Substrates

Read your Assay clearly

Substrates are used to generate the read-out signal of the ELISA assay. The signal generated is reflective of all the previous steps and the strength of the substrate itself. The substrate reacts with the peroxidase detection enzyme (HRP or AP) and the optical density of the resulting color reaction is read with a spectrophotometric plate reader.

ICT's Substrates may help you to enhance the sensitivity of your ELISA, reduce background noise, and provide linearity for enhanced reproducibility.

TMB (3,3’,5,5’-tetramethylbenzidine), ABTS (2.2’-azino-di[3-ethylbenzthi-azoline sulfonate (6)] and pNPP (para-Nitrophenylphosphate) substrates are ideal for use with kinetic assays and can also be used with a STOP solution for use in endpoint ELISAs. The relative sensitivity of a substrate is affected by the formulation of the solution as well as the length of incubation of the enzyme-substrate reaction, and the titer of the target molecule in the assay. ImmunoChemistry Technologies provides 5 substrates for use in ELISA microwell formats and 2 substrates for use with nitrocellulose, PVDF membranes, or dot-blot assays. Each substrate is provided as 100mL.

ICT offers 7 reliable substrates:

TMB One Component HRP Microwell Substrate (SUB1)

Our most popular substrate. SUB1 provides the reference for performance of all the other HRP substrates offered. SUB1 is ideal for most ELISAs where the target is in the ng-pg range. We recommend starting with this formulation when developing an assay to estimate the level of sensitivity. Then, if necessary, change formulations to increase or decrease sensitivity when optimizing your assay. Absorbance for TMB = 370 or 620 - 650 nm.is

TMB One Component HRP Microwell Substrate (SUB1)

TMB Super Sensitive One Component HRP Microwell Substrate (SUB2)

Our most sensitive TMB buffer. SUB2 is approximately 40-fold more sensitive than our most popular substrate - SUB1. SUB2 is useful when detecting very low levels of a target molecule, with samples that must be highly diluted (1:10,000), to amplify the signal when using antibodies with low binding capacity, with samples that exhibit high steric hindrance, and to shorten the incubation time of the assay. Absorbance for TMB = 370 or 620 - 650 nm.

TMB Super Sensitive One Component HRP Microwell Substrate (SUB2)

TMB Slow Kinetic One Component HRP Microwell Substrate (SUB3)

Exhibits roughly 25% less sensitivity than SUB1. Lower sensitivity substrates are ideal for ELISAs where the test samples contain high levels of the target molecule, for assays with long incubation periods (such as overnight incubations), and for assays that simply do not require a high level of sensitivity. Absorbance for TMB = 370 or 620 - 650 nm.

TMB Slow Kinetic One Component HRP Microwell Substrate (SUB3)

ABTS One Component HRP Microwell Substrate (SUB4)

Use SUB4 for samples with high levels of the target molecule; for samples that do not have to be diluted very far (1:100); for assays with long incubation periods, (such as overnight incubations); and for assays that simply do not require a high level of sensitivity. Absorbance for ABTS = 405 - 410 nm.

ABTS One Component HRP Microwell Substrate (SUB4)

pNPP One Component AP Microwell Substrate (SUB5)

Used in ELISA assays to detect alkaline phosphatase-conjugated molecules. This pNPP substrate formulation contains a stabilizer to extend the shelf life of the substrate on your benchtop. Absorbance for pNPP = 405 - 420 nm.

AP One Component pNPP Microwell Substrate (SUB5)

TMB One Component HRP Membrane Substrate (SUB6)

Useful for immunoblotting applications where HRP-conjugated molecules are used for detection. SUB6 reacts with HRP yielding a dark blue reaction product. The formulation of this substrate does not contain any aprotic solvents. Absorbance for TMB = 370 or 620 - 650 nm.

TMB One Component HRP Membrane Substrate (SUB6)

BCIP/NBT One Component AP Membrane Substrate (SUB7)

Use SUB 7 with BCIP(5-bromo-4-Chloro-3’-Indolyphosphate p-Toluidine Salt) and NBT (Nitro-Blue Tetrazolium Chloride) for immunoblotting applications where alkaline phosphatase-conjugates molecules are used for detection. NBT serves as the oxidant and BCIP is the AP substrate. SUB7 reacts with AP yielding a bluish-purple reaction product.

BCIP/NBT One Component AP Membrane Substrate (SUB7)

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