Manganese formate dehydrate Mn(HCO2)2·2H2O, sodium acetate (NaAc), ethanol (C2H5OH), methanol (CH3OH), acetic acid (HAc), glucose (Glu), sodium chloride (NaCl), potassium chloride (KCl), ascorbic acid (AA), and dopamine (DA) were purchased from Sinopharm Chemical Reagent (Shanghai, China). TMB (3,3′,5,5′-tetramethylbenzidine) was obtained from Sigma-Aldrich (USA). ZnCl2, CaCl2, and MgCl2 were bought from Sangon Biotech (Shanghai, China). l -serine (Ser), glycine (Gly), l -histidine dihydrochloride (His), l -threonine (Thr), tryptophan (Try), l -arginine (Arg), cysteine (Cys) and bovine protein serum (BSA) were acquired from BBI Life Sciences (Shanghai, China). Glutathione (GSH) was purchased from Adamas Reagent (Shanghai, China). IgG was purchased from Solarbio (Beijing, China).
Tmb 3 3 5 5 tetramethylbenzidine
Manufactured by Merck Group
Sourced in United States
TMB (3,3′,5,5′-tetramethylbenzidine) is a chromogenic substrate commonly used in enzyme-linked immunosorbent assays (ELISAs) and other colorimetric detection methods. It is a sensitive and stable substrate that undergoes a color change upon enzymatic conversion, typically resulting in a blue or green color development.
Automatically generated - may contain errors
Lab products found in correlation
Nunc immuno maxisop 96 well plates, by Corning (2 mentions)
Goat anti chicken horseradish peroxidase antibody, by Abcam (2 mentions)
El311sx, by Agilent Technologies (2 mentions)
Dopamine da, by Sinopharm (1 mentions)
Glutathione (gsh), by Adamas (1 mentions)
Cacl2, by Sangon (1 mentions)
Zncl2, by Sangon (1 mentions)
Mgcl2, by Sangon (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Synergy h4 hybrid multi mode microplate reader, by Agilent Technologies (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
H2so4, by Merck Group (1 mentions)
Il 13, by Thermo Fisher Scientific (1 mentions)
Interferon γ, by Thermo Fisher Scientific (1 mentions)
Cuso4, by Sinopharm (1 mentions)
Prism 8, by GraphPad (1 mentions)
Hrp conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti mouse igg secondary antibody, by Southern Biotech (1 mentions)
13 protocols using tmb 3 3 5 5 tetramethylbenzidine
1
Manganese-Based Colorimetric Biosensing
2
ELISA Assay for Tetanus Antibody Detection
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ELISA microplates were coated with 100 μL of TeNT 5 μg/mL in PBS at 4 °C overnight. After blocking with 1% BSA (RT, 2 h), candidate antibodies were added (2-fold dilutions, from 50 to 0.4 ng/mL, in PBS with 1% BSA) and incubated (37 °C, 1 h). The plate was washed with 0.05% Tween 20 in PBS (PBS-T) before incubation with peroxidase-conjugated goat anti-human IgG antibody (Southern Biotech, Birmingham, AL, USA). TMB 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich, Darmstad, Germany) at 100 µg/mL and 0.0045% hydrogen peroxide (Millipore, Burlington, MA, USA) in acetate/citrate buffer, pH 6.0 was used as substrate. The reaction was stopped with 2 M H2SO4, and the absorbance was recorded at 450 nm. Serum from a vaccinated donor (approval by CEP/ICB/USP by Plataforma Brasil, number 1.515.313) was used as a control.
3
Enzyme-Linked Immunosorbent Assay for Viral Antibodies
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Sucrose gradient-purified viruses were diluted in PBS and added to Nunc-Immuno MaxiSop 96-well plates (Corning, NY, USA) at 16 hemagglutinating units (HAU) per well. After incubation overnight at 4 °C, samples in wells were blocked with PBS-nonfat dry milk. Antisera against the F/98 virus in chicken were then added in serial twofold dilutions with PBS containing 0.05% Tween-20, and incubated for 3 h at 37 °C. After washing, goat anti-chicken horseradish peroxidase antibody (Abcam, Cambridge, MA) was added and allowed to incubate for 1.5 h at 37 °C. After washing, TMB (3,3′,5,5′-Tetramethylbenzidine) (Sigma, St. Louis, MO, USA) substrate was added, and the reaction was stopped by adding H2SO4. Absorbance was recorded at 450 nm using an automated ELISA plate reader (model EL311SX; Biotek, Winooski, VT) [22 (link)].
4
Enzyme-Linked Immunosorbent Assay (ELISA)
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96-well polystyrene plates were coated overnight at 4°C with OVAp or BSA at 2 μg/mL in carbonate buffer (pH 9.6). The coated plates were incubated with a blocking solution (2% BSA in PBS) for 2 h at 37°C. These wells were washed five times with PBST (PBS containing 0.05% Tween 20), and then, 100 μL diluted mouse serum was added to the wells and incubated for 2 h at 37°C. After five times washes with PBST, the plates were incubated with horseradish peroxidase-labeled goat anti-mouse IgG antibodies (SouthernBiotech, USA) for 1 h at 37°C. The reaction was developed with 100 μL TMB (3,3′,5,5′-tetramethylbenzidine, Sigma-Aldrich, USA) for 5 min and stopped with 100 μL 2 M H2SO4. The OD was measured at 450 nm by Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek, USA).
5
ELISA Assay for Viral ORF2 Protein
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Ninety-six well microtiter plates (Nunc, Thermo Fisher Scientific, Massachusetts, United States) were coated with 5 μl of culture supernatant mixed with 95 μl of 100 mM sodium bicarbonate buffer (pH 9.6) and kept at 4°C overnight. The plates were washed thrice in 200 μl/well of wash buffer (PBS + 0.1% Tween20, pH 7.4) and blocked with 200 μl/well of blocking buffer (PBS + 1% BSA) at 37°C for 2 h. Subsequently, plates were washed with wash buffer and incubated with anti-ORF2 rabbit polyclonal antibody (Nair et al., 2016 (link)) at a dilution of 1:1000 in assay buffer (PBS + 0.1% Tween20, 0.2% BSA) at 37°C for 2 h. Next, plates were incubated with HRP conjugated anti-rabbit IgG in assay buffer for 1 h at 37°C and washed three times in wash buffer. HRP activity was measured by colorimetry using TMB 3,3′,5,5′-tetramethylbenzidine, (Sigma, St. Louis, MO, United States) as the substrate. Values were measured at A450 using a multimode microplate reader (Synergy HT, BioTek, Vermont, United States).
6
Myeloperoxidase Activity Measurement
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After determining the hemoglobin content, the supernatant was stored in protease inhibitor cocktail (Sigma Aldrich) at −80 °C. The pellet was resuspended in 2 mL sodium phosphate buffer (pH 5.4). 300 μL was transferred to tubes and supplemented with 600 μL of 0.5% w/v HTAB (hexadecyltrimethylammonium bromide, Sigma) diluted in phosphate buffer (pH 5.4). After, samples were centrifuged at 10,000 × g for 10 minutes at 4 °C and the supernatant was used. Finally, 100 μL of 0.003% hydrogen peroxide and 100 μL TMB (3,3′,5,5′-tetramethylbenzidine, Sigma) at 4 mM diluted in DMSO (dimethyl sulfoxide, Merck) were added in a novel tube followed by 200 μL of the sample supernatant and incubated for 1 minute. The reaction was stopped by adding 100 μL of 4 M H2SO4 (sulfuric acid, Merck). 200 μL of the final solution was added to 96-well plate and spectrophotometric analysis was performed at 450 nm. Results are expressed as the MPO index (absorbance/mg wet weight of the implant).
7
Measuring RSV-specific Antibody Isotypes
8
Protocol for Oligonucleotide and Reagent Preparation
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The oligonucleotides were synthesized by Shanghai Sangon Biotechnology Co. (Shanghai, China) and the sequences are given in Table S1.† All the DNAs were dissolved in distilled water as stock solutions and quantified by UV-vis absorption spectroscopy, using extinction coefficients (ε 260 nm, M–1 cm–1): A = 15 400, G = 11 500, C = 7400, T = 8700. NMM (N-methylmesoporphyrin IX) was purchased from J&K (Beijing, China) and the stock solution of NMM (50 μM) was prepared with dimethyl sulfoxide (DMSO) and stored at –20 °C in darkness. TMB (3,3′,5,5′-tetramethylbenzidine) was obtained from Sigma Aldrich (USA) and dissolved in DMSO as a stock solution (20.804 mM). Tris(tris(hydroxymethyl)aminomethane) and CuSO4 were purchased from Sinopharm Chemical Regent Co. (Shanghai, China). GSH (glutathione) was provided by Shanghai Sangon Biotechnology Co. (Shanghai, China). The water used in the experiments was purified using a Millipore system. Other chemicals were of reagent grade and used without further purification.
9
ELISA Analysis of Influenza Antibodies
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The ELISA was performed as previously described [29 (link)], and sucrose gradient-purified viruses were diluted in PBS and added to Nunc-Immuno MaxiSop 96-well plates (Corning, NY, USA) at 16 HAU per well. After overnight incubation at 4 °C, the samples in wells were blocked with PBS-nonfat dry milk, and the antisera against the F/98 virus, the rF/HAS127N virus or the rF/HAA180V virus in chickens were then added in serial twofold dilutions with PBS containing 0.05% Tween 20 and incubated for 3 h at 37 °C.
After washing, goat anti-chicken horseradish peroxidase antibody (Abcam, Cambridge, MA) was added and incubated for 1.5 h at 37 °C, TMB (3,3′,5,5′ tetramethylbenzidine) (Sigma, St. Louis, MO, USA) substrate was added, and the reaction was stopped by adding H2SO4. The absorbance was recorded at 450 nm using an automated ELISA plate reader (model EL311SX; Biotek, Winooski, VT, USA), and the area under the curve (AUC) of either virus was assessed for virus-Ab binding above that of the corresponding negative control with GraphPad Prism 8 software (San Diego, CA, USA).
After washing, goat anti-chicken horseradish peroxidase antibody (Abcam, Cambridge, MA) was added and incubated for 1.5 h at 37 °C, TMB (3,3′,5,5′ tetramethylbenzidine) (Sigma, St. Louis, MO, USA) substrate was added, and the reaction was stopped by adding H2SO4. The absorbance was recorded at 450 nm using an automated ELISA plate reader (model EL311SX; Biotek, Winooski, VT, USA), and the area under the curve (AUC) of either virus was assessed for virus-Ab binding above that of the corresponding negative control with GraphPad Prism 8 software (San Diego, CA, USA).
10
SARS-CoV-2 RBD-specific Antibody Detection
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To detect SARS-CoV-2 RBD-specific antibodies in immunized sera collected from mice or macaques, plates were coated with SARS-CoV-2 RBD proteins (Sino Biological: wild-type, Cat. No# 40592-V08H; Alpha strain, Cat. No# 40592-V08H82; Beta strain, Cat. No# 40592-V08H84; Gamma strain, Cat. No# 40592-V08H86; Delta strain, Cat. No# 40592-V08H91; Omicron strain, Cat. No# 40592-V08H121) at a concentration of 1 μg/ml, followed by sequential addition of serially diluted serum samples and HRP-conjugated anti-mouse IgG (1:5000) antibodies (Invitrogen, Cat. No# A16066) or HRP-conjugated anti-monkey IgG (1:5000) antibodies (Invitrogen, Cat. No# PA1-84631) for 1 h at 37 °C. The plates were sequentially incubated with the substrate TMB (3,3’,5,5’-tetramethylbenzidine) (Sigma, Cat. No# T0440) and then H2SO4 (1 N) to stop the reaction. The absorbance at 450 nm was measured on a microplate reader.
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