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Substrates

Colorimetric substrates for horseradish peroxidase (HRP) or alkaline phosphatase (AP) detection. Substrates are used to generate the read-out signal of the ELISA or membrane assay.

Format	Conjugate	Substrate	Absorbance
Microwell	HRP	TMB 1-Component HRP Microwell Substrate SUB1, our most popular substrate. This TMB substrate for HRP detection provides the reference for performance of all the other TMB substrates offered. SUB1 TMB substrate is ideal for most ELISAs where the target is in the ng-pg range. We recommend starting with this formulation to estimate the level of sensitivity needed in the assay.	370 or 620-650 nm
Microwell	HRP	TMB Super Sensitive 1-Component HRP Microwell Substrate SUB2, our most sensitive TMB substrate. SUB2 TMB substrate is approximately 40-fold more sensitive than 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.	370 or 620-650 nm
Microwell	HRP	TMB Slow Kinetic 1-Component HRP Microwell Substrate SUB3 exhibits roughly 25% less sensitivity than SUB1. Lower sensitivity TMB 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.	370 or 620-650 nm
Microwell	HRP	ABTS 1-Component HRP Microwell Substrate SUB4 is a good choice of ELISA HRP detection substrate for assays with: samples containing high levels of the target molecule; samples that do not require much dilution (i.e. past 1:100); long incubation periods (such as overnight incubations); and for assays that simply do not require a high level of sensitivity.	405-410 nm
Microwell	AP	pNPP 1-Component AP Microwell Substrate SUB5 for alkaline phosphatase detection (AP) in ELISA/EIA microwell formats. This formulation of pNPP substrate contains a stabilizer to extend its benchtop shelf life.	405-420 nm
Membrane	HRP	TMB 1-Component HRP Membrane Substrate SUB6 is useful for immunoblotting applications where HRP-conjugated molecules are used for detection. SUB6 TMB membrane substrate reacts with HRP, yielding a dark blue reaction product. This TMB membrane substrate formulation does not contain any aprotic solvents.	370 or 620-650 nm
Membrane	AP	BCIP/NBT 1-Component AP Membrane Substrate SUB7 contains 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 AP membrane substrate reacts with AP, yielding a bluish-purple reaction product.	370 nm