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Tmb peroxidase substrate

Manufactured by Rockland Immunochemicals
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TMB (3,3',5,5'-Tetramethylbenzidine) is a chromogenic substrate for peroxidase enzymes. It is a commonly used reagent in enzyme-linked immunosorbent assay (ELISA) and other immunoassay techniques to detect and quantify the presence of target analytes.

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Lab products found in correlation

Igf 1 standards, by Abcam (2 mentions) Goat antirabbit igg1 conjugated to hrp, by Southern Biotech (2 mentions) Rabbit anti igf 1, by Abcam (2 mentions) Synergy h1, by Agilent Technologies (2 mentions) Goat anti mouse igg2a hrp, by Southern Biotech (1 mentions) Anti igg2a hrp, by Southern Biotech (1 mentions) Meb 38, by BioLegend (1 mentions)

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TMB ELISA peroxidase substrate (9 citations) TMB substrate (2 citations)

4 protocols using tmb peroxidase substrate

1

IGF-1 Sandwich ELISA Protocol

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IGF-1 standards (Abcam, Cambridge, UK) and sera samples were diluted in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, 3 mM NaH3, pH9.6) and allowed to bind to Costar (3590) high-binding EIA/RIA plates overnight. Subsequently, plates were washed 3 times in PBS-0.1% Tween-20 (PBST), blocked with 3% milk in PBST (PTM) for 1 h and incubated in primary antibody, rabbit anti-IGF-1 in PTM 1:2000 (Abcam, Cambridge, UK) for 2 h at 37 °C. Next, plates were washed 3 times in PBST and incubated in secondary antibody goat antirabbit IgG1 conjugated to HRP in PTM 1:4000 (Southern Biotechnology, Birmingham, AL) at 37 °C for 1 h. Plates were then washed in PBST, incubated in TMB peroxidase substrate (Rockland) for 10 min at room temperature, followed by stop solution (2 N H2SO4). Absorbance was read at 450 nm.
Park J., Yan G., Kwon K.C., Liu M., Gonnella P.A., Yang S, & Daniell H. (2019). Oral delivery of novel human IGF-1 bioencapsulated in lettuce cells promotes musculoskeletal cell proliferation, differentiation and diabetic fracture healing. Biomaterials, 233, 119591.
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2

Quantifying Anti-IgE Binding Activity

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To assess binding activity of anti-IgE samples wells of a 96 well microplate (high binding, Greiner Bio-one) were coated with human IgE (60 μl of 5 μg/ml anti-OVA clone 11B6) overnight at 4 °C. The next the day plate was washed with PBS-Tween (PBS with 0.5ml tween-20/L) and blocked with 1% BSA (100μl) at room temperature for 1h. Serial dilutions of anti-IgE (8), anti-IgE-N3 (10b), CD33L-anti-IgE (10a) (50 μl) were added and incubated at 37 °C for 1.5h. The plate was then washed again with PBS-Tween and secondary antibody (50 μl) anti-IgG2a-HRP (1:1000, v/v, goat anti-mouse IgG2a-HRP, Southern Biotech, Cat. No. 1081–05) was added. After washing the plate, the ELISA was developed using TMB peroxidase substrate (75μl/well, Rockland) for 4 min and quenched with 2M H2SO4 (75μl/well) and absorbance was measured at 450nm using plate reader (Synergy H1, BioTek). EC50 curve was generated using GraphPad Prism (8.4.3).
Islam M., Arlian B.M., Pfrengle F., Duan S., Smith S.A, & Paulson J.C. (2022). Suppressing Immune Responses using Siglec Ligand Decorated Anti-Receptor Antibodies. Journal of the American Chemical Society, 144(21), 9302-9311.
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3

IGF-1 Sandwich ELISA Protocol

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IGF-1 standards (Abcam, Cambridge, UK) and sera samples were diluted in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, 3 mM NaH3, pH9.6) and allowed to bind to Costar (3590) high-binding EIA/RIA plates overnight. Subsequently, plates were washed 3 times in PBS-0.1% Tween-20 (PBST), blocked with 3% milk in PBST (PTM) for 1 h and incubated in primary antibody, rabbit anti-IGF-1 in PTM 1:2000 (Abcam, Cambridge, UK) for 2 h at 37 °C. Next, plates were washed 3 times in PBST and incubated in secondary antibody goat antirabbit IgG1 conjugated to HRP in PTM 1:4000 (Southern Biotechnology, Birmingham, AL) at 37 °C for 1 h. Plates were then washed in PBST, incubated in TMB peroxidase substrate (Rockland) for 10 min at room temperature, followed by stop solution (2 N H2SO4). Absorbance was read at 450 nm.
Park J., Yan G., Kwon K.C., Liu M., Gonnella P.A., Yang S, & Daniell H. (2019). Oral delivery of novel human IGF-1 bioencapsulated in lettuce cells promotes musculoskeletal cell proliferation, differentiation and diabetic fracture healing. Biomaterials, 233, 119591.
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4

Anti-TNP-IgE Antibody Quantification Protocol

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The half-life of anti-TNP-IgE was determined as previously described.(8 ) Microplates (Greiner Bio-one, #655081) were coated with TNP31BSA (Biosearch Technology, 10 μg/mL in 50 μL of PBS/well, overnight). The next day, the plates were washed with PBS-T (PBS containing 0.05% Tween-20, v/v) 5 times, blocked with PBS containing 1% BSA (w/v, >2 hours, RT) and washed with PBS-T 5 times. Mice were bled prior treatment, 6 or 24 hours after treatment. Serum was serially diluted in PBS containing 1%BSA and loaded onto plates at 4 °C overnight. Serially diluted anti-TNP-IgE (MEB38, Biolegend) was loaded as a standard. The next day, after the plates were washed 5 times with PBS-T, plates were incubated with Biotin-anti-mouse IgE (clone RME-1, at 2 μg/mL in 50 μL, RT, >1 hour), and strepavidin-HRP (Biolegend, #405210, 1 μg/mL, >30 minutes, RT). Plates were then washed with PBS-T 5 times. All ELISAs were developed using TMB Peroxidase Substrate (75 μL/well, Rockland), quenched with 2M H2SO4 (75 μL/well), and A450 were measured using a plate reader (Synergy H1, BioTek).
Duan S., Arlian B.M., Nycholat C.M., Wei Y., Tateno H., Smith S.A., Macauley M.S., Zhu Z., Bochner B.S, & Paulson J.C. (2021). Nanoparticles displaying allergen and Siglec-8 ligands suppress IgE-FcεRI mediated anaphylaxis and desensitize mast cells to subsequent antigen challenge. Journal of immunology (Baltimore, Md. : 1950), 206(10), 2290-2300.
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