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AgriseraTMB HRP Substrate

AS15 TMB-HRP | TMB based, especially formulated with extreme sensitivity, HRP substrate for microwell application.
- 10ml trial size is restricted to one unit per customer -
AgriseraTMB HRP Substrate in the group Detection Reagents / ELISA at Agrisera AB (Antibodies for research) (AS15 TMB-HRP)

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Product Information

Storage Stable for 3 years at +2-8° C or at room temperature up to six months, and can also be frozen. Storge at room temperature for 1 month results in ca. 2 % activity loss. Protect the substrate from exposure to sunlight.
Tested applications Instructions for a 96 well micro titer plate 1. Equilibrate the substrate to the assay temperature before use. 2. Complete all required incubations with antibodies and HRP labeled reagents. 3. Wash the plate at least 4 times with phosphate buffered saline or Tirs buffered saline containing 0.1% Tween-20. THen shake and blot all residual buffer from plate wells. 4. Add 100 µl of the TMB solution to each well and incubate 5-30 minutes, depending upon the activity of the HRP probe. If color develops too rapidly, zero order kinetics will not prevail. Dilution of antibody, or HRP-labeled reagent may be required. As alternative Agrisera TMB/Diluent pack can be used. 5. To stop the reaction use:-equial volume of 0.3 M sulfuric acid (100 µl). The yellow chromogen in the stopped reaction should be read at 450 nm. This will provide highest sensitivity of detection. -Monitoring the assay kinetics at may be done at 650 nm as a function of time. The reaction can be stopped to preserve the blue chromogen using an equal volume, 100 µl of 0.1% sodium fluoride or 0.15% sodium dodecyl sulfate. The blue chromogen in the stopped reaction should be measured at 650 nm. Adjustments of time, reagent volume and temperature require standardization by the end user. In case of any further questions please contact us.

Reactivity

Application examples

Additional information

Additional information One component ready to use reagent. It contains 2.5 mMol L-1 TMB and Hydrogen Peroxide in a buffer at pH 3.6. It also contains non-toxic stabilizers.

How to work with this reagent? 
  • Pour out amount of reagent which is needed for your assay. 
  • Do not our back any unused substrate back to the original bottle. 
  • Any trace metals, HRP or other contaminant may be reason for the substrate to turn blue.

Related products

Related products AS15 TMB-HRP(100 ml), AS15 TMB-HRP-1L(1L); AS15 Diluent(100 ml) - diluent for TMB-HRP to obtain flexible kinetic range; 

To test another kinetic range we recommend: to combine Agrisera TMB HRP substrate with AS15TMB Dilutent  at a ratio 60 % substrate and 40 % diluent.

Background

Background 3,3',5,5'-Tetramethylbenzidine, horseradish peroxidase (AS15 TMB-HRP) is a sensitive reagent that is ready to use for the quantitative detection of HRP bound to a solid phase or in free solution. It is designed to detect and quantify specific peptide/protein/hormone in a complex mixture in enzyme linked immunosorbent assay (ELISA). It utilizes the enzymatic reaction triggered by horse raddish peroxidase (HRP) in which a chromogenic, chemiluminiescent or chemifluorescent substrates are oxidized resulting in color change, chemiluminiscence or chemifluorescence respectively.

A one-electron oxidation product is formed in the presence of HRP and hydrogen peroxide.  This is a cation free radical, it is blue in color with adsorption maximum at 653 nm. The a reaction with  HRP, hydrogen peroxide or acidification of the radical with acid yields the diimine terminal oxidation product that adsorbs light at 450 nm. The extinction coefficient of the radical ( E653 nm = 3.9 x 104 mol-1 cm-1 ) and diimine ( E450 nm = 5.9 x 104 mol-1 cm-1 ) provides a very sensitive system for this assay.

After incubations with antibodies or HRP labeled reagents, TMB solution is added. As an alternative TMB can also be spiked with a different volume of buffered HRP to create dilution series of this substrate, for example 50 %, 70%. This will change sensitivity of this product and can make it more suitable for certain assays, depending what kinetics are required. If you require to adjust sensitivity of your assay, please check Agrisera TMB/Diluent pack. 

The oxidation of TMB by HRP produces a blue reaction product, measured at 650 nm. Color formation can be recorded as a function of time or the reaction can be stopped using an equal volume of 0.3 M sulfuric acid after a fixed interval. Increased sensitivity is achieved by converting the blue radical to the yellow diimine by addition of acid. The resulting yellow chromogen is measured immediately at 450 nm.

Product citations

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