c ata l o g u e

OF

SUBSTRATES

Biochemistry
Immunology
A p p l i w a re : S u b s t r a t e s
Contents
1 Chromogenic Substrates 3
2 Fluorogenic Substrates 7
3 Bioluminescent Substrates 9
4 Enzyme/Cross Reference List 10
5 How to use selected Enzyme Substrates 14
6 Immunological substrates 16

About Appliware
The Acros Organics catalogue of fine chemicals lists over 16,000 different compounds
including products with typical applications.
This Appliware brochure lists only a selection of biochemical and immunological subtrates.
For packsizes and prices please consult the Acros Organics catalogue, catalogue on
CD-ROM or register at our website (www.acros.be).
For more information and availability please contact your local Acros Organics distributor
or e-mail to infodesk@be.acros.com.

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A p p l i w a re : S u b s t r a t e s

1 Chromogenic Substrates

Indoxyl Substrates
e.g. 5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside (X-gal, x-β-D-gal)
The same basic reaction applies to the following chromogenic indigo substrate derivatives:
Substrates color of precipitate absorption λmax [nm]
X blue 615
Y blue 680
Magenta magenta 565
Salmon salmon 540
Iodo purple 575
Green green 665
Br Br
CH2OH Cl Cl

OH O O
O
beta-Ga lactos idase oxidation H
OH N N N
CH 2OH
H H
OH O OH O
OH

OH
Br Cl
x-gal
absorbs at 615 nm
OH bue precipitate

BLUE I
Substrates for Glycosidases
Code Product Synonym
33691 X-beta-D-galactosaminide 5-Bromo-4-chloro-3-indoxyl-N-acetyl-beta-D-galactosaminide
34048 X-beta-D-glucosaminide 5-Bromo-4-chloro-3-indoxyl-N-acetyl-beta-D-glucosaminide
34049 X-beta-D-cellobioside 5-Bromo-4-chloro-3-indoxyl-beta-D-cellobioside
33695 X-beta-D-fucoside 5-Bromo-4-chloro-3-indoxyl-beta-D-fucopyranoside
34050 X-alpha-D-gal 5-Bromo-4-chloro-3-indoxyl-alpha-D-galactopyranoside
22777 X-gal, X-beta-D-gal 5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside
34051 X-alpha-D-glc 5-Bromo-4-chloro-3-indoxyl-alpha-D-glucopyranoside
22872 X-beta-D-glc 5-Bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside
33701 X-beta-D-glcUA.CHX 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt
33688 X-beta-D-glcUA.Na 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, Na salt
34052 X-beta-D-xyl 5-Bromo-4-chloro-3-indoxyl-beta-D-xylopyranoside

Substrates for Esterases

Code Product Synonym
33690 X-butyrate 5-Bromo-4-chloro-3-indoxyl butyrate
33689 X-caprylate 5-Bromo-4-chloro-3-indoxyl caprylate
34053 X-palmitate 5-Bromo-4-chloro-3-indoxyl palmitate

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Substrates for Phosphatases
Code Product Synonym
34054 X-phos.diAMPD 5-Bromo-4-chloro-3-indoxyl phosphate, di (2-amino-2-methyl-l, 3-propanediol) salt
33676 X-phos.diNa 5-Bromo-4-chloro-3-indoxyl phosphate, disodium salt
34055 X-phos.p-tol 5-Bromo-4-chloro-3-indoxyl phosphate, p-toluidine salt
33723 X-phos.K 5-Bromo-4-chloro-3-indoxyl phosphate, potassium salt

BLUE II
Substrates for Glycosidases
Code Product Synonym
33687 Y-beta-D-gal 3-Indoxyl-beta-D-galactopyranoside
33717 Y-beta-D-glc 3-Indoxyl-beta-D-glucopyranoside
33711 Y-beta-D-glcUA.CHX 3-Indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt
34031 Y-beta-D-glcUA.Na 3-Indoxyl-beta-D-glucuronic acid, sodium salt

Substrates for Phosphatases

Code Product Synonym
34032 Y-phos.diAMPD 3-Indoxyl phosphate, di(2-amino-2-methyl-1,3-propanediol) salt
33708 Y-phos.diNa 3-Indoxyl phosphate, disodium salt
34033 Y-phos.p-tol 3-indoxyl phosphate, p-touidine salt

Substrates for Sulfatases

Code Product Synonym
33706 Y-sulfate.K 3-indoxyl sulfate, potassium salt

MAGENTA
Substrates for Glycosidases
Code Product Synonym
34035 Magenta-beta-D-glucosaminide 5-Bromo-6-chloro-3-indoxyl-N-acetyl-beta-D-glucosaminide
33735 Magenta-beta-D-gal 5-Bromo-6-chloro-3-indoxyl-beta-D-galactopyranoside
34036 Magenta-beta-D-glc 5-Bromo-6-chloro-3-indoxyl-beta-D-glucopyranoside
33693 Magenta-beta-D-glcUA 5-Bromo-6-chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt

Substrates for Esterases

Code Product Synonym
34037 Magenta-butyrate 5-Bromo-6-chloro-3-indoxyl butyrate
33689 Magenta-caprylate 5-Bromo-6-chloro-3-indoxyl caprylate
34038 Magenta-palmitate 5-Bromo-6-chloro-3-indoxyl palmitate

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A p p l i w a re : S u b s t r a t e s
Substrate for Phosphatases
Code Product Synonym
33729 Magenta-phos.p-tol 5-Bromo-6-chloro-3-indoxyl phosphate, p-toluidine salt

Substrate for Sulfatases

Code Product Synonym
33724 Magenta-sulfate.K 5-Bromo-6-chloro-3-indoxyl sulfate, potassium salt

SALMON
Substrates for Glycosidases
Code Product Synonym
34039 Salmon-beta-D-gal 6-Chloro-3-indoxyl-beta-D-galactopyranoside
34040 Salmon-beta-D-glc 6-Chloro-3-indoxyl-beta-D-glucopyranoside
34041 Salmon-beta-D-glcUA.CHX 6-Chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt

Substrates for Esterases

Code Product Synonym
34042 Salmon-butyrate 6-Chloro-3-indoxyl butyrate
34043 Salmon-caprylate 6-Chloro-3-indoxyl caprylate
34044 Salmon-palmitate 6-Chloro-3-indoxyl palmitate

Substrates for Phosphatases

Code Product Synonym
34045 Salmon-phos.p-tol 6-Chloro-3-indoxyl phosphate, p-toluidine salt

PURPLE
Code Product Synonym
34046 Iodo-beta-D-gal 5-Iodo-3-indoxyl-beta-D-galactopyranoside

GREEN
Code Product Synonym
34047 Green-beta-D-gal N-Methylindoxyl-beta-D-galactopyranoside

Oxidizing Agent
Code Product Synonym
41592 NBT Nitrotertazolium blue chloride
15833 TNBT Tetranitrobluetetrazolium chloride

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2- and 4-Nitrophenyl (ONP and PNP) substrates
e.g. 4-Nitrophenyl phosphate, disodium salt (PNP-phos)
Na
O
P O
H
O O Na O
4-Nitrophenyl phosphate, 4-Nitrophenol
disodium salt Alkaline Phosphatase absorbs at 405 nm
yellow solution
H3PO 4
N N
O O O O

The same basic reaction applies for all 2- and 4-Nitrophenyl (ONP and PNP) substrates.

Substrates for Glycosidases

Code Product Synonym
34056 PNP-alpha-D-galactosaminide 2-Nitrophenyl-N-acetyl-alpha-D-galactosaminide
33694 PNP-beta-D-galactosaminide 4-Nitrophenyl-N-acetyl-beta-D-galactosaminide
22939 ONP-alpha-D-glucosaminide 2-Nitrophenyl-N-acetyl-alpha-D-glucosaminide
34057 ONP-beta-D-glucosaminide 2-Nitrophenyl-N-acetyl-beta-D-glucosaminide
22940 PNP-alpha-D-glucosaminide 4-Nitrophenyl-N-acetyl-alpha-D-glucosaminide
22941 PNP-beta-D-glucosaminide 4-Nitrophenyl-N-acetyl-beta-D-glucosaminide
33710 PNP-beta-D-cellobiose 4-Nitrophenyl-beta-D-cellobioside
34058 PNP-alpha-L-fucoside 4-Nitrophenyl-alpha-L-fucopyranoside
33685 PNP-beta-D-fucoside 4-Nitrophenyl-beta-D-fucopyranoside
12882 ONP-beta-D-gal 2-Nitrophenyl-beta-D-galactopyranoside
33733 PNP-alpha-D-gal 4-Nitrophenyl-alpha-D-galactopyranoside
22929 PNP-alpha-D-gal 4-Nitrophenyl-beta-D-galactopyranoside
34059 ONP-beta-D-glc 2-Nitrophenyl-beta-D-glucopyranoside
34060 MNP-beta-D-glc 3-Nitrophenyl-beta-D-glucopyranoside
33715 PNP-alpha-D-glc 4-Nitrophenyl-alpha-D-glucopyranoside
30311 PNP-beta-D-glc 4-Nitrophenyl-beta-D-glucopyranoside
33679 PNP-beta-D-glcUA 4-Nitrophenyl-beta-D-glucuronic acid
34061 PNP-beta-D-glcUA.Na 4-Nitrophenyl-beta-D-glucuronic acid, sodium salt
34062 PNP-beta-D-lac 4-Nitrophenyl-beta-D-lactopyranoside
33696 PNP-alpha-D-maltoside 4-Nitrophenyl-alpha-D-maltopyranoside
33680 PNP-alpha-D-mannoside 4-Nitrophenyl-alpha-D-mannopyranoside
34063 PNP-beta-D-mannoside 4-Nitrophenyl-beta-D-mannopyranoside
34064 PNP-beta-D-xyl 4-Nitrophenyl-beta-D-xylopyranoside

Substrate for Esterases

Code Product Synonym
34065 PNP-palmitate 4-Nitrophenyl palmitate

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Substrates for Phosphatases
Code Product Synonym
34066 2(PNP)-phos.Na Bis(4-nitrophenyl)phosphate, sodium salt
22799 PNP-phos.diCHX 4-Nitrophenyl phosphate, dicyclohexylammonium salt
12886 PNP-phos.diNa 4-Nitrophenylphosphate, disodium salt

Horseradish Peroxidase / TMB System

Code Product Synonym
22928 TMB 3,3',5,5'-tetramethylbenzidine, free base
22999 TMB.diHCl 3,3',5,5'-tetramethylbenzidine dihydrochloride

2 Fluorogenic Substrates

Methylumbelliferyl (4-MU-) Substrates

e.g. 4-Methylumbelliferyl-beta-D-glucuronic acid (MUG, 4-MU-b-D-glcUA)
O O
H

O O O O beta-Glucuronidase O O O
H
OH O O
H
OH
O OH
OH
Methylumbelliferone
4-Methylumbelliferyl- OH
emits at 460 nm
beta-D-glucuronic acid OH water soluble, fluorescent blue
OH

The same basic reaction applies for all 4-Methylumbelliferyl substrates.

Substrates for Glycosidases

Code Product Synonym
33712 4-MU-beta-D-galactosaminide 4-Methylumbelliferyl-N-acetyl-beta-D-galactosaminide
33714 4-MU-beta-D-glucosaminide 4-Methylumbelliferyl-N-acetyl-beta-D-glucosaminide
34067 4-MU-alpha-L-arabinoside 4-Methylumbelliferyl-alpha-L-arabinopyranoside
34068 4-MU-beta-D-cellobiose 4-Methylumbelliferyl-beta-D-cellobioside
33716 4-MU-alpha-D-gal 4-Methylumbelliferyl-alpha-D-galactopyranoside
33721 4-MU-beta-D-gal 4-Methylumbelliferyl-beta-D-galactopyranoside
36699 4-MU-alpha-D-glc 4-Methylumbelliferyl-alpha-D-glucopyranoside
33702 4-MU-beta-D-glc 4-Methylumbelliferyl-beta-D-glucopyranoside
33722 MUG, 4-MU-beta-D-glcUA 4-Methylumbelliferyl-beta-D-glucuronic acid
33707 4-MU-alpha-D-mannoside 4-Methylumbelliferyl-alpha-D-mannopyranoside
34070 4-MU-beta-D-mannoside 4-Methylumbelliferyl-beta-D-mannopyranoside
33727 4-MU-beta-D-xyl 4-Methylumbelliferyl-beta-D-xylopyranoside

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Substrates for Esterases
Code Product Synonym
34071 4-MU-nonanoate 4-Methylumbelliferyl nonanoate
33698 4-MU-palmitate 4-Methylumbelliferyl palmitate

Substrates for Phosphatases

Code Product Synonym
34072 2(4-MU)-phos Bis(4-methylumbelliferyl)phosphate
41504 4-MU-phos 4-Methylumbelliferyl phosphate, free acid
34073 4-MU-phos.diAMPD 4-Methylumbelliferyl phosphate, di(2-amino-2-methyl-1,3 propanediol) salt
34074 4-MU-phos.diCHX 4-Methylumbelliferyl phosphate, dicyclohexylammonium salt
33704 4-MU-phos.diNa 4-Methylumbelliferyl phosphate, disodium salt

Substrate for Sulfatases

Code Product Synonym
34074 4-MU-sulfate.K 4-Methylumbelliferylsulfate, potassium salt

7-Amido-4-methylcoumarin (AMC) Substrates

e.g. Lysine 7-Amido-methylcoumarin (H-lys-AMC)

H
H
H N O O
N Aminopeptidase B N O O
H O H
Lysine

Lysine 7-amido-4-methylcoumarin 7-Amino-4-methylcoumarin

water soluble, fluorescent blue

The same basic reaction applies for all 7-Amido-4-methylcoumarin (AMC) substrates.

Code Product Synonym

34077 Ac-Lys-AMC.AcOH N-Acetyl-L-lysine 7-amido-4-methylcoumarin, acetate salt
33737 H-Ala-AMC.TFA L-Alanine 7-amido-methylcoumarin, trifluoroacetate salt
33730 H-Arg-AMC.HCl L-Arginine 7-amido-4-methylcoumarin hydrochloride
34078 H-Arg-Arg-AMC.HCl L-Arginyl-L-arginine 7-amido-4-methylcoumarin hydrochloride
33692 H-Asp-AMC L-Aspartic acid 7-amido-4-methylcoumarin
33731 N-alpha-CBZ-Arg-AMC.HCl N-alpha-CBZ-L-Arginine 7-amido-4-methylcoumarin hydrochloride
34079 N-CBZ-Gly-Pro-AMC N-CBZ-Glycyl-L-proline 7-amido-4-methylcoumarin
34080 H-Cit-AMC.HBr L-Citrulline 7-amido-4-methylcoumarin hydrobromide
34081 H-Cit-AMC.TFA L-Citrulline 7-amido-4-methylcoumarin, trifluoroacetate salt
33732 H-Glu-AMC gamma-L-Glutamic acid 7-amido-4-methylcoumarin
33677 Glutaryl-gGy-Arg-AMC L-Glutaryl-glycyl-arginine 7-amido-4-methylcoumarin
33681 H-Gly-AMC.HBr L-Glycine 7-amido-4-methylcoumarin hydrobromide

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34082 H-Gly-Gly-AMC.HCl Glycyl-glycine 7-amido-4-methylcoumarin hydrochloride

34083 Gly-Phe-AMC Glycyl-L-phenylalanine 7-amido-4-methylcoumarin
34084 Gly-Pro-AMC.HBr Glycyl-L-proline 7-amido-4-methylcoumarin hydrobromide
34085 H-His-AMC L-Histidine 7-amido-4-methylcoumarin
34086 H-Hyp-AMC L-Hydroxyproline 7-amido-4-methylcoumarin
34087 H-lle-AMC.TFA L-Isoleucine 7-amido-4-methylcoumarin, trifluoroacetate salt
30310 H-Leu-AMC L-Leucine 7-amido-4-methylcoumarin
33728 H-Leu-AMC.HCl L-Leucine 7-amido-4-methylcoumarin hydrochloride
34088 H-Lys-AMC.AcOH L-Lysine 7-amido-4-methylcoumarin, acetate salt
34089 H-Met-AMC.AcOH L-Methionine 7-amido-4-methylcoumarin, acetate salt
33736 H-Met-AMC.TFA L-Methionine 7-amido-4-methylcoumarin, trifluoroacetate salt
34090 H-Orn-AMC.Cab L-Ornithine 7-amido-4-methylcoumarin, carbonate salt
34091 H-Phe-AMC.TFA L-Phenylalanine 7-amido-4-methylcoumarin, trifluoroacetate salt
33682 H-Pro-AMC.HBr L-Proline 7-amido-4-methylcoumarin hydrobromide
33726 H-Pyr-AMC L-Pyroglutamic acid 7-amido-4-methylcoumarin
33683 H-Ser-AMC.HCl L-Serine 7-amido-4-methylcoumarin hydrochloride
34092 H-Ser-Tyr-AMC L-Seryl-L-tyrosine 7-amido-4-methylcoumarin
34093 H-Trp-AMC L-Tryptophan 7-amido-4-methylcoumarin
34094 H-Trp-AMC L-Tyrosine 7-amido-4-methylcoumarin
34095 H-Val-AMC L-Valine 7-amido-4-methylcoumarin
34096 H-Val-AMC.TFA L-Valine 7-amido-4-methylcoumarin trifluoroacetate salt

3 Bioluminescent Substrates

Firefly Luciferase Substrates

e.g. Luciferin Firefly, free acid, synth.
O Firefly Luciferase
O S N H O
H Mg
2+ S N O
O H
+ ATP + O2 + AMP + PP i + CO2
N S hv = 560 nm N S
yellow-green flash
Luciferin Oxyluciferin

The same basic reaction occurs when Luciferase is added to the system or Luciferase is
expressed by a reporter gene.
Code Product
33705 Luciferin Firefly, free acid (synth.)
33678 Luciferin Firefly, sodium salt (synth.)
34097 Luciferase, recombinant

Apoaequorin- and Renilla Luciferase-Substrate: Coelenterazine

The different wavelengths compared to applications with Firefly Luciferase make double
reporter gene applications possible.

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Code Product
34098 Coelenterazine
34099 2-(4-Dehydroxy)coelenterazine

4 Enzyme/Cross Reference List

Glycosidases

Enzyme Code Substrate

alpha-L-Arabinosidase 34067 4-Methylumbelliferyl-alpha-L-arabinopyranoside
beta-Cellobiosidase 34049 5-Bromo-4-chloro-3-indoxyl-beta-D-cellobioside
34068 4-Methylumbelliferyl-beta-D-cellobioside
33710 4-Nitrophenyl-beta-D-cellobioside
alpha-L-Fucosidase 34100 2-Naphthyl-alpha-L-fucopyranoside
34101 4-Methylumbelliferyl-alpha-L-fucopyranoside
34058 4-Nitrophenyl-alpha-L-fucopyranoside
beta-D-fucosidase 33695 5-Bromo-6-chloro-3-indoxyl-beta-D-fucopyranoside
34102 4-Methylumbelliferyl-beta-D-fucopyranoside
33685 4-Nitrophenyl-beta-D-fucopyranoside
beta-L-Fucosidase 33695 5-Bromo-4-chloro-3-indoxyl-beta-L-fucopyranoside
34103 4-Methylumbelliferyl-beta-L-fucopyranoside
34104 4-Nitrophenyl-beta-L-fucopyranoside
alpha-Galactosaminidase 34056 2-Nitrophenyl-N-acetyl-alpha-D-galactosaminide
beta-Galactosaminidase 33691 5-Bromo-4-chloro-3-indoxyl-N-acetyl-beta-D-galactosaminide
33712 4-Methylumbelliferyl-N-acetyl-beta-D-galactosaminide
33694 4-Nitrophenyl-N-acetyl-beta-D-galactosaminide
alpha-Galactosidase 34050 5-Bromo-4-chloro-3-indoxyl-alpha-D-galactopyranoside
33716 4-Methylumbelliferyl-alpha-D-galactopyranoside
34105 1-Naphthyl-alpha-D-galactopyranoside
33733 4-Nitrophenyl-alpha-D-galactopyranoside
beta-Galactosidase 34106 4-Aminophenyl-beta-D-galactopyranoside
22777 5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside
33735 5-Bromo-6-chloro-3-indoxyl-beta-D-galactopyranoside
33738 5-Bromo-3-indoxyl-beta-D-galactopyranoside
34039 6-Chloro-3-indoxyl-beta-D-galactopyranoside
34107 6-Fluoro-3-indoxyl-beta-D-galactopyranoside
34108 8-Hydroxyquinoline-beta-D-galactopyranoside
33687 3-Indoxyl-beta-D-galactopyranoside
34046 5-Iodo-3-indoxyl-beta-D-galactopyranoside
34047 N-Methylindoxyl-beta-D-galactopyranoside
33721 4-Methylumbelliferyl-beta-D-galactopyranoside
34109 Naphthol AS-Bl-beta-D-galactopyranoside
22942 2-Naphthyl-beta-D-galactopyranoside
12882 2-Nitrophenyl-beta-D-galactopyranoside
22929 4-Nitrophenyl-beta-D-galactopyranoside
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A p p l i w a re : S u b s t r a t e s
Enzyme Code Substrate
alpha-Glucosaminidase 22939 2-Nitrophenyl-N-acetyl-alpha-D-glucosaminide
22940 4-Nitrophenyl-N-acetyl-alpha-D-glucosaminide
beta-Glucosaminidase 34048 5-Bromo-4-chloro-3-indoxyl-N-acetyl-beta-D-glucosaminide
34035 5-Bromo-6-chloro-3-indoxyl-N-acetyl-beta-D-glucosaminide
33714 4-Methylumbelliferyl-N-acetyl-beta-D-glucosaminide
34057 2-Nitrophenyl-N-acetyl-beta-D-glucosaminide
22941 4-Nitrophenyl-N-acetyl-beta-D-glucosaminide
alpha-Glucosidase 34051 5-Bromo-4-chloro-3-indoxyl-alpha-D-glucopyranoside
36699 4-Methylumbelliferyl-alpha-D-glucopyranoside
34110 2-Naphthyl-alpha-D-glucopyranoside
33715 4-Nitrophenyl-alpha-D-glucopyranoside
beta-Glucosidase 22872 5-Bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside
34036 5-Bromo-6-chloro-3-indoxyl-beta-D-glucopyranoside
34040 6-Chloro-3-indoxyl-beta-D-glucopyranoside
33717 3-Indoxyl-beta-D-glucopyranoside
33702 4-Methylumbelliferyl-beta-D-glucopyranoside
34111 1-Naphthyl-N-acetyl-glucosaminide
34112 1-Naphthyl-beta-D-glucopyranoside
34059 2-Nitrophenyl-beta-D-glucopyranoside
34060 3-Nitrophenyl-beta-D-glucopyranoside
30311 4-Nitrophenyl-beta-D-glucopyranoside
30302 Phenyl-beta-D-galactopyranoside
27082 Phenyl-beta-D-glucopyranoside
beta-Gluconidase 33701 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, CHX salt
33693 5-Bromo-6-chloro-3-indoxyl-beta-D-glucuronic acid, CHX salt
33688 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, sodium salt
34041 6-Chloro-3-indoxyl-beta-D-glucuronic acid, CHX salt
34113 8-Hydroxyquinoline-beta-D-glucuronide
33711 3-Indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt
34031 3-Indoxyl-beta-D-glucuronic acid, sodium salt
33722 4-Methylumbelliferyl-beta-D-glucuronic acid
33679 4-Nitrophenyl-beta-D-glucuronic acid
34061 4-Nitrophenyl-beta-D-glucuronic acid, sodium salt
33697 Phenyl-beta-D-glucuronic acid
beta-Lactosidase 34062 4-Nitrophenyl-beta-D-lactopyranoside
alpha-Maltosidase 33696 4-Nitrophenyl-beta-D-maltopyranoside
alpha-Mannosidase 34114 2-(6-Bromonaphthyl)-alpha-D-mannopyranoside
33707 4-Methylumbelliferyl-alpha-D-mannopyranoside
33680 4-Nitrophenyl-alpha-D-mannopyranoside
beta-Mannosidase 34070 4-Methylumbelliferyl-beta-D-mannopyranoside
34063 4-Nitrophenyl-beta-D-mannopyranoside
beta-Xylosidase 34052 5-Bromo-4-chloro-3-indoxyl-beta-D-xylopyranoside
34115 2-(6-Bromonaphthyl)-beta-D-xylopyranoside
33727 4-Methylumbelliferyl-beta-D-xylopyranoside
34064 4-Nitrophenyl-beta-D-xylopyranoside

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Esterases

Enzyme Code Substrate

Carboxyl Esterase 33690 5-Bromo-4-chloro-3-indoxyl butyrate
33689 5-Bromo-4-chloro-3-indoxyl caprylate
Cholinesterase 34116 S-butyrylthiocholine iodide
Esterase (misc.) 34117 5-Bromo-4-chloro-3-indoxyl-3-acetate
34118 5-Bromo-6-chloro-3-indoxyl-3-acetate
33690 5-Bromo-4-chloro-3-indoxyl butyrate
34037 5-Bromo-6-chloro-3-indoxyl butyrate
33689 5-Bromo-4-chloro-3-indoxyl caprylate
34119 5-Bromo-6-chloro-3-indoxyl caprylate
34053 5-Bromo-4-chloro-3-indoxyl palmitate
34038 5-Bromo-6-chloro-3-indoxyl palmitate
34120 6-Chloro-3-indoxyl-3-acetate
34042 6-Chloro-3-indoxyl butyrate
34043 6-Chloro-3-indoxyl caprylate
34044 6-Chloro-3-indoxyl palmitate
15666 3-Indoxyl-3-acetate
34071 4-Methylumbelliferyl nonanoate
33698 4-Methylumbelliferyl palmitate
34065 4-Nitrophenyl palmitate

Phosphatases

Enzyme Code Substrate

Alkaline Phosphatase
Phosphatase (misc.)
12
33676 5-Bromo-4-chloro-3-indoxyl phosphate, disodium salt
34055 5-Bromo-4-chloro-3-indoxyl phosphate, p-toluidine salt
33729 5-Bromo-6-chloro-3-indoxyl phosphate, p-toluidine salt
34045 6-Chloro-3-indoxyl phosphate, p-toluidine salt
34032 3-Indoxyl phosphate, di(2-amino-2-methyl-1,3-propanediol) salt
33708 3-Indoxyl phosphate, disodium salt
34033 3-Indoxyl phosphate, p-toluidine salt
41504 4-Methylumbelliferyl phosphate, free acid
34073 4-Methylumbelliferyl phosphate, diAMPD salt
34074 4-Methylumbelliferyl phosphate, dicyclohexylammonium salt
33704 4-Methylumbelliferyl phosphate, disodium salt trihydrate
34072 Bis(4-methylumbelliferyl) phosphate
34054 5-Bromo-4-chloro-3-indoxyl phosphate, diAMPD salt
33723 5-Bromo-4-chloro-3-indoxyl phosphate, p-tol salt
34121 1-Naphthyl phosphate, calcium salt
22689 1-Naphthyl phosphate, sodium salt
34122 2-Naphthyl phosphate, monosodium salt
22799 4-Nitrophenyl phosphate, dicyclohexylammonium salt
12886 4-Nitrophenyl phosphate, disodium salt
34123 Phenolphtalein diphosphate, tetrasodium salt
for packsizes and prices see http://www.acros.be or our catalogue on CD-Rom
A p p l i w a re : S u b s t r a t e s

Peptidases

Enzyme Code Substrate

Cathepsin
Dipeptidyl Aminopeptidase II
g-Glutamyl Transpeptidase
Pyroglutamyl Peptidase
Peptidase (misc.)
33730 L-Arginine 7-amido-4-methylcoumarin hydrochloride
34124 L-Lysyl-L-alanine-4-methoxy-beta-napthylamide hydrobromide
33732 gamma-L-Glutamic acid 7-amido-4-methylcoumarin
22871 L-gamma-glutamyl-4-nitroanilide monohydrate
33726 L-Pyroglutamic acid 7-amido-4-methylcoumarin
34125 L-Pyroglutamic acid 2-naphthylamide
34077 N-Acetyl-L-lysine 7-amido-4-methylcoumarin, acetate salt
33737 L-Alanine 7-amido-methylcoumarin, trifluoroacetate salt
33730 L-Arginine 7-amido-4-methylcoumarin hydrochloride
34078 L-Arginyl-L-arginine 7-amido-4-methylcoumarin hydrochloride
33692 L-Aspartic acid 7-amido-4-methylcoumarin
33731 N-alpha-CBZ-L-arginine 7-amido-4-methylcoumarin HCl
34079 N-CBZ-glycyl-L-proline 7-amido-4-methylcoumarin
34080 L-Citrulline 7-amido-4-methylcoumarin hydrobromide
34081 L-Citrulline 7-amido-4-methylcoumarin, trifluoroacetate salt
33732 gamma-L-glutamic acid 7-amido-4-methylcoumarin
33677 L-Glutaryl-glycyl-arginine 7-amido-4-methylcoumarin
33681 L-Glycine 7-amido-4-methylcoumarin hydrobromide
34082 Glycyl-glycine 7-amido-4-methylcoumarin hydrochloride
34083 Glycyl-L-phenylalanine 7-amido-4-methylcoumarin
34084 Glycyl-L-proline 7-amido-4-methylcoumarin hydrobromide
34085 L-Histidine 7-amido-4-methylcoumarin
34086 L-Hydroxyproline 7-amido-4-methylcoumarin
34087 L-Isoleucine 7-amido-4-methylcoumarin, trifluoroacetate salt
30310 L-Leucine 7-amido-4-methylcoumarin
33728 L-Leucine 7-amido-4-methylcoumarin hydrochloride
34088 L-Lysine 7-amido-4-methylcoumarin, acetate salt
34089 L-Methionine 7-amido-4-methylcoumarin, acetate salt
33736 L-Methionine 7-amido-4-methylcoumarin, trifluoroacetate salt
34090 L-Ornithine 7-amido-4-methylcoumarin, carbonate salt
34091 L-Phenylalanine 7-amido-4-methylcoumarin, TFA salt
33682 L-Proline 7-amido-4-methylcoumarin hydrobromide
33726 L-Pyroglutamic acid 7-amido-4-methylcoumarin
33683 L-Serine 7-amido-4-methylcoumarin hydrochloride
34092 L-Seryl-L-tyrosine 7-amido-4-methylcoumarin
34093 L-Tryptophan 7-amido-4-methylcoumarin
34094 L-Tyrosine 7-amido-4-methylcoumarin
34095 L-Valine 7-amido-4-methylcoumarin
34096 L-Valine 7-amido-4-methylcoumarin trifluoroacetate salt

contact us for availability 13

A p p l i w a re : S u b s t r a t e s
Sulfatases

Enzyme Code Substrate

Arylsulfatase 33723 5-Bromo-4-chloro-3-indoxyl sulfate, potassium salt
33724 5-Bromo-6-chloro-3-indoxyl sulfate, potassium salt
33706 3-Indoxyl sulfate, potassium salt
34074 4-Methylumbelliferylsulfate, potassium salt
Sulfatases (misc.) 34126 Phenolphthalein disulfate, tripotassium salt

5 How to use selected Enzyme Substrates

Substrate Reaction Product Color

X-gal or X-glcUA Blue
Magenta-gal or Magenta-glcUA Magenta
Salmon-gal or Salmon-glcUA Salmon
Preparation of the substrates: Solubilize in dimethylformamide (DMF) or dimethylsul-
foxide (DMSO) at 25-50 mg/ml. This will then be the stock solution for further dilution
into the final media. There is no need for sterilization of the stock solutions when they are
made in DMF or DMSO.
Concentration of substrate in agar: The recommended starting concentrations of these
substrates are listed below, but the concentration should be optimized for each application.
Substrate Final Concentration in Media
X-gal or X-glcUA 50 µg/ml
Magenta-gal or Magenta-glcUA 100 µg/ml
Salmon-gal or Salmon-glcUA 150 µg/ml
Types of agar media: Minimal media are preferable, but other media will work. Due to
catabolite repression of beta-Glucuronidase and beta-Galactosidase by glucose, it is pre-
ferable to use an alternative carbon source such as glycerol or succinate (7).
Adding substrates to the agar: Substrates should be added to the molten agar after it has
cooled down to 80ºC. The X- and Magenta-substrates, but not the Salmon-substrates, can
also be added to the media prior to autoclaving.
Stability of substrates in prepared agar plates: Prepared plates with these substrates
have been found to be stable when stored for up to several months at 4ºC. In general the
X-substrates are the most stable (< 6 months), followed by the Magenta-substrates (up to
3 months). The Salmon-substrates are the least stable. Additionally, the beta-
Galactosidase substrates are less stable than the beta-Glucuronidase substrates.
pH effects: It has been observed that E. coli colonies growing on M9 minimal media
containing either Magenta- or Salmon-substrates can show a diffuse halo of the colored
product around the colony, rather than discrete localization of the color within the colony.
The unadjusted pH of this medium is approximately pH 6.5. Lowering the pH to pH 6.0
resulted in even greater diffusion, whereas raising the pH to pH 7.0 or pH 7.5 resulted in
the color being localized within the colonies.
14 for packsizes and prices see http://www.acros.be or our catalogue on CD-Rom
A p p l i w a re : S u b s t r a t e s
Important considerations in using gus and lac substrates: Bacterial strains which pos-
sess the beta-Glucuronidase or beta-Galactosidase enzymes may or may not also possess
the corresponding permease. The function of a specific permease is to transport the enzy-
me substrates across the bacterial membrane. For example, beta-Glucuronidase substrates
are transported across the E. coli membrane via the glucuronide permease (3,5,7). The nor-
mal laboratory E. coli strain, K12 and its many derivatives, express beta-Glucuronidase,
but lack a functional glucuronide permease. As a result, K12 strains are unable to active-
ly take up gus substrates or inducers and appear to be gus negative in spite of the fact that
they do contain the gus enzyme. However, almost all wild isolates of E. coli do have both
an activity beta-Glucuronidase and an active glucuronide permease, and hence show
strong gus activity (3,5,7). There is a similar permease responsible for transporting beta-
Galactosidase substrates across the bacterial membrane, although this is not required for
the uptake of indoxyl-galactoside substrates or the inducer IPTG (4).
Potential uses for these substrates in the detection and identification of bacteria:
There are at least two broad areas of research in which there are many potential uses. The
first is in environmental microbiology, where possession of beta-Galactosidase and/or
beta-Glucuronidase activity is used as a diagnostic test for many bacteria (1,4 and see refe-
rences in 7). Secondly, the gusA and lacZ genes encoding beta-Glucuronidase and beta-
Galactosidase respectively are now being developed as microorganisms (3,6 and refe-
rences in 7). (by Richard Jefferson, Ph.D. and Kate Wilson, Ph.D.)

References:
1. Brenner, D.J. (1984) Enterobacteriaceae. In "Bergey's Manual of Systematic Bacterilolgy" (N.R.
Krieg, ed.), Vol1, pp 408-423. Williams and Wilkins, Baltimore/London.
2. Jefferson, R.A. (1989) The gus reporter gene system. Nature 342: 837-838.
3. Lam, S.T., Ellis, D.M. and Ligon, J.M. (1990) Genetic approaches for studying rhizosphere coloniza-
tion. Plant and Soil, 192, 11-18
4. Parker, M.T. and Duerden B.I. (eds) (1990) Topley and Wilson's Principles of Bacteriology, Virology
and Immunity. Vol2: Systematic Bacteriology. Edward Arnold, London.
5. Stoeber, F. (1961) Etudes des propriétés et de la biosynthese de la glucuronidase et de la glucuroni-
de-permease chez Escherichia coli. These de Docteur des Sciences, Paris.
6. Wilson, K.J., Giller, K.E. and Jefferson, R.A. (1991) beta-Glucuronidase (gus) operon fusions as a
tool for studying plant-microbe interactions. In: "Advances in Molecular Genetics of Plant-Microbe
Interacitons", Vol1, ed. H. Hennecke and D.P.S. Verma, pp 226-229. Kluwer Academic Publishers,
Dordrecht, The Netherlands.
7. Wilson, K.J. Hughes, S.G. and Jefferson, R.A. (1992) The Escherichia coli gus operon in E. coli and
the occurence and use of gus in other bacteria. In: "Gus Protocols: Using the gus Gene as a Reporter
of Gene Expression". Ed. S. Gallagher. Academic Press, New York. pp 7-23.

contact us for availability 15

A p p l i w a re : S u b s t r a t e s

6 Immunological substrates
Acros Organics offers a high quality range of substrates as ready to use tablets and
liquids for the immunological detection of HRP and AP. Quality of water is essential in
immunological detection. Acros Organics offers high grade water and a revolutionary
support for water free of heavy metals (for more information, ask the Affinity
Chromatography brochure).

Enzyme: HRP (Horse Radish Peroxidase)

Product Formulation Code Product Packsize Suggested Colour

application

TMB liquid (ready to use) 32805 TMB QUICK 30 ml ELISA (high sensitivity) YELLOW (450nm)
(with stop reagent)

100 ml BLUE (665nm)

(no stop reagent)

liquid (ready to use) 32803 TMB EASY 30 ml ELISA (high sensitivity) YELLOW (450nm)
(with stop reagent)

100 ml BLUE (665nm)

( no stop reagent)

OPD fizzing tablet

(containing buffer) 32797 OPD EASY 100x2 mg/tablet ELISA YELLOW (492nm)
(with stop reagent)

tablet 32802 OPD EASY 100x10 mg/tablet

(without buffer)

DAB fizzing tablet

(containing buffer pH 7,0) 32800 DAB EASY 50x10 mg /tablet HISTOCHEMISTRY BROWN

fizzing tablet
(containing buffer pH 5,0) 32799 DAB EASY 50x10 mg /tablet BLOTTING BROWN

AEC fizzing tablet

(containing buffer pH 5,1) 34199 AEC EASY 10x5 mg /tablet HISTOCHEMISTRY RED

Urea
Peroxide fizzing tablet
Tablet (replaces peroxide addition) 32807 PeroxideEASY 50x5,68 mg/tablet HRP substrates

Enzyme: Alkaline Phosphatase

Product Formulation Code Product Packsize Suggested Colour

application
FAST RED tablet (without buffer) 34207 FAST RED on request HISTOCHEMISTRY RED
BLOTTING

1. TMB EASY/QUICK substrate

TMB EASY (3,3’ tetramethylbenzidine) is an aqueous formulation without any harmful organic sol-
vents for maximized safety and minimized waste.
TMB QUICK (3,3’ tetramethylbenzidine) is recommended for very fast assay reactions, giving a
higher signal than other standard TMB solutions.

How to use:
Substrate as well as hydrogen peroxide are included. No other reagents should be added.
Dilution is not recommended. Store at 4°C.

16 for packsizes and prices see http://www.acros.be or our catalogue on CD-Rom

A p p l i w a re : S u b s t r a t e s
1. Desired amount of substrate is poured into a sealed container and allowed to reach room temperatu-
re in the dark.
2. Add 100 µl/microtiterwell substrate solution. Time of development is 5-30 min.
3. In kinetic assays the blue color is read at 655 nm. For endpoint assays equal volumes of 0.2 M sulphuric
acid is added and the absorbance is read at 450 nm. The obtained yellow solution are stable for 1 hour.

2. DAB EASYtablets for immunoblotting/for immunohistochemistry

DAB EASY (3,3’-diaminobenzidine tetrahydrochloride ) tablets for immunoblot-
ting contain also the citrate buffer giving you the proper pH(5.0) for immunoblot-
ting when dissolved in water.
DAB EASY (3,3’-diaminobenzidine tetrahydrochloride ) tablets for immunohisto-
chemistry contain also the phosphate buffer giving you the proper pH(7.0) for
immunohistochemistry when dissolved in water.
Short term storage at 4°C, long term storage at -20°C. Keep the lid tightly closed
during storage of the tablets and do not remove the drying capsule from the container.

How to use:
1. Remove the container from the refrigerator and allow to stand at room temperatu-
re for 15 minutes. DO NOT USE METAL FORCEPS DURING HANDLING OF THE TABLETS!
2. Dissolve the tablet, containing 10 mg DAB, in 10 ml of distilled water at room temperature.
3. Some fine particular matter will remain in the suspension even after the solution is thoroughly
mixed. This will not effect results, however if preferred the solution can be filtered using Whatman
no.2 paper.
4. Add 4 Urea Peroxide tablets to 10 ml of the substrate solution. (alternatively add 40 µl 30% hydro-
gen peroxide.) This solution should be used within 2 hours.

3. OPD EASYtablets for ELISA

OPD EASY (ortho-phylenediamine) tablets for ELISA contain also the citrate buffer giving you the
proper pH(5.2) for immunoblotting when dissolved in water.
Short term storage at 4°C, long term storage at –20°C. Keep the lid tightly closed during storage of the
tablets and do not remove the drying capsule from the container.

How to use:
1. Remove the container from the refrigerator and allow to stand at room temperature for 15 minutes.
DO NOT USE METAL FORCEPS DURING HANDLING OF THE TABLETS!
2. Dissolve 5 tablets, containing 2 mg OPD, in 12.5 ml of distilled water at room temperature.
3. Add 2 Urea Peroxide tablets to 10 ml of the substrate solution. (alternatively add 12.5 µl 30% hydro-
gen peroxide.) This solution should be used within 1/2 hour.

4. AEC EASYtablets
AEC EASY (3-amino-9-ethyl carbazole) tablets for immunohistochemistry contain also the citrate buf-
fer giving you the proper pH(5.1) for immunohistochemistry when dissolved in water.
Short term storage at 4°C, long term storage at –20°C. Keep the lid tightly closed during storage of the
tablets and do not remove the drying capsule from the container.

How to use:
1. Remove the container from the refrigerator and allow to stand at room temperature for 15 minutes.
DO NOT USE METAL FORCEPS DURING HANDLING OF THE TABLETS!
2. Add 1 ml of ethanol 96% to 1 tablet. Allow to rest for 3 minutes, add distilled water to a total volu-
me of 10 ml at room temperature. The solution should be protected from direct sunlight.
3. Add 1 Urea Peroxide tablets to 10 ml of the substrate solution. ( alternatively add 10 µl 30% hydro-
gen peroxide.) This solution should be used within 1 hour.
contact us for availability 17
A p p l i w a re : S u b s t r a t e s
5. UREA PEROXIDE Tablets
UREA PEROXIDE tablets are a simple and convenient method of adding peroxide. To ease the use
tablets are fizzing and dissolve in 5 minutes.
Each tablet contains 5.68 mg of urea-peroxide. When dissolved in 10 ml substrate solution of substrate
of 6.0 µmol Urea-peroxide/ml.
DO NOT USE METAL FORCEPS DURING HANDLING OF THE TABLETS!

Short term storage at 4°C, long term storage at -20°C. Keep the lid tightly closed during storage of the
tablets and do not remove the drying capsule from the container.

6. FAST RED Tablets

Fast red tablets usefull in immunohistochemistry an immunoblotting contain 0.5 mg naphthol substrate,
2 mg Fast Red chromogen and 0.4 mg levimasole.
Short term storage at 4°C, long term storage at -20°C. Keep the lid tightly closed during storage of the
tablets and do not remove the drying capsule from the container.

How to use:
1. Remove tablets from refrigerator allowing 30 minutes to equilibrate to room temperature.
2. Prepare necessary substrate buffer (0.1M TRIS/HCL pH8.2).12.1 g TRIS, add distilled water to 800
ml, adjust with 4 M HCL up to pH 8.2,add distilled water to final total volume of 1 Liter.
3. Use gloves and plastic forceps to handle tablets !
4. Add 1 tablet to 2 ml substrate buffer, shake for 3 minutes.
5. Some fine particular matter will remain in the suspension even after the solution is thoroughly mixed.
This will not effect results, however if preferred the solution can be filtered using Whatman no.2 paper.
6. This solution should be used within 1/2 hour.

7. DAB tablets
DAB (3,3’-diaminobenzidine tetrahydrochloride ) tablets are available in different sizes (2 and 10
mg/tablet).
Short term storage at 4°C, long term storage at -20°C. Keep the lid tightly closed during storage of the
tablets and do not remove the drying capsule from the container.

How to use:
1. Remove tablets from refrigerator allowing 15 minutes to equilibrate to room temperature.
2. DO NOT USE METAL FORCEPS DURING HANDLING OF THE TABLETS!
3. Prepare the necessary substrate buffer. Several buffer systems exist. DO NOT USE BUFFERS
CONTAINING SODIUM AZIDE which is an inhibitor of HRP.
4. Dissolve 1 tablet in 10 ml of buffer (1 mg DAB/ ml). Some fine particular matter will remain in the
suspension even after this DAB solution is thoroughly mixed. This will not effect results.
5. Add 20µl 30% hydrogen peroxide to 2 ml DAB solution. This solution should be used within 2 hours.
6. The remaining DAB solution that does not contain hydrogen peroxide can be stored in the dark for
up to 5 days at 4 °C.

8. OPD tablets
OPD EASY (ortho-phenylenediamine) are available in different sizes (2 and 10 mg/tablet) and guaran-
tee a background as low as A490nm <0.05 in a concentration of 0.8 mg/ml (recommended substrate
buffer : 0.05M citric acid/sodiumphosphate buffer).
Short term storage at 4°C, long term storage at -20°C. Keep the lid tightly closed during storage of the
tablets and do not remove the drying capsule from the container.
DO NOT USE METAL FORCEPS DURING HANDLING OF THE TABLETS !

18 for packsizes and prices see http://www.acros.be or our catalogue on CD-Rom

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