Shop All Enzyme Substrates

EnzChek™ Cellulase Substrate, blue fluorescent, 339/452 Invitrogen™

The EnzChek® cellulase substrate was developed for simple and rapid quantitation of cellulase. The assay can be completed in 30 minutes or less. Simply mix the enzyme with the EnxChek® cellulase substrate working solution, incubate, and read the results using excitation/emission maxima ~339/452 nm. In contrast to other more complex, multistep assays, the EnzChek® cellulase substrate is perfect for use in a high-throughput environment. This fluorescence substrate is highly sensitive, with a detection limit as low as 40 µU/mL cellulase. It is also possible to use this substrate in a colorimetric assay, albeit with reduced sensitivity.

ELF™ 97 Phosphatase Substrate (ELF™ 97 Phosphate) - Contains 2 mM Azide Invitrogen™

The ELF® 97 phosphatase substrate upon hydrolysis produces a bright and photostable yellow-green fluorescent precipitate at the site of enzymatic activity. This fluorescent precipitate has several unique spectral characteristics, including an extremely large Stokes shift that makes it easily distinguishable from endogenous fluorescence.

SuperSignal™ West Dura Extended Duration Substrate Thermo Scientific™

Thermo Scientific SuperSignal West Dura Extended Duration Substrate is a luminol-based enhanced chemiluminescence (ECL) horseradish peroxidase (HRP) substrate with stable light output for mid-femtogram level detection for western blot analysis.

SuperSignal West Dura Extended Duration Substrate characteristics include:
Sensitivity: mid-femtogram
Stability: 24-hr working solution stability; 1-year kit stability at room temperature
Compatibility: nitrocellulose and PVDF membranes
Signal duration: 24 hours
Recommended primary antibody concentration: 1:1,000–1:50,000 dilution (0.02–1.0 µg/mL)
Recommended secondary antibody concentration: 1:50,000–1:250,000 dilution (4–20 ng/mL)

Compare and learn more about our western blot chemiluminescent substrates ›
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SuperSignal West Dura Extended Duration Substrate for HRP is optimized for high sensitivity and long signal duration, making it ideal for cooled CCD camera-based detection systems. Unlike substrates with signals that decline to barely detectable levels in 30–60 minutes, the signal produced with SuperSignal West Dura chemiluminescent substrate is stable for 24 hours, allowing multiple film or camera exposures.

CCF4-AM

CCF4-AM, our LiveBLAzer™ substrate, is a lipophilic, esterified form of the CCF4 substrate which allows it to readily enter cells. Upon entry, cleavage by endogenous cytoplasmic esterases rapidly converts CCF4-AM into its negatively charged form, CCF4, which is retained in the cytosol. CCF4 is a Fluorescence Resonance Energy Transfer (FRET) substrate which consists of a cephalosporin core linking 7-hydroxycoumarin to fluorescein. This product is to be used as a FRET-based substrate for β-lactamase as a sensitive reporter of mammalian gene expression in the GeneBLAzer® technology.

EnzChek™ Lipase Substrate, green fluorescent, 505/515 Invitrogen™

The nonfluorescent EnzChek® lipase substrate, in the presence of lipases, produces a green-fluorescent product (excitation/emission maxima ~507/515) for accurate lipase detection in solution.

Fluorescein-IKK Substrate

A fluorescein-labeled peptide substrate that is primarily used with the IkappaB kinase (IKK) family.

For use in combination with the LanthaScreen® Tb-IkappaBalpha [pSer32] antibody (PV3562) to evaluate inhibitors in medium to high throughput screening applications.

Galacton-Star™ Substrate (50X Concentrate) Invitrogen™

Galacton-Star Substrate is a chemiluminescent substrate designed for the rapid, sensitive detection of β-galactosidase in cell lysates. The chemiluminescent assay exhibits over three orders of magnitude greater sensitivity compared to colorimetric β-galactosidase assays. Light emission from Galacton-Star Substrate reaches a maximum in 60-90 minutes and remains constant for at least 1 hour. After incubation at room temperature, the signal is measured in a luminometer.

As few as 10 femtograms of β-galactosidase (20,000 molecules) are detectable. High sensitivity makes this substrate ideal for detection of weak expression and for transfection normalization with other sensitive reporter gene assays.

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.

LiveBLAzer™ FRET-B/G Loading Kit with CCF4-AM

Background:
GeneBLAzer® cell-based assays utilize the membrane-permeant ester forms (CCF2-AM and CCF4-AM) of the negatively charged fluorescent beta-lactamase substrates, CCF2 and CCF4. These lipophilic esters readily enter the cell, where cleavage by endogeneous cytoplasmic esterases rapidly converts them into their negatively charged forms, thereby trapping them in the cytosol.

Detection of GeneBLAzer® assays is FRET-based. Each substrate is labeled with two fluorophores that form an efficient FRET pair. In the absence of beta-lactamase activity, exciting the coumarin at 409 nm in the intact CCF2 molecule results in FRET to the fluorescein, which emits a green fluorescence signal at 518 nm (Figure 1). In the presence of beta-lactamase activity, however, cleavage of CCF2 spatially separates the two dyes and disrupts FRET, so that exciting the coumarin at 409 nm now produces a blue fluorescence signal at 447 nm. This blue signal can be readily observed under a microscope and can also be detected as an increase in the blue channel readout on fluorescent
microplate readers.

The CCF2-AM and CCF4-AM substrates are essential assay components for the GeneBLAzer® platform. These substrates are fully compatible with flow cytometry, speeding the time to clone selection. Ratiometric analysis of the blue and green signals reduces well-to-well variation due to differences in cell numbers and substrate loading, leading to high Z´-factor values and low coefficients of variation (CVs).

CCF2-AM and CCF4-AM differ by two carbons in the bridge linking the coumarin moiety to the lactam ring. Both are in the membrane-permeable, esterified forms, and can be used for assays in intact cells. CCF4-AM has better solubility properties (soluble for >24 hours) than CCF2-AM and is thus best suited for screening applications. In addition, CCF4-AM has slightly better FRET and thus slightly lower background than CCF2-AM.

CCF2-FA is essentially the CCF2 substrate without the esters found in the AM version. CCF2-FA is de-esterified and used in cell lysate applications, bypassing loading across the cell membrane and de-esterification steps. Cell lysates are the preferred method for applications using cells that contain a cell wall. CCF2-FA can also be used as a control to acquire the excitation and emission spectra for CCF2-AM and CCF4-AM.

LanthaScreen™ Tb-DUB Substrate

Deubiquitinating enzymes (DUBs) play housekeeping functions by maintaining pools of active ubiquitin, and regulatory function by rescuing specific target proteins from degradation by the proteasome. The LanthaScreen® Tb-DUB Substrate is a general substrate for assaying DUB activity in a high throughput assay format.

X-Gal Thermo Scientific™

Thermo Scientific X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside) is an inert chromogenic substrate for beta-galactosidase which hydrolyzes X-Gal into colorless galactose and 4-chloro-3-brom-indigo, forming an intense blue precipitate. Induction of the lacZ gene with IPTG leads to the hydrolysis of X-Gal and to the development of blue colonies (see Supporting data).

Applications

• Blue/white colony screening to distinguish recombinant (white) from non-recombinant (blue) colonies (see Reference 1)
• Visualization of beta-galactosidase reporter gene expression in transfected eukaryotic cells
• Detection of beta-galactosidase activity in immunological and histochemical procedures

Note
• Preparation of a 20 mg/mL stock solution in dimethylformamide (DMF) or dimethylsulfoxide (DMSO) is recommended for X-Gal. DMF dissolves some plastic materials. The direct addition of DMF containing solution to plastic Petri dishes should be avoided.
• DMF is toxic. Allow X-Gal solution containing dimethylformamide to completely dry before plating bacteria on agar plates.

Related Products
X-Gal
X-Gal Solution, ready-to-use

LiveBLAzer™ FRET-B/G Loading Kit with CCF2-AM

Background:
GeneBLAzer® cell-based assays utilize the membrane-permeant ester forms (CCF2-AM and CCF4-AM) of the negatively charged fluorescent beta-lactamase substrates, CCF2 and CCF4. These lipophilic esters readily enter the cell, where cleavage by endogeneous cytoplasmic esterases rapidly converts them into their negatively charged forms, thereby trapping them in the cytosol.

Detection of GeneBLAzer® assays is FRET-based. Each substrate is labeled with two fluorophores that form an efficient FRET pair. In the absence of beta-lactamase activity, exciting the coumarin at 409 nm in the intact CCF2 molecule results in FRET to the fluorescein, which emits a green fluorescence signal at 518 nm (Figure 1). In the presence of beta-lactamase activity, however, cleavage of CCF2 spatially separates the two dyes and disrupts FRET, so that exciting the coumarin at 409 nm now produces a blue fluorescence signal at 447 nm. This blue signal can be readily observed under a microscope and can also be detected as an increase in the blue channel readout on fluorescent microplate readers.

The CCF2-AM and CCF4-AM substrates are essential assay components for the GeneBLAzer® platform. These substrates are fully compatible with flow cytometry, speeding the time to clone selection. Ratiometric analysis of the blue and green signals reduces well-to-well variation due to differences in cell numbers and substrate loading, leading to high Z´-factor values and low coefficients of variation (CVs).

CCF2-AM and CCF4-AM differ by two carbons in the bridge linking the coumarin moiety to the lactam ring. Both are in the membrane-permeable, esterified forms, and can be used for assays in intact cells. CCF4-AM has better solubility properties (soluble for >24 hours) than CCF2-AM and is thus best suited for screening applications. In addition, CCF4-AM has slightly better FRET and thus slightly lower background than CCF2-AM.

CCF2-FA is essentially the CCF2 substrate without the esters found in the AM version. CCF2-FA is de-esterified and used in cell lysate applications, bypassing loading across the cell membrane and de-esterification steps. Cell lysates are the preferred method for applications using cells that contain a cell wall. CCF2-FA can also be used as a control to acquire the excitation and emission spectra for CCF2-AM and CCF4-AM.

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

Thermo Scientific Pierce 1-Step Slow TMB ELISA Substrate Solution 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. It is formulated for kinetic studies where rapid reaction is secondary to accuracy.

Features of 1-Step Slow TMB ELISA Substrate Solution :

• 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.

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1-Step™ Ultra TMB-ELISA Substrate Solution
Pierce™ TMB Substrate Kit
1-Step™ Turbo TMB-ELISA Substrate Solution

CDP-Star™ Substrate (0.25 mM Ready-To-Use) Invitrogen™

The CDP-Star® 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 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.

PNPP Substrate Thermo Scientific™

Thermo Scientific Pierce PNPP (p-nitrophenyl phosphate) is a colorimetric, soluble substrate of alkaline phosphatase for use in ELISA applications.

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.

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1-Step™ PNPP Substrate Solution
Pierce™ PNPP Substrate Kit
PNPP Substrate Tablets
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