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Pierce™ DAB Substrate Kit Thermo Scientific™

The Thermo Scientific Pierce DAB Substrate Kit enables chromogenic detection of horseradish peroxidase (HRP) activity based the action of 3,3'-diaminobenzidine (DAB) in Western blot and tissue staining methods.

DAB (3,3'-diaminobenzidine tetrahydrochloride; MW = 214.1) reacts with HRP in the presence of peroxide to yield an insoluble brown-colored product at locations where peroxidase-conjugated antibodies are bound to samples. The brown precipitate is insoluble in alcohol and other organic solvents, making it an excellent substrate for immunohistochemical staining that requires the use of traditional counterstains and mounting media. The Pierce DAB Substrate Kit includes a 10X solution of DAB and Stable Peroxide Substrate Buffer. Crystalline DAB (powder) is also available.

Features DAB Substrate:

HRP substrate—for detection of horseradish peroxidase activity on solid media, including nitrocellulose and PVDF membranes and fixed tissue samples
Chromogenic—no special equipment needed for visualization; the DAB reaction produces brown bands or spots at sites of reaction with HRP-conjugated antibodies or probes
Package options—choose powder for custom formulation with buffer and peroxide or a complete kit of preformulated solutions for immediate use

Features of the Pierce DAB Substrate Kit:

Easy-to-use—dilute 10X stock solution to 1X in provided stable peroxide buffer
Safer—preformulated DAB solution minimizes safety concerns with this chemical
Complete—includes convenient solution of stable hydrogen peroxide buffer (Part No. 34062)
Stable—store refrigerated at 4°C for at least one year; freezer storage is not necessary

DAB has been used in a variety of applications, including Western blotting, immunohistochemistry and electron microscopy. Many enhancement methods have been reported for DAB involving different buffer conditions, the addition of metal ions and post-treatment applications (Ref. 1-7; also see the Pierce Metal Enhanced DAB Substrate Kit, Part No. 34065). One disadvantage of DAB compared to other substrates is the need to use the working solution immediately because it begins a reaction process upon the addition of hydrogen peroxide. This results in increased background color of the substrate. However, the brown bands resulting from DAB reactions do not fade as quickly as the color-products of other precipitating HRP substrates.

Related Products
DAB Substrate Powder (3,3'-diaminobenzidine tetrahydrochloride)

Histone H3 Peptide (1-20)

Matched and validated lipid and peptide substrates are available to complement the Adapta® Universal Kinase Assay. The assays developed with these reagents allow for primary or secondary screening of kinase inhibitors.

GFP-4E-BP1

4E-BP1 expressed as a fusion with GFP (Green Fluorescent Protein) to act as a physiological substrate for FRAP1 (mTor).

For use in combination with the LanthaScreen® Tb-anti-4E-BP1 [pThr46] Antibody (PV4757) to evaluate inhibitors in medium to high throughput screening applications. An optimized mTor assay buffer (PV4794) is also available.

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

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.

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

This concentrated CDP-Star® Substrate is a chemiluminescent alkaline phosphatase substrate 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.

CSPD™ Substrate (25 mM Concentrate) Invitrogen™

This concentrated CSPD® substrate is a chemiluminescent alkaline phosphatase substrate 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.

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.

Vivid™ DBOMF Substrate

Vivid® Substrates are blocked dyes that yield a minimal fluorescence signal until oxidation occurs. Cytochrome P450 metabolism at either of two potential oxidation sites releases the highly fluorescent product. They have superior fluorescence, solubility, and kinetic properties compared to conventional fluorogenic probes. This results in higher sensitivity, greater signal-to-noise ratio, and better assay reproducibility. When used in combination with CYP450 BACULOSOMES® Plus Reagents as part of the Vivid® CYP450 Screening Kits, the Vivid® Substrates enable rapid, robust detection of cytochrome P450 inhibitors. This particular substrate (Vivid ® DBOMF Substrate) yields a product that emits a green fluorescence.

More About Vivid® CYP450 Screening Kits
Vivid® CYP450 Screening Kits are high-throughput fluorescence-based assays for detection of enzyme-drug interactions and CYP450 inhibition. Vivid® CYP450 Screening Kits include Vivid® Substrate, Vivid® Fluorescent Standard, reaction buffer, Vivid® Regeneration System, NADP+, and CYP450 BACULOSOMES® Plus Reagent. CYP450 BACULOSOMES® Plus Reagents are microsomes prepared from insect cells expressing a human CYP450 isozyme and human cytochrome P450 reductase. CYP450 BACULOSOMES® Plus Reagents offer a distinct advantage over human liver microsomes in that only one CYP450 isozyme is expressed, thereby preventing metabolism by other CYP450s.

>> View all Vivid® CYP450 Screening Kits

Applications: high-throughput screening of enzyme-drug interactions, compound profiling for drug inhibition of cytochrome P450 isozymes, generation of predictive SAR models to guide compound acquisition

For Research Use Only. Not for any animal or human therapeutic or diagnostic use.

DQ™ Gelatin From Pig Skin, Fluorescein Conjugate - Special Packaging Invitrogen™

Our DQ gelatin is a fluorogenic substrate that can be used to detect protease activity in vitro with high sensitivity. This substrate, formerly available only in our popular EnzChek Gelatinase/Collagenase Assay Kit (E-12055), consists of highly quenched, fluorescein-labeled gelatin. Upon proteolytic digestion, its bright, green fluorescence is revealed and can be used to measure enzymatic activity. The fluorescence increase can be monitored with a fluorescence microplate reader or fluorometer.

SuperSignal™ West Pico PLUS Chemiluminescent Substrate Thermo Scientific™

Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate is an enhanced chemiluminescent (ECL) horseradish peroxidase (HRP) substrate that enables picogram to high femtogram-level protein detection by western blot analysis.

SuperSignal West Pico PLUS Chemiluminescent Substrate characteristics include:
Sensitivity: pictogram to femtogram sensitivity
Stability: 8-hr working solution stability; 1-year kit stability at room temperature
Compatibility: nitrocellulose and PVDF membranes
Signal duration: 6 to 24 hours
Recommended primary antibody concentration: 1:1,000–1:5,000 dilution (0.2–1.0 µg/mL)
Recommended secondary antibody concentration: 1:20,000–100,000 dilution (10–50 ng/mL)

Compare and learn more about our western blot chemiluminescent substrates ›
View all HRP substrates available ›

SuperSignal West Pico PLUS substrate is designed to provide excellent signal intensity and sensitivity for western blotting with HRP conjugates. The intensity of the light emission combined with the exceptional duration allows for the acquisition of multiple exposures to more easily obtain publication-quality blot images. SuperSignal West Pico PLUS substrate is compatible with different membranes, blocking reagents, and a wide range of antibody dilutions, making it an ideal choice for most western blotting applications.

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

This concentrated CDP-Star® Substrate is a chemiluminescent alkaline phosphatase substrate 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.

GFP-STAT3

STAT3 expressed as a fusion with GFP (Green Fluorescent Protein) to act as a physiological substrate for the Janus family of tyrosine kinases.

For use in combination with the LanthaScreen® Tb-anti-STAT3 [pTyr705] Antibody (PV4846) to evaluate inhibitors in medium to high throughput screening applications.

X-Gal (5-Bromo-4-Chloro-3-Indolyl β-D-Galactopyranoside) Invitrogen™

X-Gal is a widely used chromogenic substrate for β-galactosidase. It yields a dark blue precipitate at the site of enzymatic activity. X-Gal is useful for numerous histochemical and molecular biology applications, including detection of lacZ activity in cells and tissues. In contrast to β-glucuronidase as a gene marker, β-galactosidase can be fixed in cells and tissues with glutaraldehyde without loss of activity and detected with high resolution using X-Gal.


• Chromogenic substrate
• Cell permeant
• β-galactosidase activity can still be detected using X-Gal after fixation


Additional Information About Glycosidase Enzymes
Glycosidase enzymes exhibit very high selectivity for hydrolysis of their preferred sugars. For example, β-galactosidase rapidly hydrolyzes β-D-galactopyranosides but usually does not hydrolyze either the anomeric α-D-galactopyranosides or the isomeric β-D-glucopyranosides. Endogenous glycosidase activity is frequently used to characterize strains of microorganisms and to selectively label organelles of mammalian cells; defects in glycosidase activity are characteristic of several diseases.

In addition, glycosidases are important reporter gene markers. Specifically, lacZ, which encodes β-galactosidase, is extensively used as a reporter gene in animals and yeast, whereas the β-glucuronidase (GUS) gene is a popular reporter gene in plants. Glycosidase substrates are also used in conjunction with glycosidase-conjugated secondary detection reagents in immunohistochemical techniques and enzyme-linked immunosorbent assays (ELISAs). See all of our Molecular Probes® fluorogenic and chromogenic glycosidase substrates.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

1-Step™ NBT/BCIP Substrate Solution Thermo Scientific™

Thermo Scientific Pierce NBT and BCIP Substrates are powders and ready-made solutions of nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate for chromogenic blot and IHC staining with alkaline phosphatase probes.

The combination of NBT (nitro-blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt) yields an intense, insoluble black-purple precipitate when reacted with alkaline phosphatase, a popular enzyme conjugate for antibody probes. Pierce NBT and BCIP powders are available individually and in two convenient, ready-to-use solutions (with and without a levamisole suppressor of endogenous phosphatase activity).

Features:

AP substrate—for detection of alkaline phosphatase (alk-phos) activity on solid media, including nitrocellulose and PVDF membranes and fixed tissue samples
Chromogenic—no special equipment needed for visualization; produces purple-black precipitate that is easy to photograph
Package options—choose individual NBT and BCIP powders, or preformulated, ready-to-use solutions with or without levamisole suppressor

Highlights of Pierce NBT/BCIP 1-Step Solution (Part No. 34042):
• Best for immunoblotting applications
• Ready-to-use single component
• Sensitive with low background and minimial assay-to-assay variability

Highlights of Pierce NBT/BCIP plus Suppressor 1-Step Solution (Part No. 34070):
• Best for immunohistochemistry applications
• Ready-to-use single component
• Contains 1 mM levamisole to inhibit endogenous phosphatase activity, while yielding an intense dark purple precipitate in the presence of calf intestinal phosphatase

Related Products
1-Step™ NBT/BCIP plus Suppressor Substrate Solution
NBT Substrate Powder (nitro-blue tetrazolium chloride)
BCIP Substrate Powder (5-bromo-4-chloro-3'-indolyphosphate p-toluidine)
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