogallin units/mg, dissolved in 2 ml of ddH2O] to MRS agar. The TMB plates were then inoculated with each LAB and incubated anaerobically at 35°C for 48 hours before transfer to aerobic conditions at room temperature (RT). Blue colonies were observed after incubation at RT for 1, 24 or 90 hours.
9
Visualizing Peroxidase Activity in Arabidopsis Stems
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 16-to 18-cm tall Col-0 stems were sampled as per developmental analyses and incubated in 100 mM TMB (Acros Organics) for 1 h in the dark, with gentle shaking. For TMB 1 H 2 O 2 analyses, cross-sections were incubated in 100 mM TMB and 100 mM H 2 O 2 for 1 h in the dark, with gentle shaking. PRX enzymatic activity could be visualized by formation of a blue precipitate. Stem sections were rinsed twice in water, imaged on the Leica DMR compound microscope, and repeated for five biological replicates.
To test for protein function, stems were boiled in a 100°C water bath for 10 minutes, then incubated in TMB, as above. To inhibit PRXs, stems were pretreated in 100 mM SHAM (Sigma-Aldrich) for 1 h with shaking, then incubated in TMB. To remove endogenous H 2 O 2 , stems were incubated in 300 U mL 21 bovine liver catalase (Sigma) for 2 h with shaking, then incubated with TMB. Control experiments were performed with at least three biological replicates with consistent results.
To test for protein function, stems were boiled in a 100°C water bath for 10 minutes, then incubated in TMB, as above. To inhibit PRXs, stems were pretreated in 100 mM SHAM (Sigma-Aldrich) for 1 h with shaking, then incubated in TMB. To remove endogenous H 2 O 2 , stems were incubated in 300 U mL 21 bovine liver catalase (Sigma) for 2 h with shaking, then incubated with TMB. Control experiments were performed with at least three biological replicates with consistent results.
10
Immobilized HRP for Colorimetric Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Using an all-liquid fluidic device configured with silicone oil (viscosity η = 10 cSt) as the oil phase, HRP in MES buffer (pH 6.5, 100 µg mL−1) was pumped through the channel at a flow rate of 1 mL h−1 for 10 min (Supplementary Fig. 16 ). After 15 min aging for enzyme immobilization, pure MES buffer was pumped through the channel at a flow rate of 1 mL h−1 for 10 min to remove the free HRP. TMB (10 times diluted from commercial TMB from Sigma, pH 6.5) or 4-AAP/phenol peroxidase substrates (5 mM 4-AAP, 25 mM phenol, and 1 mM H2O2 in MES buffer, pH 6.5) were introduced. Two separate needles were connected to the inlet of the channel to avoid enzyme and substrate contamination. Conversion of substrates into colored products were observed along the length of the channel (Fig. 3g ), which tracked color changes monitored exogenously in a cuvette (Supplementary Figs. 17 , 18 ).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!