Log In Sign up
The largest database of trusted experimental protocols

Tetramethylbenzidine substrate solution

Manufactured by Merck Group
Visit Supplier
Sourced in United States

Tetramethylbenzidine substrate solution is a laboratory reagent used as a chromogenic substrate in various analytical and diagnostic applications. It is commonly employed in enzyme-linked immunosorbent assay (ELISA) and other colorimetric detection methods. The solution contains the chemical compound tetramethylbenzidine, which undergoes a color change reaction when exposed to certain enzymes or oxidizing agents.

Automatically generated - may contain errors

Lab products found in correlation

Multiskan go, by Thermo Fisher Scientific (2 mentions) 96 well polystyrene plate, by Thermo Fisher Scientific (1 mentions) Hrp conjugated goat anti mouse igg or iga antibody, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Goat anti mouse igg, by Merck Group (1 mentions) Optimax, by Molecular Devices (1 mentions) Ist 9, by Abcam (1 mentions) Automated plate washer, by Agilent Technologies (1 mentions) Streptavidin hrp, by R&D Systems (1 mentions) Carbonate bicarbonate buffer, by Merck Group (1 mentions) Fluostar omega plate reader, by BMG Labtech (1 mentions)

6 protocols using tetramethylbenzidine substrate solution

1

ELISA for Influenza Virus Antibodies

Check if the same lab product or an alternative is used in the 5 most similar protocols
Influenza virus antigen-specific antibodies were measured by ELISA. Briefly, polystyrene 96-well plates (Nunc, China) were coated with whole ITIV antigens (HA, 1 μg/mL per strain) in 0.1 M bicarbonate buffer (pH 9.5) and incubated overnight at 4°C. The plates were washed with PBS-0.05% Tween-20 (PBST) and then blocked with PBS-1% bovine serum albumin (BSA, Sigma-Aldrich, USA) for 2 h at 37°C. After washing with PBST, 100 μL of the samples serially diluted with PBS-0.1%BSA was added, and the plates were incubated for 2 h at 37°C. After washing with PBST, an HRP-conjugated goat anti-mouse IgG or IgA antibody (Sigma-Aldrich, USA) was added at a 1:5000 dilution and incubated overnight at 4°C. A color reaction was developed using tetra-methyl benzidine substrate solution (Sigma-Aldrich, USA) at 37°C for 1 h. The reaction was stopped by adding 0.05 mL of 0.5 M H2SO4 per well, and the optical densities (ODs) were read at 450 nm using a microplate reader (Thermo Scientific Multiskan GO, USA). The antibody titers were expressed as the reciprocal of the highest dilution of sample for which the OD was 5×ODmean background, and the geometric mean titer (GMT) was calculated.
Ren S.T., Zhang X.M., Sun P.F., Sun L.J., Guo X., Tian T., Zhang J., Guo Q.Y., Li X., Guo L.J., Che J., Wang B, & Zhang H. (2017). Intranasal Immunization Using Mannatide as a Novel Adjuvant for an Inactivated Influenza Vaccine and Its Adjuvant Effect Compared with MF59. PLoS ONE, 12(1), e0169501.
+ Open protocol
+ Expand
2

ELISA Quantification of Serum SP-D

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
The levels of SP‐D were determined in serum as a measure of intravascular leakage (Gaunsbaek et al., 2013 (link)). Diluted serum specimens were subjected to an enzyme linked immunosorbent assay (ELISA; R&D Systems) for measurement of SP‐D levels. Briefly, microwells were coated with 4 μg/ml capture antibody overnight at 4°C. After washing the wells, the nonspecific sites were blocked with 1% BSA solution in Dulbecco's phosphate‐buffered saline (DPBS) for 1 h at room temperature. The wells were washed and incubated with the mouse SP‐D standard (62.5–4000 pg/ml) and serum specimens (1:10 and 1:50) diluted in 1% BSA solution in DPBS, overnight at 4°C. The next day, the wells were washed and incubated with 125 ng/ml detection antibody. The immune complex was then incubated with 1:40 diluted streptavidin‐horse radish peroxidase for 2 h at room temperature, and developed by adding tetramethylbenzidine substrate solution (Sigma). The reaction was stopped by adding 2 N H2SO4. Finally, the optical density was read spectrophotometrically at 450 and 540 nm, and subtracted per the manufacturer's instructions to correct for optical imperfections in the plate.
Chowdhury A.A., Godbole N.M., Chataut N., Kosanke S., Rodgers K, & Awasthi S. (2022). Effects of SPA4 peptide on lipopolysaccharide‐disrupted lung epithelial barrier, injury, and function in a human cell system and mouse model of lung injury. Physiological Reports, 10(13), e15353.
+ Open protocol
+ Expand
3

Indirect ELISA for Antibody Titer

Check if the same lab product or an alternative is used in the 5 most similar protocols
Indirect ELISA was used to determine the titer of mouse serum and ascites according to the protocol described previously with a few modifications (Xu et al. 2015 (link)). Briefly, microplates were sensitized at 4 °C overnight with the affinity-purified GST label protein at 5 μg/mL. The sensitized plates were incubated with test culture supernatants from hybridoma cells at 37 °C for 1 h, with goat anti-mouse IgG (H + L) conjugated with horseradish peroxidase (HRP) secondary antibodies (Sigma) at a 1:5,000 dilution at 37 °C for 1 h, followed by color development upon addition of tetramethylbenzidine substrate solution (Sigma). Color development was terminated with 2 M H2SO4, and the absorbance value of OD450nm was recorded.
Wu Q., Ma Z., Pan Q., Liu T., Zhang Y., Xin J, & Xu Q. (2024). A candidate competitive ELISA based on monoclonal antibody 3A8 for diagnosis of contagious bovine pleuropneumonia. Applied Microbiology and Biotechnology, 108(1), 290.
+ Open protocol
+ Expand
4

Quantifying Polymer-Specific IgM Responses by ELISA

Check if the same lab product or an alternative is used in the 5 most similar protocols
The production of IgM against each polymer was evaluated by ELISA. Polymer-lipid conjugates were dissolved in ethanol and added to 96-well flat-bottom polypropylene plates (PlateOne) (50 μL, 20 nmol polymer-lipid/well). The wells were air-dried overnight. Serum samples were diluted with PBS-C (PBS, 0.5% casein) at a 1:25 dilution to identify samples with positive and negative serum IgM, or serially from 1:25 to 1:12800 to determine reciprocal endpoint titers. Diluted serum samples were added to the plate and the wells were incubated at 37°C for two hours and then washed six times with PBS-T (PBS, 0.1% Tween-20). Horseradish peroxidase-conjugated goat anti-rat IgM mu chain secondary antibody (Abcam) was then diluted 1:1000 in PBS-C and 100 μL was added to each well and incubated for 30 minutes at 37 °C. Following six washes with PBS-T, 100 μL of a tetramethylbenzidine substrate solution (Sigma Aldrich) was added to wells and incubated for 30 minutes at room temperature. The reaction was stopped with 100 μL of 0.5 M H2SO4 and the yellow product was monitored at 450 nm (Optimax, Molecular Devices, Sunnyvale, CA). Titer was defined as the reciprocal dilution of antisera yielding an optical density twice that of background. Samples were assayed in duplicate. Data analyses were performed using GraphPad Prism.
Kierstead P.H., Okochi H., Venditto V.J., Chuong T.C., Kivimae S., Fréchet J.M, & Szoka F.C. (2015). The Effect of Polymer Backbone Chemistry on the Induction of the Accelerated Blood Clearance in Polymer Modified Liposomes. Journal of controlled release : official journal of the Controlled Release Society, 213, 1-9.
+ Open protocol
+ Expand
5

Cellular Fibronectin-EDA ELISA Protocol

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Cellular Fn-EDA levels in the plasma were measured by sandwich enzyme-linked immunosorbent assay (ELISA) as described.(16 (link)) Briefly, microtiter plates were coated overnight at 4°C with primary antibody for cellular Fn-EDA (IST-9, 10 μg/mL, Abcam) diluted in 50 mM sodium carbonate buffer. 50 μl of plasma samples (diluted 1:2 in PBS) were incubated for 2 h in the coated wells at RT. After five washes biotinylated secondary antibody to FN (2 μg/ml diluted in blocking buffer) was added to wells and incubated for 1 h at RT. Following five washes avidin HRP solution (1:1000) in blocking buffer was added to wells and incubated for 30 minutes. Microtiter plates were washed five times, before adding 3, 3′, 5, 5′-tetramethylbenzidine substrate solution (Sigma) to the wells and the colorimetric reaction was stopped with H2SO4 (2M) after 10 min. Results were read in an ELISA microplate reader at A450 nm. Human cellular Fn (Sigma) was used for standards.
Chorawala M.R., Prakash P., Doddapattar P., Jain M., Dhanesha N, & Chauhan A.K. (2018). Deletion of extra domain A of fibronectin reduces acute myocardial ischemia/reperfusion injury in hyperlipidemic mice by limiting thrombo-inflammation. Thrombosis and haemostasis, 118(8), 1450-1460.
+ Open protocol
+ Expand
6

Quantitative ELISA for Antigen Detection

Check if the same lab product or an alternative is used in the 5 most similar protocols
Wells of reactive ELISA plates (Fisher Scientific) were coated, in triplicate, with 50ul of antigens (see 2.3.1) diluted to 1μg/ml in carbonate-bicarbonate buffer (Sigma-Aldrich), alongside a calibration human IgG standard curve (25μg/ml to 0.2μg/ml) and refrigerated at 4°C overnight. The following morning, wells were aspirated and washed 3 times with 0.05% Tween-20 in PBS using an automated plate washer (BioTek) and blocked for 1 hour with 100μl of 3% BSA. Plates were then aspirated and washed again (as above) before 50μl of serum diluted at 1:8,000 in PBS was added and incubated for 1 hour. After a repeated aspiration and washing, 50μl of biotinylated anti-human IgG diluted to 1:20,000 in PBS was added for 1 hour. Plates were then aspirated and washed again, before 50μl of Streptavidin-HRP (R&D systems) at 1:40 in PBS was added to each well and incubated for 15 minutes.
After a final aspiration and wash, 50μl of tetramethylbenzidine substrate solution (Sigma-Aldrich) was added to each well for 10 minutes, before the reaction was stopped with 1N solution of H2SO4 (sulphuric acid). Plates were then scanned at 450nm on a FLUOstar Omega plate reader (BMG Labtech).
Ayling K., Bowden T., Tighe P., Todd I., Dilnot E.M., Negm O.H., Fairclough L, & Vedhara K. (2017). The application of protein microarray assays in psychoneuroimmunology. Brain, behavior, and immunity, 59.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!