 s(HS)TMB (#sTMB) s(HS)TMB, soluble (High Sensitivity) TMB Substrate for ELISA, #sTMB, is a peroxidase substrate containing 3,3\',5,5\'-tetramethylbenzidine (TMB) and hydrogen peroxide in an aqueous buffer formulation. This product is supplied as a one component ready-to-use reagent. Intact/unreacted substrate is clear colorless to slightly yellowish in appearance. Reaction with peroxidase results in converting substrate into soluble blue (~ 650 nm) product which after stopping with strong acid further turns to super-oxidized soluble yellow product. Yellow product can be measured at 450 nm (measuring at 450 nm vs. 620-650 nm reference wavelength will additionally improve readout pattern in ELISA). s(HS)TMB is optimal for 15-min. substrate development reaction in ELISA. Shaker mode will improve overall performance. s(HS)TMB substrate yields exceptionally high signals with HRP conjugates and therefore is particularly recommended for Ultra-Sensitive ELISA tests. It does not contain high density components, is not viscous, has no detergent or colloidal activity. It does not foam and does not stick to hydrophobic surfaces. This allows easy and precise dispensing: standard PP and HD-PE pipette tips repel s(HS)TMB restlessly, almost as it were pure water. After stopping, fixed reaction remains stable during several following hours, also under direct light. There is no fading, neither increase in color. This concerns both low signals (blank, background, negative control) and high positive signals (no precipitation in wells with OD up to 3.500) and makes later postponed reading in ELISA-spectrophotometer unproblematic. s(HS)TMB is stable two years at +2°C/+8°C or one year at ambient room temperature (15 °C/25°C). Both storage regimens are equally applicable depending on desired duration of storage. Within first year, storage in refrigerator will neither improve, nor worsen product performance. +4°C is however a preferred storage regimen providing better safety in cutting off adverse influence of higher temperatures. Warm up s(HS)TMB substrate, if stored at +4°C, to assay temperature (optimum = 17°C- 18°C) prior to use. Avoid exposure to light, air and extreme temperatures. For repackaging bulk product use only clean intact amber Nunc/Nalge(n) or Wheaton HD- PE bottles. Bottle substrate in light-protected (weak diffused light) and clean area gravimetrically relying on d20°C = 1 g/ml. Alternatively use peristaltic pump with certified compatible tubing (e.g. Norprene or Cole-Parmer/Millipore C-Flex tubing). Do not re-filter. For dispensing smaller volumes, use close-design syringe dispensers (e.g. CombiTips of Eppendorf). s(HS)TMB substrate does not contain volatile/aprotic organic solvents and is non caustic and non corrosive to plastic, glass and metal materials used in automatic dispensing and robotic ELISA instumentation. It is a completely water base product added with non-toxic proprietary stabilizers and enhances that are not health-hazardous. This product is non- hazardous, non-toxic, non-carcinogenic, environmentally safe unrestricted product. s(HS)TMB, stable soluble 1-component TMB s(HS)TMB Weakener is complementary reagent. s(HS)TMB Weakener (#sTMB-W) Some customers who do not really need very high analytical sensitivity in their test (typical case is determination of specific antibodies in indirect antigen sandwich ELISA with anti- species HRP conjugate) or formerly used in their systems weaker TMB products and do wish to retain signal levels they used to have, ask us whether it would be possible to lower signals with s(HS)TMB through e.g. diluting it with blank substrate buffer. Although it is generally not recommended to dilute TMB substrates, instead of producing tailored weaker TMB we have decided to add our TMB product line with s(HS)TMB Weakener, which is essentially the same basic formulation as s(HS)TMB containing no TMB and hydrogen peroxide. Now users who wish to reduce signals in their system when switching over to our s(HS)TMB can adjust performance to previous level by simply mixing s(HS)TMB with Weakener. This blank optimization TMB diluent offers a possibility of achieving desired OD values while maintaining overall stability of prepared mixture at a level of undiluted s(HS)TMB. s(HS)TMB Weakener, ready-to-use esTMB is a newer, enhanced product that gives 30-to-40% p(HS)TMB (#pTMB) p(HS)TMB is a precipitating peroxidase substrate containing 3,3\',5,5\'-tetramethylbenzidine (TMB) and hydrogen peroxide in an aqueous buffer formulation. This product is supplied as a one component ready to use reagent. Intact/unreacted substrate is clear colorless to slightly yellowish in appearance. Positive reaction results in precipitating of insolublepermanent dark blue product at the sites of enzymatic HRP activity (where detecting HRP- labeled anti-analyte is bound). This product is originally designed for using in 3D ImmunoFiltration (Flow-Through) assays. p(HS)TMB is stable two years at +2°C/+8°C or one year at ambient room temperature (15°C/25°C). Both storage regimens are equally applicable depending on desired duration of storage. Within first year, storage in refrigerator will neither improve, nor worsen product performance. +4°C is however a preferred storage regimen providing better safety in cutting off adverse influence of higher temperatures. Warm up p(HS)TMB substrate, if stored at +4°C, to assay temperature (optimum = 17°C- 18°C) prior to use. Avoid exposure to light, air and extreme temperatures. For repackaging bulk product use only clean intact amber Nunc/Nalge(n) or Wheaton HD- PE bottles. Bottle substrate in light-protected (weak diffused light) and clean area gravimetrically relying on d20°C = 1 g/ml. Alternatively use peristaltic pump with certified compatible tubing (e.g. Norprene or Cole-Parmer/Millipore C-Flex tubing). Do not re-filter. For dispensing smaller volumes, use close-design syringe dispensers (e.g. CombiTips of Eppendorf). p(HS)TMB substrate does not contain volatile organic solvents and is non caustic and non corrosive to plastic, glass and metal materials. It is a completely water base product added with non-toxic proprietary stabilizers and enhances that are not health-hazardous. This product is non-hazardous, non-toxic, non-carcinogenic, environmentally safe unrestricted product. p(HS)TMB, stable precipitating 1-component TMB pricing ep(HS)TMB is a newer, enhanced product that gives 20-to-30%  ep(HS)TMB-mA is a novel, one component stable enhanced precipitating TMB Substrate especially designed for the microArray applications. It produces smaller, clearly shaped colored 1e-oxidized TMB precipitate at the sites of the HRP activity. Colored product features higher density and perfect adhesion and grip on the porous (3D) and non-porous (2D) surfaces of the different solid phase materials used as reactive matrices/supports in the diverse heterogeneous binding assays of the modern MicroChip format. ep(HS)TMB-mA is a High Sensitivity product applicable in: - Microchips / planar multiplex arrays - fine micro-blotting - ELISPOT assays ep(HS)TMB, enhanced stable precipitating 1-component TMB Substrate for High Sensitivity microArray applications, 100% aqueous formula, ready-to-use #epTMB-mA pricing Stop/Elution Reagent (#SER) Chemical purity of acid used in stopping enzymatic reaction with TMB substrate is important factor that also influences overall performance of the ELISA test. Divalent Fe++, Ni++, Co++, Cd++, Cr++, Cu++, Mn++, Mg++ cations, if present in stop reagent, may provoke huge backgrounds as a result of post-enzymatic (when HRP is already dead) catalytic reaction where elevated blank signals become visible also through larger negative control and/or cut-off values, even if measured shortly after stopping with dirty acid reagent. That much evident picture is an extreme. None less, in practice, a possible impact of Fe++/Ni++/Co++-associated post-enzymatic reaction in actually observed backgrounds is just a matter of quantitative dependence. It might be quite possible that a couple of decades of microOD units in your ELISA system are due to not optimal stopping reagent. In colorimetric test, each 10 micro OD units in lower OD range, i.e. with blanks, negative controls and cut-offs, are often critical. Adding TMB substrate formulation with strong Fe++/Ni++/Co++-sequestering agents, as Diethylenetriamine penta-methylenephosphonic acid(DTPMPA) and other phosphonates (multivalent derivatives of methylene phosphonic acid), also known as effective peroxide stabilizers, can compensate for metal ion contamination of Stop Reagent. This however weakens substrate developmental strength in reaction with peroxidase label through interference with iron in Protohematin IX and therefore is not rational method compared to exclusion metal ions in Stop Reagent. Our Stop/Elution Reagent is essentially Ultra-pure 8% Sulfuric Acid containing no detectable bivalent metal ions. Therefore post-enzymatic oxidizing of TMB after stopping substrate reaction is absolutely excluded. #SER guarantees stability of developed color reaction with s(HS)TMB during three hours at room temperature, not protected from light and air, providing for a possibility of dependable later measuring ELISA plates which is practicable in running larger series involving many tests. This adds security and convenience in working with intensive test flows and on the other hand improves robustness of the each individual test in labor intensive series. Besides, #SER has been extra validated as eluting reagent optimal for 3D-ImmunoFiltratio tests with PolyHRP/p(HS)TMB detection. Oxidized in enzymatic reaction with HRP label cationic TMB product will be in situ precipitated by polyanionic polyelectrolyte polymer present in p(HS)TMB formulation with formation of dark blue electrostatic complex deposited onto the surface of porous 3D device filter matrix. Precipitated TMB product can be eluted from device filters by applying strong acid Elution Reagent #SER which destroys electrostatic complex and simultaneously, within same elution event, further converts blue TMB product into super-oxidized final yellow product. Eluted/stopped product remains soluble and can be measured exactly as in conventional ELISA test in standard ELISA reader at 450/620 nm. This creates option of complementary read-out in 3D- ImmunoFiltration test. Said option might be practicable when running larger series arranged in compatible with 8x12 microwell ELISA plate geometry format with elution directly in standard serocluster 8x12 microplates and measuring in common ELISA reader. #SER elutes in a minute, and OD readout values in eluted substrate product remain stable within 3 hours after elution. Stop/Elution Reagent for stopping |