Immunodetection

enzymatic substrates

 

 

  1. Chromogenic soluble substrates for Peroxidase (for ELISA)

Selection Guide of Enzymatic labels and Substrates

  1. Chromogenic soluble substrates for Alkaline Phosphatase (for ELISA)

  1. Chromogenic precipitating substrates for Alkaline Phosphatase (Blotting - IHC)

  1. Chromogenic soluble substrates for Peroxidase in immunoblotting / IHC

 

1 - Chromogenic soluble substrates for Peroxidase (for ELISA)

TMB (3.3',5,5'-tetramethylbenzidine):

TMB, «Standard» solution - TMB, «Check» solution - TMB, «Aqueous» solution

TMB is the most popular chromogenic peroxidase substrate :

  • Produces upon reaction with peroxidae a blue colour, which is measured at 370nm or 652nm, and a yellow colour (stopped reaction after adding an acid (measured at 450nm).

  • Sensitivity is greater than classic substrates like OPD and ABTS, with very low background.

  • Non-carcinogenic.

TMB, «Standard» solution

UP664780

200ml

TMB, «Check» solution

UPS08170

100ml

-

 UP664781

500ml

NEW

 UPS08171 500ml
- UP664782 1L -6 - -

The original formulation, ideal For classic applications

  • Highest sensitivity

  • Stability > 18 months at +4°C

Technical Sheet Security Sheet

Red staining. Recommended for routine assays, to control better failure in reagent distribution

  • Includes a red dye that do not interfere with reaction nor final reading

  • High sensitivity

  • Stability > 24 months at +4°C

  • Visual or optical controle of deposits

Technical Sheet

TMB, «Aqueous» solution

UPS08180

200ml

-NEW

 UPS08181

500ml

Uptima TMB solutions are optimized chromogenic for peroxidase, designed for ELISA techniques, manual or automatic systems. They contain 3,3’,5,5’-tetramethylbenzidine (TMB), hydrogene peroxide (H2O2), and proprietary catalyzing and stabilizing agents.

  • Highest sensitivity

  • Reproducible lots

  • Ready-to-use
- UPS08182 1L

Recommanded in particular for diagnostic kits

  • Non-hazardous, non-volatile, non-organic, non-toxic (does not contain DMF, DMSO) : 100% water based formulation to maximize the safety (no regulation concerns).

  • Highest sensitivity

  • Stability > 24 months at +4°C

Technical Sheet

Related Products

TMB / HRP Stop solution

NEW

UPS29590

250ml

To be used with TMB reagents

Stops the reaction, change the color to deep yellow,

increasing the sensivity 2 to 3 fold

 

ABTS (2.2-Azino-di(3-EthylBenzthiazoline Sulfonic acid):

ABTS, ready to use solution - ABTS UltraPure, powder

Produces a blue-green staining, which is measured between 405 and 410 nm

ABTS, ready to use solution

UP732550

100ml

ABTS UltraPure, powder 

UP423876

1g

-

 UP732551

500ml

UP423877 10g

  • Formulation stable at room temperature

  • Highest sensitivity

Di-ammonium salt, >98% pure, soluble in water

  • Suitable for ELISA

 

2- Chromogenic soluble substrates for Alkaline Phosphatase (for ELISA)

pNPP (p-nitrophenyl phosphate, disodium salt)

PnPP, ready to use solution - PnPP UltraPure, powder - PnPP, tablets of 30mg

Soluble substrate giving an intense yellow colour measured at 405 nm (reaction may be stopped with NaOH).

pNPP, ready to use solution

UP664790

200ml

pNPP UltraPure, powder

UP89562C

25g

UP664791

qsp 500ml

Economic

UP89562D

100g

-

UP664792

qsp      1L

pNPP, tablets of 30mg

UP732500

100tab

  • Highest sensitivity

  • Stability > 1year at +4°C

  • Convenience (no buffer to prepare, nor powder to dissolve !)

Technical Sheet

UP732501 1000tab

Convenient : no weighing !

For 96 wells microplates ELISA

 

3 - Chromogenic precipitating substrates for Alkaline Phosphatase (Blotting - IHC)

BCIP (5-bromo-4-choro-3’indolylphosphate)

BCIP/NBT ready to use solution - BCIP/INT ready to use solution - BCIP, T salt, UltraPure powder - BCIP, Na salt, powder

Most commonly used precipitating substrate for AP, because it is easy to use and sensitive.

BCIP/NBT ready to use solution

UP099851

100 ml

BCIP/INT ready to use solution

UP096051

100ml

An one component chromogenic substrate for immobilized alkaline phosphatase suitable for immunoblotting and immunohistology.

Produces a dark blue colour.

Technical Sheet

An one component chromogenic substrate for immobilized alkaline phosphatase suitable for immunohistochemistry.

The brick-red end product is clearly visible against a blue hematoxylin background, avoiding the misleading results that can arise from observing NBT.

Detects sub-nanogram levels of mouse IgG.

Technical Sheet

BCIP, T, UltraPure powder

UP10904E

100mg

BCIP, Na salt powder

UP38047A

1g

UP10904F

500mg

5-Bromo-4-Chloro-3-indolyl-phosphate, p-toluidine salt

C8H6BrClO4PC7H9N

CAS 6578-06-9

MW 433.6

Purity > 99 %

To be used with NBT for blotting techniques (Southern, Northern, Dot, Western), produces on deep purple color.

5-Bromo-4-Chloro-3-Indolyl-phosphate, disodium salt

C8H4BrClNO4PNa7

MW 370.43

Blue insoluble substrate of alkaline phosphatase

INT (iodonitrotetrazolium chloride)

Precipitating substrate for alkaline phosphatase,

and soluble substrate for glucose oxidase (with PMS)

INT UltraPure, powder

UP020611

1g

C19H13NO21CI

CAS 146-68-9

MW 505, 71

Purity > 99 %

NBT (Nitro Blue Tetrazolium Chloride)

NBT UltraPure, powder

UP143456

500mg

UP143457

1g

C40H30N10O6Cl2

CAS 298-83-9

MW 817,6

E(260nm, MetOH) >54000, enhances the color of BCIP staining

 

4 - Chromogenic soluble substrates for Peroxidase in immunoblotting / IHC

TMB (3.3.5.5’-tetramethylethylenediamine)

Produces a dark blue stain, stable 90 mn in DMSO, precipitating on nitrocellulose or nylon membranes. Excellent sensitivity, not toxic.

TMB UltraPure Grade, powder

UP15426D

1g

UP15426E

5g

Suitable for ELISA and Immunoblotting, >98% pure, soluble in water

DAB (3.3diaminobenzidine)

DAB, ready to use solution - DAB, tablets - DAB, UltraPure powder - Op-Metal Enhanced DAB kit

The most sensitive chromogenic substrate for peroxidase. Insoluble staining in alcohol and water, brown or black depending on the use.

DAB produces a stable water/alcohol insoluble reddish brown product. This effect is enhanced in our DAB kit UP67962 by modifying the DAB solution with the addition of metal ions to produce a more dense reaction forming a localized deep blue/black precipitate.

DAB, ready to use solution

UP732320

500 ml

Op-Metal Enhanced DAB kit

NEW

679921

300ml

Suitable for immunoblotting and immunohistochemistry ; brown colour; supplied as a 50 x concentrate with a 10 x concentrated dilution buffer.

Contains:

  • DAB Substrate

  • Peroxide Substrate

  • Metal Enhancer

A stable solution developed to give enhanced color reaction with low background

The reagents supplied are sufficient for making 300ml working solution. The kit allows to stainin reddish/brown, and/or in blue/ black, as the enhancement with metal is optionnal.

Literature

1. (1981) J. Histochem. Cytochem. Vol.29, No.6:775

2. (1989) J. Clin, Pathol, 42:875-880

DAB, tablets

UP732310

50x5mg

UP732311

100x5mg

Avoids to weight this carcinogenic compound

Technical Sheet

DAB, UltraPure, powder

UP01012G

5g

UP01012H

10g

Suitable for electronic microscopy with metallic additives, or for immunoblotting with 4-CN (black stain).

4-CN (4-chloro-1-naphtol)

A classical peroxidase substrate for blotting.

4-CN UltraPure, powder

UP007490

25g

UP007491

50g

Purity >99.8% blue-purple colour, suitable for double labeling, or associated with DAB

Uptilight HRP blotting kit

UP99619

Ultra-sensitive ! Chemiluminescent substrate

 

Selection Guide of Enzymatic labels and Substrates

Labels

Substrate

Pros

Cons

Applications

HRP

 

TMB, ABTS, OPD

Chemiluminescence

Rapid kinetic

Versatile detection system

Light sensitive coloration

Need to stop the colour formation

ELISA

-

CN, DAB, AEC

-

-

-

Back ground: blood samples

Less staining stability than AP

ImmunoBlotting

ImmunoHistology

Chemiluminescence

The most sensitive

Require radiographie or scanner equipment

IB

AP

pNPP

-

Linear kinetic

Often more sensitive

Less economic than peroxidas

-

ELISA

--

BCIP / NBT

FastRed

(for chromogenic detections)

Stability of staining

Back ground: some samples

High molecular weight conjugates

ImmunoBlotting

ImmunoHistology

 

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