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Rhodamine 110, bis-(p-Tosyl-L-Glycyl-L-Prolyl-L-Arginine Amide) (Invitrogen™)

This Molecular Probes® bisamide derivative of rhodamine 110 (R110) is a sensitive and selective substrate for assaying proteases in solution or inside living cells. These fluorogenic substrates contain an amino acid or peptide covalently linked to each of R110’s amino groups. Upon enzymatic cleavage, the nonfluorescent bisamide substrate is converted first to the fluorescent monoamide and then to R110, with a further increase in fluorescence. The substrate can be used to continuously measure enzyme activity in cell extracts and purified enzyme preparations using a fluorometer or fluorescence microplate reader, or can be used for intracellular protease assays with analysis by flow cytometry or fluorescence microscopy.

Substrate Specifications:
• Substrate name: (p-tosyl-Gly-Pro-Arg)2-R110
• Useful to measure: Thrombin
• Ex/Em of R110 product: (498/521 nm)

Properties of the R110 Reaction Product
The R110 cleavage product has spectral properties similar to those of fluorescein, making it compatible with standard fluorescein filter sets. Because of the large extinction coefficient of R110, signal to background ratios are very good. And, unlike fluorescein, the fluorescence intensity of the rhodamine 110 reaction product is constant from pH 3–9. We also offer an R110 reference standard for use in your assays.

We’ll Make a Custom R110 Substrate for You
If you can’t find what you’re looking for in our stocked list, we’ll prepare a custom R110 substrate for you. Our custom services are efficient and confidential, and we stand by the quality of our work. We are ISO 9001:2000 certified.

For more information about these products, read Detecting Peptidases and Proteases—Section 10.4 in the Molecular Probes® Handbook.

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

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 ›
View all HRP substrates available ›

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.

PED-A1 (N-((6-(2,4-DNP)Amino)Hexanoyl)-1-(BODIPY™ FL C5)-2-Hexyl-Sn-Glycero-3-Phosphoethanolamine) (Phospholipase A1 Selective Substrate) (Invitrogen™)

Molecular Probes® phospholipase substrates are dye labeled phospholipids used to monitor phospholipase A (PLA) activity in purified enzyme preparations, cell lysates, and living cells. Two structural variations of these substrates are available, which confer specificity for PLA1 and PLA2. Fluorescently labeled versions of the cleavage product generated from these substrates are also available for use as standards for evaluating percentage substrate conversion in enzymatic reactions.

Fluorogenic Phospholipase A Substrates Specifications:

• Label (Ex/Em): PED-A1
• Specificity: PLA1
• Cleavage product: D3834
• Lyophilized product can be dissolved in DMSO for use
• Fluorescence is measured either in microplate assays or using flow cytometry

Find More Probes for Lipid Metabolism and Fatty Acid Analogs
Review Fatty Acid Analogs and Phospholipids—Section 13.2 and Probes for Lipid Metabolism and Signaling—Section 17.4 in the Molecular Probes® Handbook for more information on these products.

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

Fluorescein Di-β-D-Glucopyranoside (FDGlu) (Invitrogen™)

Fluorescein di-β-D-glucopyranoside is one of the most sensitive substrates for glucosidases. The nonfluorescent substrate is sequentially hydrolyzed by β-glucosidase, first to fluorescein monoglucoside and then to highly fluorescent fluorescein. Enzyme-mediated hydrolysis of fluorescein di-β-D-glucopyranoside can be followed by the increase in either absorbance or fluorescence.

Pierce™ TMB Substrate Kit (Thermo Scientific™)

Thermo Scientific Pierce TMB ELISA Substrates detect 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.

Features of Thermo Scientific Pierce 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
1-Step™ Ultra TMB-ELISA Substrate Solution
1-Step™ Turbo TMB-ELISA Substrate Solution
1-Step™ Slow TMB-ELISA Substrate Solution

SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific™)

Thermo Scientific SuperSignal West Femto Maximum Sensitivity Substrate is an ultra-sensitive enhanced chemiluminescent (ECL) substrate for low femtogram protein level detection by western blot analysis with horseradish peroxidase (HRP) enzyme.

SuperSignal West Femto Maximum Sensitivity Substrate characteristics include:
Sensitivity: low-femtogram (mid-zeptomole)
Stability: 8-hr working solution stability, 1-year kit stability at 4°C
Compatibility: nitrocellulose and PVDF membranes
Signal duration: up to 8 hours
Recommended primary antibody concentration: 1:5,000–1:100,000 dilution (10–200 ng/mL)
Recommended secondary antibody concentration: 1:100,000–1:500,000 dilution (2–10 ng/mL)

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

SuperSignal West Femto Maximum Sensitivity Substrate is designed for low femtogram-level detection by western blot analysis. When combined with optimized antibody concentrations and blocking buffers, SuperSignal West Femto substrate enables detection of target proteins in amounts that are too small to be seen with typical ECL substrates.

PI(5)P Lipid Kinase Substrate (Invitrogen™)

The PI(5)P Lipid Kinase Substrate has been specifically optimized for use with Type II PIP kinases (e.g., PIP4K2A). The substrate consists of solubilized phosphatidylinositol 5-phosphate (PI(5)P) in an aqueous buffer, and is used in combination with our Adapta™ Universal Kinase Assay to help identify unique kinase inhibitors.

• Validated for use with Thermo Fisher Scientific lipid kinases
• Available in multiple pack sizes for convenient use
• Also available in our SelectScreen™ Kinase Profiling Service

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.

1-Step™ Turbo TMB-ELISA Substrate Solution (Thermo Scientific™)

Thermo Scientific Pierce 1-Step Turbo TMB-ELISA Substrate Solution rapidly 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. Reactions are typically complete between five and thirty minutes after addition to HRP-Conjugates.

Features of 1-Step Turbo 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.

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

Pierce™ ECL Western Blotting Substrate (Thermo Scientific™)

Thermo Scientific Pierce ECL Western Blotting Substrate is a value-priced, entry-level horseradish peroxidase (HRP) substrate for enhanced chemiluminescence (ECL) that directly replaces costlier products without the need to re-optimize conditions.

Pierce ECL Plus Western Blotting Substrate characteristics include:
Sensitivity: low picogram
Stability: 1-hr working solution stability, 1-year kit stability at 4°C
Compatibility: nitrocellulose and PVDF membranes
Signal duration: 1 to 2 hours
Recommended primary antibody concentration: 1:1,000–1:5,000 dilution (0.2–10 µg/mL)
Recommended secondary antibody concentration: 1:1,000–1:15,000 dilution (0.07–1.0 µg/mL)

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

Pierce ECL Western Blotting Substrate provides reliability and performance equivalent to other standard ECL substrates for detection of HRP enzyme activity. It enables the detection of picogram amounts of antigen and allows for easy detection of HRP using photographic or other imaging methods.

Because the luminol and peroxide reagent formulations are identical to other commercially available substrate products, one can switch to Pierce ECL without needing to optimize probing conditions or incubation protocols. Pierce ECL Western Blotting Substrate provides performance identical to the original Amersham ECL Western Blotting Detection Reagent from GE Healthcare.

Red/Green BODIPY™ PC-A2 (1-O-(6-BODIPY™ 558/568-Aminohexyl)-2-BODIPY™ FL C5-Sn-Glycero-3-Phosphocholine) (Ratiometric Phospholipase A2 Substrate) (Invitrogen™)

Molecular Probes® phospholipase substrates are dye labeled phospholipids used to monitor phospholipase A (PLA) activity in purified enzyme preparations, cell lysates, and living cells. Two structural variations of these substrates are available, which confer specificity for PLA1 and PLA2. Fluorescently labeled versions of the cleavage product generated from these substrates are also available for use as standards for evaluating percentage substrate conversion in enzymatic reactions.

Fluorogenic Phospholipase A Substrates Specifications:

• Label (Ex/Em): Red/Green BODIPY® PC-A2 (~488/530,590 nm) (can be used in dual emission fluorescence ratio detection)
• Specificity: PLA2
• Cleavage product: D3834
• Lyophilized product can be dissolved in DMSO for use
• Fluorescence is measured either in microplate assays or using flow cytometry.

Find More Probes for Lipid Metabolism and Fatty Acid Analogs
Review Fatty Acid Analogs and Phospholipids—Section 13.2 and Probes for Lipid Metabolism and Signaling—Section 17.4 in the Molecular Probes® Handbook for more information on these products.

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

DQ™ Ovalbumin - Special Packaging (Invitrogen™)

DQ ovalbumin is a fluorogenic substrate for proteases. A strong fluorescence quenching effect is observed when proteins are heavily labeled with BODIPY dyes. Upon hydrolysis of the DQ ovalbumin to single, dye-labeled peptides by proteases, this quenching is relieved, producing brightly fluorescent products.

Alexa Fluor™ 647-Poly-GT Peptide Substrate

A general AlexaFluor647-labeled peptide substrate consisting of a polymer of glutamate, and tyrosine that is used to monitor tyrosine kinase acitivty.
For use in combination with the LanthaScreen® Eu-PY20 Antibody (PV5696) to evaluate inhibitors in medium to high throughput screening applications.

1-Step™ ABTS Substrate Solution (Thermo Scientific™)

ABTS Substrate is a water-soluble peroxidase substrate that yields a measurable green end product for use in ELISA methods.

ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) is a water-soluble HRP substrate that yields a green end product upon reaction with peroxidase. The green product has two major absorbance peaks, 410nm and 650nm. ABTS is less sensitive than OPD and TMB in ELISA applications. It is less readily oxidized, and its color development is slower (approximately 20 minutes). This may be advantageous if unacceptable background results from the use of the OPD or TMB substrates due to higher sensitivities.

Related Products
ABTS Substrate Tablets

DiFMUP (6,8-Difluoro-4-Methylumbelliferyl Phosphate) (Invitrogen™)

DiFMUP is a fluorinated 4-methylumbelliferyl phosphate (MUP) derivative that has a lower pKa than that of MUP, making DiFMUP an excellent substrate for continuously assaying acid phosphatases at low pH. The reaction product of DiFMUP has excitation/emission maxima of ~358/450 nm. DiFMUP is also available packaged in ten vials of 10 mg each (D-22065).