Substrates for ELISA and Immunoblotting
Substrates are used to generate the read-out signal of an immunoassay utilizing an enzyme-labeled detection antibody. ICT offers several varieties of fluorometric-based alkaline phosphatase and HRP substrates for ELISA and immunoblotting applications, including TMB substrates for HRP detection with varying levels of sensitivity.
|Microwell||HRP||TMB 1-Component HRP Microwell Substrate (SUB1), our most popular TMB substrate. This TMB substrate for HRP detection provides the reference for performance of all the other TMB substrates offered. TMB 1-Component HRP Microwell Substrate is ideal for most HRP detection ELISAs where the target is in the ng-pg range. We recommend starting with this TMB 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, is approximately 40-fold more sensitive than SUB1 TMB substrate. This sensitive substrate for ELISA 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 TMB substrate. Lower sensitivity TMB substrates are ideal for HRP detection 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 HRP substrate for HRP detection-based 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 alkaline phosphatase substrate contains a stabilizer to extend its benchtop shelf life.||405-420 nm|
||HRP||TMB 1-Component HRP Membrane Substrate (SUB6) is useful for immunoblotting applications where HRP-conjugated molecules are used for detection. TMB membrane substrates react with HRP, yielding a dark blue reaction product. Our TMB 1-Component HRP Membrane Substrate formulation does not contain any aprotic solvents.||370 or 620-650 nm|
||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-conjugated molecules are used for detection. BCIP/NBT 1-Component AP Membrane Substrate reacts with alkaline phosphatase, yielding a bluish-purple reaction product. NBT serves as the oxidant and BCIP is the alkaline phosphatase substrate.