Tetramethylbenzidine solution

Manufactured by Merck Group

Sourced in United States

Tetramethylbenzidine (TMB) solution is a colorimetric substrate used in various analytical and diagnostic applications. It is a commonly used reagent in enzyme-linked immunosorbent assays (ELISA) for the detection and quantification of specific analytes. The solution provides a chromogenic reaction when catalyzed by peroxidase enzymes, resulting in a blue color change that can be measured spectrophotometrically.

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Lab products found in correlation

Cetyltrimethylammonium bromide, by Merck Group (2 mentions) Superblock t20 pbs blocking buffer, by Thermo Fisher Scientific (2 mentions) Peroxidase conjugated secondary antibody, by Vector Laboratories (2 mentions) Multiskan skyhigh photometer, by Thermo Fisher Scientific (2 mentions) Human apoe elisa kit, by Abcam (1 mentions) Extravidin, by Merck Group (1 mentions) Microplate reader, by Molecular Devices (1 mentions) Skim milk, by Merck Group (1 mentions) Sodium bicarbonate, by Merck Group (1 mentions) Sodium carbonate, by Merck Group (1 mentions) 2n sulfuric acid, by Merck Group (1 mentions) Sc 40 hrp, by Santa Cruz Biotechnology (1 mentions) Infinite m200 plate reader, by Tecan (1 mentions) Horseradish peroxidase conjugated secondary antibody, by Vector Laboratories (1 mentions) Elisa plate reader, by Bio-Rad (1 mentions) Synergy ht, by Agilent Technologies (1 mentions) Polystyrene microplates, by Thermo Fisher Scientific (1 mentions) 1 n sulfuric acid, by Merck Group (1 mentions) Spectramax plus 384 microplate reader, by Molecular Devices (1 mentions)

Spelling variants of this product

Tetramethylbenzidine (167 citations) 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (9 citations) 3,3′,5,5′-tetramethylbenzidine substrate solution (6 citations) Tetramethylbenzidine substrate solution (6 citations) Tetramethylbenzidine (TMB) solution (6 citations) Tetramethylbenzidine (TMB) substrate solution (5 citations) 3,3′,5,5′-tetramethylbenzidine solution (4 citations) 3,3′,5,5′-tetramethylbenzidine (TMB) solution (4 citations)

17 protocols using tetramethylbenzidine solution

ELISA for Anti-rMLP15 IgG Detection

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The immunoreactivity of anti-rMLP15 IgG in patient sera was assessed using a modified ELISA protocol of Lima et al. (2017 (link)). In polystyrene 96-well ELISA plates (Kasvi™), 1 µg/well of rMLP15 in 100 μL of 0.1 M sodium carbonate buffer (pH 9.6) was coated and incubated for 16–18 h at 4 °C. Blocking was done with 200 µL/well of 5% skim milk in PBS-T for 2 h at 37 °C after washing the plates thrice with 0.05% PBS-T. Thereafter, the plates were washed three times with PBS-T and 100 µL/well of patient sera (diluted 1:50 in 5% skim milk in 0.05% PBS-T) was added. The plates were incubated at 37 °C and after three PBS-T washes, 100 μL/well of anti-human IgG peroxidase-conjugated antibody (Sigma-Aldrich, St. Louis, MO, USA) was added, diluted 1:10,000 in PBS-T, and further incubated at 37 °C for 1 h. The chromogenic reaction was developed by the addition of 100 μL/well of tetramethylbenzidine solution (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 37 °C, and the reaction was stopped with 100 μL/well of 2 N sulfuric acid. Absorbance at 450 nm was measured with a spectrophotometer (Bio-Rad, Hercules, CA, USA) and mean OD values were calculated from the serum samples assayed in triplicate.

Barbosa M.D., de Sousa I.B., Simionatto S., Borsuk S, & Marchioro S.B. (2019). Recombinant polypeptide of Mycobacterium leprae as a potential tool for serological detection of leprosy. AMB Express, 9, 201.

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ELISA for C4d and ApoE in RA SF

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ELISA to measure C4d in 5% SF of RA patients was performed as previously described for pooled serum (37 ) with minor adaptations, instead of ortho-phenylenediamine tablets the assay was performed using tetramethylbenzidine solution (Sigma-Aldrich) and colorimetric development was measured at 450 nm. Human ApoE ELISA kit (#ab233623, Abcam) was used following the manufacturer’s instructions with minor adaptations. Dilutions of SF (1:32000) were used and were kept on ice until shortly before the assay to prevent decay.

Vogt L.M., Kwasniewicz E., Talens S., Scavenius C., Bielecka E., Ekdahl K.N., Enghild J.J., Mörgelin M., Saxne T., Potempa J, & Blom A.M. (2020). Apolipoprotein E triggers complement activation in joint synovial fluid of rheumatoid arthritis patients by binding C1q. Journal of immunology (Baltimore, Md. : 1950), 204(10), 2779-2790.

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Quantifying Brain Myeloperoxidase Activity

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As described previously [37 (link)], the ipsilateral brain cortex was homogenized in 50 mM potassium phosphate buffer, centrifuged, and then resuspended in 0.5% cetyltrimethylammonium bromide (Sigma-Aldrich) in potassium phosphate buffer. After centrifugation, 40 μL of supernatant was incubated with 100 μL tetramethylbenzidine solution (Sigma-Aldrich) in a 96-well plate and the reaction was stopped with 100 μL 2 N HCl. The absorbance (OD) at 450 nm was measured after 10 min incubation in the dark. MPO activity was in equivalent units by comparison with a reference curve generated using purified MPO (Sigma-Aldrich) according to the manufacturer’s instructions. All samples for completion and analysis of ELISA were performed by two researchers blinded to experimental groups.

Shi G., Liu L., Cao Y., Ma G., Zhu Y., Xu J., Zhang X., Li T., Mi L., Jia H., Zhang Y., Liu X., Zhou Y., Li S., Yang G., Liu X., Chen F., Wang B., Deng Q., Zhang S, & Zhang J. (2023). Inhibition of neutrophil extracellular trap formation ameliorates neuroinflammation and neuronal apoptosis via STING-dependent IRE1α/ASK1/JNK signaling pathway in mice with traumatic brain injury. Journal of Neuroinflammation, 20, 222.

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Enzyme-Linked Immunosorbent Assay Protocol

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Enzyme-linked immunosorbent assay was performed as previously described.^{61 (link)} Briefly, 96-well enzyme-linked immunosorbent assay plates (Nalge Nunc International, Rochester, NY, USA) were coated with 1 μg/ml of capture antibody (Ab62) in 50 mM carbonate buffer (pH 9.6) at 4 °C overnight. After washing with PBS with 0.05% Tween 20 (PBST), SuperBlock T20 (PBS) Blocking Buffer (Thermo Scientific, Rockford, IL, USA) was added to each well. After incubation for 1 h at room temperature with shaking, plates were washed five times in PBST. Samples and standards were incubated at room temperature for 2.5 h with shaking. After washing with PBST, 1 μg/ml of biotinylated Ab62 in blocking buffer was added to each well. After incubation for 1.5 h at room temperature, the plates were washed with PBST. Avidin-conjugated peroxidase (ExtrAvidin, Sigma) was added to each well. After incubation for 1 h at room temperature, plates were washed with PBST. One hundred microliters of 3,3′,5,5′-tetramethylbenzidine solution (Sigma) were added to each well and incubated for 15 min with shaking. To stop the reaction, 50 μl of 2 N H₂SO₄ was added to each well. The absorbance was measured at 450 nm.

Bae E.J., Yang N.Y., Lee C., Kim S., Lee H.J, & Lee S.J. (2015). Haploinsufficiency of cathepsin D leads to lysosomal dysfunction and promotes cell-to-cell transmission of α-synuclein aggregates. Cell Death & Disease, 6(10), e1901-.

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Apolipoprotein Binding and Complement Activation Assays

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Deposition assays with pooled normal human serum and complement binding assays were performed as described previously (12 (link)) with minor adaptations. Purified human recombinant ApoE (Novoprotein Apo ε3, and BioPure, Biovision Apo ε2, ε3, and ε4), ApoB (Athens research and technology), and ApoD (Novoprotein) were coated at 10 μg/mL in PBS. For C1q deposition from serum, heat aggregated IgG was coated as a positive control at 1.0 μg/mL because of its relatively strong signal, and for components C4b, C3b, and C9, heat aggregated IgG was coated at 2.5 μg/mL, unless otherwise specified in the graph. C1q was purified from plasma as described (24 (link)). For FH binding, ApoE was coated at 1 μg/mL in CBB buffer consisting of 50 mM HEPES, 100 mM NaCl, pH 7.4 (25 (link)), blocking was performed in 5% BSA (Fraction V, AppliChem, Saveen and Werner AB) in CBB, and FH binding was performed in CBB. FH and C4BP were expressed and purified as described previously (26 (link), 27 ). FB depleted serum and FB were purchased from Quidel (Triolab AB) and CompTech, respectively. Colorimetric assays developed with ortho-phenylenediamine tablets (Kem-En-Tec) were spectrophotometrically analyzed at 490 nm, and assays where tetramethylbenzidine solution (Sigma-Aldrich) was used were analyzed at 450 nm as recommended by the manufacturers.

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Biomarker Quantification by ELISA

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The levels of creatine kinase-MB (CK-MB), aspartate transaminase (AST), lactate dehydrogenase (LDH), and interleukin (IL)-1β were detected by ELISA (R&D Systems, Minneapolis, MN, United States). The supernatant was collected and added to a 96-well plate and the standards were added. After incubation for 1.5 h, the conjugate solution was added to the wells, after which the plates were incubated for another 1.5 h. Next, tetramethylbenzidine solution (861510; Sigma-Aldrich) was added, followed by a 15 m of incubation. Finally, the stop solution was added to stop the reaction, after which the optical density was detected with a microplate reader (Molecular Devices, San Jose, CA, United States) at 450 nm.

Zhang G.L., Liu Y., Liu Y.F., Huang X.T., Tao Y., Chen Z.H, & Lai H.L. (2024). Teneligliptin mitigates diabetic cardiomyopathy by inhibiting activation of the NLRP3 inflammasome. World Journal of Diabetes, 15(4), 724-734.

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Quantifying Cortisol and Glucocorticoid Receptor Levels

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Cortisol and GR binding were measured in the gastrocnemius muscle protein samples. First, 96-well microplates were coated with the anti-cortisol diluted in the coating buffer described in Section 2.6 (Table S1) and then incubated overnight at 4 °C. After washing with TPBS, they were blocked with 5% skim milk (Sigma-Aldrich) for 6 h at room temperature, and 60 μg of the isolated protein, as described in Section 2.5.2, was loaded into each well and incubated overnight at 4 °C. The microplates were washed with TPBS and incubated with anti-GR antibody overnight at 4 °C (Table S1). After washing again with TPBS, the microplates were incubated with a peroxidase-conjugated secondary antibody (Vector Laboratories) for 4 h at room temperature. Following the last TPBS wash, a tetramethylbenzidine solution (Sigma-Aldrich) was added and allowed to incubate for 20 min at room temperature. Then, an equal volume of a stop solution of 2 N sulfuric acid (Sigma-Aldrich) was added, and the optical density at 450 nm was determined using a microplate reader (Multiskan SkyHigh Photometer; Thermo Fisher Scientific).

Batsukh S., Oh S., Rheu K., Lee B.J., Choi C.H., Son K.H, & Byun K. (2023). Rice Germ Attenuates Chronic Unpredictable Mild Stress-Induced Muscle Atrophy. Nutrients, 15(12), 2719.

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ELISA Quantification of Protein Levels

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ELISA was performed as previously described [45 (link)]. 96-well ELISA plates (Nalgene Nunc International, Rochester, NY) were coated with 1 mg/mL capture antibody (Ab62) in 50 mM carbonate buffer (pH 9.6) at 4℃ overnight. After washing with PBS with 0.05% Tween 20 (PBST), the plate was incubated with SuperBlock T20 (PBS) Blocking Buffer (Thermo Scientific, Rockford, IL) at room temperature (RT) with shaking for 1 h, and washed five times in PBST. Samples and standards were incubated at RT for 2.5 h with shaking. After washing with PBST, 1 µg/mL biotinylated Ab62 in blocking buffer was added to each well. After 1.5 h incubation at RT with shaking, the plates were washed with PBST. Avidin-conjugated peroxidase (ExtrAvidin, Sigma) was incubated for 1 h at RT. After washing with PBST, 100 µL of 3,3',5,5'-tetramethylbenzidine solution (Sigma) was added to each well and plate was incubated for 15 min with shaking. To stop the reaction, 50 µL of 2N H₂SO₄ was added to each well. The absorbance was measured at 450 nm.

Bae E.J., Lee C., Lee H.J., Kim S, & Lee S.J. (2014). ATP13A2/PARK9 Deficiency Neither Cause Lysosomal Impairment Nor Alter α-Synuclein Metabolism in SH-SY5Y Cells. Experimental Neurobiology, 23(4), 365-371.

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MPO Activity Assay in Brain Tissues

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Ipsilateral brain tissues were homogenized in 500 μL of 50 mM potassium phosphate buffer (pH 7.0). The samples were centrifuged, resuspended and subjected to three freeze-thaw cycles in 1% cetyltrimethylammonium bromide (Sigma-Aldrich) in potassium phosphate buffer. Forty microliters of supernatant was added to 100 μL tetramethylbenzidine solution (Sigma-Aldrich), and the optical density was measured at 450 nm after stopping the reaction with 2N HCl. MPO activity was expressed in equivalent units by comparison with a reference curve generated using purified MPO (Sigma-Aldrich).

Zhu X., Cao Y., Wei L., Cai P., Xu H., Luo H., Bai X., Lu L., Liu J.R., Fan W, & Zhao B.Q. (2016). von Willebrand factor contributes to poor outcome in a mouse model of intracerebral haemorrhage. Scientific Reports, 6, 35901.

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Indirect ELISA for ACTH and Cortisol

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The amounts of ACTH and cortisol were measured in the serum samples described in 2.5.1 via an indirect-ELISA. Briefly, 96-well microplates (LPS solution, Daejeon, Republic of Korea) were coated with a buffer (0.3% sodium carbonate (Sigma-Aldrich), 0.6% sodium bicarbonate (Sigma-Aldrich), and 91.9% distilled water) and left overnight at 4 °C. The coated microplates were washed with phosphate-buffered saline containing 0.1% Tween-20 (TPBS; LPS solution) and blocked with 5% skim milk (LPS solution) for 6 h at room temperature. After being washed with TPBS, 100 μg of serum was added into each well and then incubated overnight at 4 °C. After washing with TPBS, the microplates were incubated with anti-ACTH or anti-cortisol antibodies overnight at 4 °C (Table S1). Following washing with TBPS, the primary antibodies were incubated with a peroxidase-conjugated secondary antibody (Vector Laboratories, Burlingame, CA, USA) at room temperature for 4 h. After another round of washing, a tetramethylbenzidine solution (Sigma-Aldrich) was added and allowed to incubate in the dark at room temperature for 15–20 min to enable color development. To terminate the reaction, an equal volume of 2 N sulfuric acid (Sigma-Aldrich) was added. The optical density at 450 nm was measured using a microplate reader (Multiskan SkyHigh Photometer; Thermo Fisher Scientific).

Batsukh S., Oh S., Rheu K., Lee B.J., Choi C.H., Son K.H, & Byun K. (2023). Rice Germ Attenuates Chronic Unpredictable Mild Stress-Induced Muscle Atrophy. Nutrients, 15(12), 2719.

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