Shop All ELISA Substrates and Reagents

PNPP Substrate Tablets Thermo Scientific™

Thermo Scientific Pierce PNPP (p-nitrophenyl phosphate) is a colorimetric, soluble substrate of alkaline phosphatase for use in ELISA applications. This product comes in 5 mg tablets, with an inert filler as the balance.

Features of PNPP (p-nitrophenyl phosphate):

• One-component alkaline phosphatase (AP) substrate
• Absorbance maximum of 405nm
• Develops a yellow hue in solution upon reaction with AP
• Safer to use—contains a non-mercury, non-azide preservative

PNPP (p-Nitrophenyl Phosphate, Disodium Salt) is a widely used substrate for detecting alkaline phosphatase in ELISA applications. When alkaline phosphatase and PNPP are reacted, a yellow water-soluble reaction product is formed. This reaction product absorbs light at 405nm. The substrate is offered in several formats.

Related Products
1-Step™ PNPP Substrate Solution
Pierce™ PNPP Substrate Kit
PNPP Substrate

OPD Substrate Tablets (o-phenylenediamine dihydrochloride) Thermo Scientific™

Thermo Scientific Pierce OPD is a water-soluble substrate for horseradish peroxidase (HRP) that produces a yellow-orange product detectable at 492nm by ELISA plate readers.

OPD yields a water soluble yellow-orange product when reacted with peroxidase with an absorbance maximum of 492. OPD can easily be dissolved in a substrate buffer such as Stable Peroxide Substrate Buffer (Part No. 34062).

Related Products
OPD (o-phenylenediamine dihydrochloride)

SuperSignal™ ELISA Femto Substrate Thermo Scientific™

Thermo Scientific SuperSignal ELISA Femto Substrate is formulated for superior protein detection and low-end linearity in chemiluminescent ELISA applications.

Features of SuperSignal ELISA Femto Substrate:

Immediate light generation—intense signal generated immediately both at room temperature and 37°C; emits light at 425nm
Improved low-end linearity—easy detection of low quantities of proteins with high signal:noise ratios and low-end linearity of dose response curves
High sensitivity—femtogram-level detection of target proteins in ELISA
Reduction in assay time—high sensitivity allows for reduction in ELISA incubation steps
Stability—storage for six months at room temperature or a minimum of 12 months at 4°C with a six-hour working solution stability

SuperSignal ELISA Femto Substrate provides greater sensitivity, in ELISAs or any other solution-based assay, using chemiluminescent substrates for enzyme detection and quantification. These ELISAs can take place in either a test tube or a microplate and are quantified by measuring relative light units (RLU) in a luminometer.

SuperSignal ELISA Substrate uses an improved enhancer system that meet the needs of high-throughput screening (HTS) applications and diagnostic assays. SuperSignal ELISA Femto Substrate generates detectable light within one minute of addition to trace amounts of soluble HRP, saving up to 30 minutes per assay. This feature makes SuperSignal ELISA Femto Substrate ideal for HTS applications with the capability of running as many as 100,000 assays on robotic equipment.

ONPG (o-nitrophenyl-β-D-galactopyranoside) Thermo Scientific™

Thermo Scientific Pierce ONPG is the preferred colorimetric substrate for ELISA applications involving beta-galactosidase (b-Gal) as the reporter enzyme.

When using beta-Galactosidase as the label for proteins in ELISA studies, a wide variety of substrates are available, including: o-Nitrophenyl-beta-D-galactopyranosidase (ONPG), Naphthol-AS-Bl-beta-D-galactopyranosidase (Nap-Gal), and 4-Methyl-umbelliferyl-beta-D-galactopyranosidase (MUm-Gal). However, it is important to choose a substrate with adequate solubility, that uses readily available equipment, and that gives a significant reading over the background. ONPG is a superior beta-Galactosidase substrate option.(1) The product formed is completely soluble and has a high extinction coefficient at 405nm. The substrate yields a yellow product that is easily detectable in the visual range after stopping the reaction with 1M Sodium Carbonate.

ELISA-Light™ Immunoassay System with CSPD™ and Sapphire-II™ Substrate/Enhancer Solution Applied Biosystems™

The ELISA-Light™ Immunoassay System with CSPD® and Sapphire-II™ Substrate⁄Enhancer Solution is used for ultra-sensitive detection of antigens or antibodies in multiple immunoassay formats employing an alkaline phosphatase enzyme label.

• Heterogeneous assay format increases sensitivity and dynamic range and reduces interference by test compounds in the detection portion of the assay (as compared with homogeneous assay formats)
• Chemiluminescent immunoassay detection allows greater sensitivity and improved dynamic range as compared to radioimmunoassays and immunoassays that employ colorimetric or fluorescent detection
• High sensitivity and wide dynamic range permit sensitive detection with fewer assays, because sample dilution to fit the dynamic range of the assay is unnecessary
• A variety of ready-to-use substrate formulations lets the user precisely optimize signal intensity to the exact requirements of an assay
• The kinetics of the glow-assay provides a "window" during which measurements may be performed, facilitating HTS applications where assay automation is used
• Chemiluminescent immunoassay detection provides versatile applications such as sandwich immunoassays, competitive immunoassays, and DNA probe capture assays
• Non-radioactive immunoassays eliminate concerns over use of radioisotopes

Extend the Limits of Detection
The ELISA-Light™ system offers an excellent combination of high sensitivity and throughput for assays measured in a luminometer. The ELISA-Light™ system significantly extends the limits of detection of most immunoassays previously performed with colorimetric and isotopic methods.

Multiple Substrate⁄Enhancer Formulations
The kit contains reagents optimized for chemiluminescent ELISAs employing alkaline phosphatase labels and is available with a choice of ready-to-use chemiluminescent substrate formulations containing CSPD® or CDP-Star® substrate with Sapphire-II™ or Emerald-II™ luminescence enhancers. Multiple substrate⁄enhancer formulations provide options that let the user optimize specific assays or luminometers. The specific formulation that will provide the highest sensitivity for a specific assay can vary. Therefore, an ELISA sampler kit containing all four substrate⁄enhancer formulations is available to facilitate assay development.

For Research Use Only. Not for use in diagnostics procedures.

eBioscience™ ELISA Wash Buffer, 1L packets Invitrogen™

The eBioscience ELISA Wash Buffer Powder is a dried powder formulation of phosphate buffered saline with 0.05% Tween-20. Each packet is reconstituted in 1 Liter of distilled, deionized water; the resulting 1x PBS/Tween-20 buffer (pH 7.4) is suitable for washing ELISA plate microwells. 10 packets of ELISA Wash Buffer Powder are supplied, yielding a total of 10 liters of 1x Wash Buffer.

Reported Application
ELISA

ELISA-Light™ Immunoassay System with CSPD™ and Emerald-II™ Substrate/Enhancer Solution Applied Biosystems™

The ELISA-Light™ Immunoassay System with CSPD® and Emerald-II™ Substrate⁄Enhancer Solution is used for ultra-sensitive detection of antigens or antibodies in multiple immunoassay formats employing an alkaline phosphatase enzyme label.

• Heterogeneous assay format increases sensitivity and dynamic range and reduces interference by test compounds in the detection portion of the assay (as compared with homogeneous assay formats)
• Chemiluminescent immunoassay detection allows greater sensitivity and improved dynamic range as compared to radioimmunoassays and immunoassays that employ colorimetric or fluorescent detection
• High sensitivity and wide dynamic range permit sensitive detection with fewer assays, because sample dilution to fit the dynamic range of the assay is unnecessary
• A variety of ready-to-use substrate formulations lets the user precisely optimize signal intensity to the exact requirements of an assay
• The kinetics of the glow-assay provides a "window" during which measurements may be performed, facilitating HTS applications where assay automation is used
• Chemiluminescent immunoassay detection provides versatile applications such as sandwich immunoassays, competitive immunoassays, and DNA probe capture assays
• Non-radioactive immunoassays eliminate concerns over use of radioisotopes

Extend the Limits of Detection
The ELISA-Light™ system offers an excellent combination of high sensitivity and throughput for assays measured in a luminometer. The ELISA-Light™ system significantly extends the limits of detection of most immunoassays previously performed with colorimetric and isotopic methods.

Multiple Substrate⁄Enhancer Formulations
The kit contains reagents optimized for chemiluminescent ELISAs employing alkaline phosphatase labels and is available with a choice of ready-to-use chemiluminescent substrate formulations containing CSPD® or CDP-Star® substrate with Sapphire-II™ or Emerald-II™ luminescence enhancers. Multiple substrate⁄enhancer formulations provide options that let the user optimize specific assays or luminometers. The specific formulation that will provide the highest sensitivity for a specific assay can vary. Therefore, an ELISA sampler kit containing all four substrate⁄enhancer formulations is available to facilitate assay development.

For Research Use Only. Not for use in diagnostics procedures.

CSPD™ Substrate (0.25 mM Ready-To-Use) Invitrogen™

This CSPD® substrate is a chemiluminescent alkaline phosphatase substrate, ready-to-use for protein or nucleic acid blotting on nitrocellulose membranes. This substrate can also be used in solution-based assays.

This CSPD® chemiluminescent substrate for alkaline phosphatase let you detect alkaline phosphatase and alkaline phosphatase-labeled molecules with unparalleled sensitivity, speed, and ease.

• Chemiluminescent substrates provide highly sensitive replacements for the widely used fluorogenic substrate methylumbelliferyl phosphate (MUP), and the colorimetric substrate p-nitrophenyl phosphate (pNPP).
• Low background luminescence coupled with high-intensity light output enables detection of alkaline phosphatase labels with the highest possible sensitivity and signal-to-noise.

This versatile chemiluminescent substrate exhibits high sensitivity in membrane-based applications such as Southern, Northern, and Western blotting. This substrate can also be used in solution-based assays such as immunoassays, DNA probe assays, enzyme assays, and reporter gene assays. Maximum light levels are reached in approximately 10 minutes and glow emission persists for several hours.

For Research Use Only. Not for use in diagnostics procedures.

1-Step™ PNPP Substrate Solution Thermo Scientific™

Thermo Scientific Pierce PNPP (p-nitrophenyl phosphate) is a colorimetric, soluble substrate of alkaline phosphatase for use in ELISA applications. This product is a ready-to-use (1X) proprietary buffered solution of PNPP.

Features of PNPP (p-nitrophenyl phosphate):

• One-component alkaline phosphatase (AP) substrate
• Absorbance maximum of 405nm
• Develops a yellow hue in solution upon reaction with AP
• Safer to use—contains a non-mercury, non-azide preservative

Until now, the method for preparing PNPP solutions required dissolving the powder or tablets in buffer and then diluting the solution to the desired concentration. Substrate instability made it necessary to prepare it on a daily basis. Pierce 1-Step PNPP circumvents this time-consuming and variable introducing step by providing a single component PNPP substrate. This substrate is stable for 12 months at 2°C-8°C and can be stopped with conventional methods.

PNPP (p-Nitrophenyl Phosphate, Disodium Salt) is a widely used substrate for detecting alkaline phosphatase in ELISA applications. When alkaline phosphatase and PNPP are reacted, a yellow water-soluble reaction product is formed. This reaction product absorbs light at 405nm. The substrate is offered in several formats.

Related Products
Pierce™ PNPP Substrate Kit
PNPP Substrate Tablets
PNPP Substrate

CDP-Star™ Substrate (12.5 mM Concentrate) Invitrogen™

This CDP-Star™ chemiluminescent substrate for alkaline phosphatase let you detect alkaline phosphatase and alkaline phosphatase-labeled molecules with unparalleled sensitivity, speed, and ease.

• Chemiluminescent substrate provides highly sensitive replacements for the widely used fluorogenic substrate methylumbelliferyl phosphate (MUP), and the colorimetric substrate p-nitrophenyl phosphate (pNPP).
• Low background luminescence coupled with high-intensity light output enables detection of alkaline phosphatase labels with the highest possible sensitivity and signal-to-noise.

This versatile chemiluminescent substrate exhibits high sensitivity in membrane-based applications such as Southern, Northern, and Western blotting. This substrate can also be used in solution-based assays such as immunoassays, DNA probe assays, enzyme assays, and reporter gene assays. Maximum light levels are reached in approximately 10 minutes and glow emission persists for several hours.

For Research Use Only. Not for use in diagnostics procedures.

CSPD™ Substrate (0.4 mM Ready-To-Use) with Sapphire-II™ Enhancer, Size B Invitrogen™

This CSPD® substrate with Sapphire-II™ Enhancer is a chemiluminescent alkaline phosphatase substrate, ready-to-use for protein or nucleic acid blotting on nitrocellulose membranes. This substrate can also be used in solution-based assays.

This CSPD® chemiluminescent substrate for alkaline phosphatase let you detect alkaline phosphatase and alkaline phosphatase-labeled molecules with unparalleled sensitivity, speed, and ease.

• Chemiluminescent substrates provide highly sensitive replacements for the widely used fluorogenic substrate methylumbelliferyl phosphate (MUP), and the colorimetric substrate p-nitrophenyl phosphate (pNPP).
• Low background luminescence coupled with high-intensity light output enables detection of alkaline phosphatase labels with the highest possible sensitivity and signal-to-noise.

This versatile chemiluminescent substrate exhibits high sensitivity in membrane-based applications such as Southern, Northern, and Western blotting. This substrate can also be used in solution-based assays such as immunoassays, DNA probe assays, enzyme assays, and reporter gene assays. Maximum light levels are reached in approximately 10 minutes and glow emission persists for several hours. Solution assays require chemiluminescent enhancers. Membrane-based assays may also require them for increased light output and sensitivity.

For Research Use Only. Not for use in diagnostics procedures.

OPD (o-phenylenediamine dihydrochloride) Thermo Scientific™

Thermo Scientific Pierce OPD is a water-soluble substrate for horseradish peroxidase (HRP) that produces a yellow-orange product detectable at 492nm by ELISA plate readers.

OPD yields a water soluble yellow-orange product when reacted with peroxidase with an absorbance maximum of 492. OPD can easily be dissolved in a substrate buffer such as Stable Peroxide Substrate Buffer (Part No. 34062).

Related Products
OPD Substrate Tablets (o-phenylenediamine dihydrochloride)

ELISA-Light™ Immunoassay System with CDP-Star™ and Emerald-II™ Substrate/Enhancer Solution Applied Biosystems™

The ELISA-Light™ Immunoassay System with CDP-Star® and Emerald-II™ Substrate⁄Enhancer Solution is used for ultra-sensitive detection of antigens or antibodies in multiple immunoassay formats employing an alkaline phosphatase enzyme label.

• Heterogeneous assay format increases sensitivity and dynamic range and reduces interference by test compounds in the detection portion of the assay (as compared with homogeneous assay formats)
• Chemiluminescent immunoassay detection allows greater sensitivity and improved dynamic range as compared to radioimmunoassays and immunoassays that employ colorimetric or fluorescent detection
• High sensitivity and wide dynamic range permit sensitive detection with fewer assays, because sample dilution to fit the dynamic range of the assay is unnecessary
• A variety of ready-to-use substrate formulations lets the user precisely optimize signal intensity to the exact requirements of an assay
• The kinetics of the glow-assay provides a "window" during which measurements may be performed, facilitating HTS applications where assay automation is used
• Chemiluminescent immunoassay detection provides versatile applications such as sandwich immunoassays, competitive immunoassays, and DNA probe capture assays
• Non-radioactive immunoassays eliminate concerns over use of radioisotopes

Extend the Limits of Detection
The ELISA-Light™ system offers an excellent combination of high sensitivity and throughput for assays measured in a luminometer. The ELISA-Light™ system significantly extends the limits of detection of most immunoassays previously performed with colorimetric and isotopic methods.

Multiple Substrate⁄Enhancer Formulations
The kit contains reagents optimized for chemiluminescent ELISAs employing alkaline phosphatase labels and is available with a choice of ready-to-use chemiluminescent substrate formulations containing CSPD® or CDP-Star® substrate with Sapphire-II™ or Emerald-II™ luminescence enhancers. Multiple substrate⁄enhancer formulations provide options that let the user optimize specific assays or luminometers. The specific formulation that will provide the highest sensitivity for a specific assay can vary. Therefore, an ELISA sampler kit containing all four substrate⁄enhancer formulations is available to facilitate assay development.

For Research Use Only. Not for use in diagnostics procedures.

Pierce™ Stable Peroxide Substrate Buffer (10X) Thermo Scientific™

Thermo Scientific Pierce Stable Peroxide Substrate Buffer (10X) include optimized, reliable and convenient stock solutions of peroxide for HRP-based assays detection.

Features of Stable Peroxide Substrate Buffer (10X):

• Ready-to-use
• Minimize assay-to-assay variability

Buffered stable hydrogen peroxide (H2O2) is used by researchers who prefer to make their own HRP-ELISA substrates. This is easily done by adding a chromogen of choice to the buffer. Stable Peroxide Substrate Buffer has a long shelf life (12 months after receipt) and is provided as a 10-fold concentrate. A total volume of 1000 mL can be made from one 100 mL bottle of concentrate. If you use 10 mL of substrate per plate, this will allow you to make substrate for 100 plates from only one bottle of Stable Peroxide Substrate Buffer.

Related Products
Pierce™ Diethanolamine Substrate Buffer (5X)

1-Step™ Ultra TMB-ELISA Substrate Solution Thermo Scientific™

Thermo Scientific Pierce 1-Step Ultra TMB ELISA Substrate (1 L) detects horseradish peroxidase (HRP) activity, yielding a blue color (Amax = 370nm and 652nm) that changes to yellow (Amax = 450nm) upon addition of a sulfuric or phosphoric acid stop solution. 1-Step Ultra TMB is the most sensitive of the chromagenic substrates.

Features of 1-Step Ultra TMB ELISA Substrate:

• Available in traditional or ready-to-use formulations
• No additional reagents or filtering required
• Non carcinogenic
• Various levels of sensitivity and development speed to suit any assay
• No DMF or DMSO present in the reagents

TMB is the most popular chromogenic substrate for HRP detection in ELISA and is available in several formats. The Pierce 1-Step TMB Substrates are one-component substrates that require no preparation before use. Unlike other commercially available substrates, these products contain no DMF or DMSO.
Thermo Scientific Pierce 1-Step Ultra TMB yields the greatest sensitivity among the TMB substrates, followed by 1-Step Turbo TMB and 1-Step Slow TMB. The sensitivity of the 1-Step Turbo TMB is similar to OPD-based substrates formulated at approximately 1 mg/mL. 1-Step Slow TMB has intermediate sensitivity—more sensitive than ABTS, but less sensitive than o-phenylenediamine dihydrochloride (OPD) or 1-Step Turbo TMB. The 1-Step Slow TMB is an ideal substrate for kinetic studies.

TMB (3,3',5,5'-tetramethylbenzidine) is a chromogen that yields a blue color when oxidized, typically as a result of oxygen radicals produced by the hydrolysis of hydrogen peroxide by HRP. For kinetic or non-stopped ELISA assays, the TMB chromogen has maximal absorbances at 370nm and 652nm. The color then changes to yellow with the addition of sulfuric or phosphoric acid with maximum absorbance at 450nm. A green reaction product may result from partial conversion to the yellow product from the blue intermediate. TMB is very sensitive ELISA substrate and is more quickly oxidized than other HRP substrates, resulting in faster color development.

Related Products
Pierce™ TMB Substrate Kit
1-Step™ Turbo TMB-ELISA Substrate Solution
1-Step™ Slow TMB-ELISA Substrate Solution
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