arcos cat dyes substrates

Biochemistry

Immunology

c ata l o g u eO F

c ata l o g u eO F

S U B S T R A T E SS U B S T R A T E S

Appl iware : Substrates

Contents1 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 compoundsincluding products with typical applications.

This Appliware brochure lists only a selection of biochemical and immunological subtrates.

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3contact us for availability

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 615Y blue 680

Magenta magenta 565Salmon salmon 540

Iodo purple 575Green green 665

BLUE I

Substrates for GlycosidasesCode 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-glucosaminide34049 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-fucopyranoside34050 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-galactopyranoside34051 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-glucopyranoside33701 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 salt34052 X-beta-D-xyl 5-Bromo-4-chloro-3-indoxyl-beta-D-xylopyranoside

Substrates for EsterasesCode Product Synonym

33690 X-butyrate 5-Bromo-4-chloro-3-indoxyl butyrate

33689 X-caprylate 5-Bromo-4-chloro-3-indoxyl caprylate34053 X-palmitate 5-Bromo-4-chloro-3-indoxyl palmitate

beta-Galactos idase oxidation

x-gal

NN

O

Br Cl

BrCl

O

H

H

N

Cl Br

H

OO

CH2OH

OH

OH

OH

absorbs at 615 nm

bue precipitate

O OH

CH2OH

OH

OH

OH



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 salt34055 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


33687 Y-beta-D-gal 3-Indoxyl-beta-D-galactopyranoside

33717 Y-beta-D-glc 3-Indoxyl-beta-D-glucopyranoside33711 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 PhosphatasesCode 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 salt34033 Y-phos.p-tol 3-indoxyl phosphate, p-touidine salt

Substrates for SulfatasesCode Product Synonym

33706 Y-sulfate.K 3-indoxyl sulfate, potassium salt

MAGENTA


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-galactopyranoside34036 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


34037 Magenta-butyrate 5-Bromo-6-chloro-3-indoxyl butyrate

33689 Magenta-caprylate 5-Bromo-6-chloro-3-indoxyl caprylate34038 Magenta-palmitate 5-Bromo-6-chloro-3-indoxyl palmitate



Substrate for PhosphatasesCode Product Synonym

33729 Magenta-phos.p-tol 5-Bromo-6-chloro-3-indoxyl phosphate, p-toluidine salt

Substrate for SulfatasesCode Product Synonym

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

SALMON


34039 Salmon-beta-D-gal 6-Chloro-3-indoxyl-beta-D-galactopyranoside

34040 Salmon-beta-D-glc 6-Chloro-3-indoxyl-beta-D-glucopyranoside34041 Salmon-beta-D-glcUA.CHX 6-Chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt


34042 Salmon-butyrate 6-Chloro-3-indoxyl butyrate

34043 Salmon-caprylate 6-Chloro-3-indoxyl caprylate34044 Salmon-palmitate 6-Chloro-3-indoxyl palmitate


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

PURPLE


34046 Iodo-beta-D-gal 5-Iodo-3-indoxyl-beta-D-galactopyranoside

GREEN


34047 Green-beta-D-gal N-Methylindoxyl-beta-D-galactopyranoside

Oxidizing AgentCode Product Synonym

41592 NBT Nitrotertazolium blue chloride

15833 TNBT Tetranitrobluetetrazolium chloride



2- and 4-Nitrophenyl (ONP and PNP) substrates

e.g. 4-Nitrophenyl phosphate, disodium salt (PNP-phos)

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


34056 PNP-alpha-D-galactosaminide 2-Nitrophenyl-N-acetyl-alpha-D-galactosaminide

33694 PNP-beta-D-galactosaminide 4-Nitrophenyl-N-acetyl-beta-D-galactosaminide22939 ONP-alpha-D-glucosaminide 2-Nitrophenyl-N-acetyl-alpha-D-glucosaminide

34057 ONP-beta-D-glucosaminide 2-Nitrophenyl-N-acetyl-beta-D-glucosaminide22940 PNP-alpha-D-glucosaminide 4-Nitrophenyl-N-acetyl-alpha-D-glucosaminide

22941 PNP-beta-D-glucosaminide 4-Nitrophenyl-N-acetyl-beta-D-glucosaminide33710 PNP-beta-D-cellobiose 4-Nitrophenyl-beta-D-cellobioside

34058 PNP-alpha-L-fucoside 4-Nitrophenyl-alpha-L-fucopyranoside33685 PNP-beta-D-fucoside 4-Nitrophenyl-beta-D-fucopyranoside

12882 ONP-beta-D-gal 2-Nitrophenyl-beta-D-galactopyranoside33733 PNP-alpha-D-gal 4-Nitrophenyl-alpha-D-galactopyranoside

22929 PNP-alpha-D-gal 4-Nitrophenyl-beta-D-galactopyranoside34059 ONP-beta-D-glc 2-Nitrophenyl-beta-D-glucopyranoside

34060 MNP-beta-D-glc 3-Nitrophenyl-beta-D-glucopyranoside33715 PNP-alpha-D-glc 4-Nitrophenyl-alpha-D-glucopyranoside

30311 PNP-beta-D-glc 4-Nitrophenyl-beta-D-glucopyranoside33679 PNP-beta-D-glcUA 4-Nitrophenyl-beta-D-glucuronic acid

34061 PNP-beta-D-glcUA.Na 4-Nitrophenyl-beta-D-glucuronic acid, sodium salt34062 PNP-beta-D-lac 4-Nitrophenyl-beta-D-lactopyranoside

33696 PNP-alpha-D-maltoside 4-Nitrophenyl-alpha-D-maltopyranoside33680 PNP-alpha-D-mannoside 4-Nitrophenyl-alpha-D-mannopyranoside

34063 PNP-beta-D-mannoside 4-Nitrophenyl-beta-D-mannopyranoside34064 PNP-beta-D-xyl 4-Nitrophenyl-beta-D-xylopyranoside

Substrate for EsterasesCode Product Synonym

34065 PNP-palmitate 4-Nitrophenyl palmitate

NO O

OP

O Na

O

NaO

OH

NO O

Alkaline Phosphatase

4-Nitrophenyl phosphate,disodium salt

H3PO4

4-Nitrophenolabsorbs at 405 nmyellow solution




34066 2(PNP)-phos.Na Bis(4-nitrophenyl)phosphate, sodium salt

22799 PNP-phos.diCHX 4-Nitrophenyl phosphate, dicyclohexylammonium salt12886 PNP-phos.diNa 4-Nitrophenylphosphate, disodium salt

Horseradish Peroxidase / TMB SystemCode 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)

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


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-glucosaminide34067 4-MU-alpha-L-arabinoside 4-Methylumbelliferyl-alpha-L-arabinopyranoside

34068 4-MU-beta-D-cellobiose 4-Methylumbelliferyl-beta-D-cellobioside33716 4-MU-alpha-D-gal 4-Methylumbelliferyl-alpha-D-galactopyranoside

33721 4-MU-beta-D-gal 4-Methylumbelliferyl-beta-D-galactopyranoside36699 4-MU-alpha-D-glc 4-Methylumbelliferyl-alpha-D-glucopyranoside

33702 4-MU-beta-D-glc 4-Methylumbelliferyl-beta-D-glucopyranoside33722 MUG, 4-MU-beta-D-glcUA 4-Methylumbelliferyl-beta-D-glucuronic acid

33707 4-MU-alpha-D-mannoside 4-Methylumbelliferyl-alpha-D-mannopyranoside34070 4-MU-beta-D-mannoside 4-Methylumbelliferyl-beta-D-mannopyranoside

33727 4-MU-beta-D-xyl 4-Methylumbelliferyl-beta-D-xylopyranoside

OH

O

O OH

OH

OH

OH

O O

OH

O

O O

OH

OH

OH

beta-Glucuronidase O OOH

Methylumbelliferoneemits at 460 nmwater soluble, fluorescent blue

4-Methylumbelliferyl-

beta-D-glucuronic acid




34071 4-MU-nonanoate 4-Methylumbelliferyl nonanoate

33698 4-MU-palmitate 4-Methylumbelliferyl palmitate


34072 2(4-MU)-phos Bis(4-methylumbelliferyl)phosphate

41504 4-MU-phos 4-Methylumbelliferyl phosphate, free acid34073 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 salt33704 4-MU-phos.diNa 4-Methylumbelliferyl phosphate, disodium salt

Substrate for SulfatasesCode 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)

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


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 salt33730 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 hydrochloride33692 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 hydrochloride34079 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 hydrobromide34081 H-Cit-AMC.TFA L-Citrulline 7-amido-4-methylcoumarin, trifluoroacetate salt

33732 H-Glu-AMC gamma-L-Glutamic acid 7-amido-4-methylcoumarin33677 Glutaryl-gGy-Arg-AMC L-Glutaryl-glycyl-arginine 7-amido-4-methylcoumarin

33681 H-Gly-AMC.HBr L-Glycine 7-amido-4-methylcoumarin hydrobromide

ONN

H

O

OH

H

O ON

H

HAminopeptidase B

Lysine

Lysine 7-amido-4-methylcoumarin 7-Amino-4-methylcoumarinwater soluble, fluorescent blue



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

34083 Gly-Phe-AMC Glycyl-L-phenylalanine 7-amido-4-methylcoumarin34084 Gly-Pro-AMC.HBr Glycyl-L-proline 7-amido-4-methylcoumarin hydrobromide

34085 H-His-AMC L-Histidine 7-amido-4-methylcoumarin34086 H-Hyp-AMC L-Hydroxyproline 7-amido-4-methylcoumarin

34087 H-lle-AMC.TFA L-Isoleucine 7-amido-4-methylcoumarin, trifluoroacetate salt30310 H-Leu-AMC L-Leucine 7-amido-4-methylcoumarin

33728 H-Leu-AMC.HCl L-Leucine 7-amido-4-methylcoumarin hydrochloride34088 H-Lys-AMC.AcOH L-Lysine 7-amido-4-methylcoumarin, acetate salt

34089 H-Met-AMC.AcOH L-Methionine 7-amido-4-methylcoumarin, acetate salt33736 H-Met-AMC.TFA L-Methionine 7-amido-4-methylcoumarin, trifluoroacetate salt

34090 H-Orn-AMC.Cab L-Ornithine 7-amido-4-methylcoumarin, carbonate salt34091 H-Phe-AMC.TFA L-Phenylalanine 7-amido-4-methylcoumarin, trifluoroacetate salt

33682 H-Pro-AMC.HBr L-Proline 7-amido-4-methylcoumarin hydrobromide33726 H-Pyr-AMC L-Pyroglutamic acid 7-amido-4-methylcoumarin

33683 H-Ser-AMC.HCl L-Serine 7-amido-4-methylcoumarin hydrochloride34092 H-Ser-Tyr-AMC L-Seryl-L-tyrosine 7-amido-4-methylcoumarin

34093 H-Trp-AMC L-Tryptophan 7-amido-4-methylcoumarin34094 H-Trp-AMC L-Tyrosine 7-amido-4-methylcoumarin

34095 H-Val-AMC L-Valine 7-amido-4-methylcoumarin34096 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.

The same basic reaction occurs when Luciferase is added to the system or Luciferase isexpressed 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 doublereporter gene applications possible.

S

N

N

SOH O

H

O

S

N

N

SOH O

+ ATP +O2

Mg2+

hv = 560 nmyellow-green flash

Firefly Luciferase

Luciferin

+ AMP +PP i + CO2

Oxyluciferin


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-cellobioside34068 4-Methylumbelliferyl-beta-D-cellobioside

33710 4-Nitrophenyl-beta-D-cellobiosidealpha-L-Fucosidase 34100 2-Naphthyl-alpha-L-fucopyranoside

34101 4-Methylumbelliferyl-alpha-L-fucopyranoside34058 4-Nitrophenyl-alpha-L-fucopyranoside

beta-D-fucosidase 33695 5-Bromo-6-chloro-3-indoxyl-beta-D-fucopyranoside34102 4-Methylumbelliferyl-beta-D-fucopyranoside

33685 4-Nitrophenyl-beta-D-fucopyranosidebeta-L-Fucosidase 33695 5-Bromo-4-chloro-3-indoxyl-beta-L-fucopyranoside

34103 4-Methylumbelliferyl-beta-L-fucopyranoside34104 4-Nitrophenyl-beta-L-fucopyranoside

alpha-Galactosaminidase 34056 2-Nitrophenyl-N-acetyl-alpha-D-galactosaminidebeta-Galactosaminidase 33691 5-Bromo-4-chloro-3-indoxyl-N-acetyl-beta-D-galactosaminide

33712 4-Methylumbelliferyl-N-acetyl-beta-D-galactosaminide33694 4-Nitrophenyl-N-acetyl-beta-D-galactosaminide

alpha-Galactosidase 34050 5-Bromo-4-chloro-3-indoxyl-alpha-D-galactopyranoside33716 4-Methylumbelliferyl-alpha-D-galactopyranoside

34105 1-Naphthyl-alpha-D-galactopyranoside33733 4-Nitrophenyl-alpha-D-galactopyranoside

beta-Galactosidase 34106 4-Aminophenyl-beta-D-galactopyranoside22777 5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside

33735 5-Bromo-6-chloro-3-indoxyl-beta-D-galactopyranoside33738 5-Bromo-3-indoxyl-beta-D-galactopyranoside

34039 6-Chloro-3-indoxyl-beta-D-galactopyranoside34107 6-Fluoro-3-indoxyl-beta-D-galactopyranoside

34108 8-Hydroxyquinoline-beta-D-galactopyranoside33687 3-Indoxyl-beta-D-galactopyranoside

34046 5-Iodo-3-indoxyl-beta-D-galactopyranoside34047 N-Methylindoxyl-beta-D-galactopyranoside

33721 4-Methylumbelliferyl-beta-D-galactopyranoside34109 Naphthol AS-Bl-beta-D-galactopyranoside

22942 2-Naphthyl-beta-D-galactopyranoside12882 2-Nitrophenyl-beta-D-galactopyranoside

22929 4-Nitrophenyl-beta-D-galactopyranoside



alpha-Glucosaminidase 22939 2-Nitrophenyl-N-acetyl-alpha-D-glucosaminide

22940 4-Nitrophenyl-N-acetyl-alpha-D-glucosaminidebeta-Glucosaminidase 34048 5-Bromo-4-chloro-3-indoxyl-N-acetyl-beta-D-glucosaminide

34035 5-Bromo-6-chloro-3-indoxyl-N-acetyl-beta-D-glucosaminide33714 4-Methylumbelliferyl-N-acetyl-beta-D-glucosaminide

34057 2-Nitrophenyl-N-acetyl-beta-D-glucosaminide22941 4-Nitrophenyl-N-acetyl-beta-D-glucosaminide

alpha-Glucosidase 34051 5-Bromo-4-chloro-3-indoxyl-alpha-D-glucopyranoside36699 4-Methylumbelliferyl-alpha-D-glucopyranoside

34110 2-Naphthyl-alpha-D-glucopyranoside33715 4-Nitrophenyl-alpha-D-glucopyranoside

beta-Glucosidase 22872 5-Bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside34036 5-Bromo-6-chloro-3-indoxyl-beta-D-glucopyranoside

34040 6-Chloro-3-indoxyl-beta-D-glucopyranoside33717 3-Indoxyl-beta-D-glucopyranoside

33702 4-Methylumbelliferyl-beta-D-glucopyranoside34111 1-Naphthyl-N-acetyl-glucosaminide

34112 1-Naphthyl-beta-D-glucopyranoside34059 2-Nitrophenyl-beta-D-glucopyranoside

34060 3-Nitrophenyl-beta-D-glucopyranoside30311 4-Nitrophenyl-beta-D-glucopyranoside

30302 Phenyl-beta-D-galactopyranoside27082 Phenyl-beta-D-glucopyranoside

beta-Gluconidase 33701 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, CHX salt33693 5-Bromo-6-chloro-3-indoxyl-beta-D-glucuronic acid, CHX salt

33688 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, sodium salt34041 6-Chloro-3-indoxyl-beta-D-glucuronic acid, CHX salt

34113 8-Hydroxyquinoline-beta-D-glucuronide33711 3-Indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt

34031 3-Indoxyl-beta-D-glucuronic acid, sodium salt33722 4-Methylumbelliferyl-beta-D-glucuronic acid

33679 4-Nitrophenyl-beta-D-glucuronic acid34061 4-Nitrophenyl-beta-D-glucuronic acid, sodium salt

33697 Phenyl-beta-D-glucuronic acidbeta-Lactosidase 34062 4-Nitrophenyl-beta-D-lactopyranoside

alpha-Maltosidase 33696 4-Nitrophenyl-beta-D-maltopyranosidealpha-Mannosidase 34114 2-(6-Bromonaphthyl)-alpha-D-mannopyranoside

33707 4-Methylumbelliferyl-alpha-D-mannopyranoside33680 4-Nitrophenyl-alpha-D-mannopyranoside

beta-Mannosidase 34070 4-Methylumbelliferyl-beta-D-mannopyranoside34063 4-Nitrophenyl-beta-D-mannopyranoside

beta-Xylosidase 34052 5-Bromo-4-chloro-3-indoxyl-beta-D-xylopyranoside34115 2-(6-Bromonaphthyl)-beta-D-xylopyranoside

33727 4-Methylumbelliferyl-beta-D-xylopyranoside34064 4-Nitrophenyl-beta-D-xylopyranoside




Esterases


Carboxyl Esterase 33690 5-Bromo-4-chloro-3-indoxyl butyrate

33689 5-Bromo-4-chloro-3-indoxyl caprylateCholinesterase 34116 S-butyrylthiocholine iodide

Esterase (misc.) 34117 5-Bromo-4-chloro-3-indoxyl-3-acetate34118 5-Bromo-6-chloro-3-indoxyl-3-acetate

33690 5-Bromo-4-chloro-3-indoxyl butyrate34037 5-Bromo-6-chloro-3-indoxyl butyrate

33689 5-Bromo-4-chloro-3-indoxyl caprylate34119 5-Bromo-6-chloro-3-indoxyl caprylate

34053 5-Bromo-4-chloro-3-indoxyl palmitate34038 5-Bromo-6-chloro-3-indoxyl palmitate

34120 6-Chloro-3-indoxyl-3-acetate34042 6-Chloro-3-indoxyl butyrate

34043 6-Chloro-3-indoxyl caprylate34044 6-Chloro-3-indoxyl palmitate

15666 3-Indoxyl-3-acetate34071 4-Methylumbelliferyl nonanoate

33698 4-Methylumbelliferyl palmitate34065 4-Nitrophenyl palmitate

Phosphatases


Alkaline Phosphatase 33676 5-Bromo-4-chloro-3-indoxyl phosphate, disodium salt

34055 5-Bromo-4-chloro-3-indoxyl phosphate, p-toluidine salt33729 5-Bromo-6-chloro-3-indoxyl phosphate, p-toluidine salt

34045 6-Chloro-3-indoxyl phosphate, p-toluidine salt34032 3-Indoxyl phosphate, di(2-amino-2-methyl-1,3-propanediol) salt

33708 3-Indoxyl phosphate, disodium salt34033 3-Indoxyl phosphate, p-toluidine salt

41504 4-Methylumbelliferyl phosphate, free acid34073 4-Methylumbelliferyl phosphate, diAMPD salt

34074 4-Methylumbelliferyl phosphate, dicyclohexylammonium salt33704 4-Methylumbelliferyl phosphate, disodium salt trihydrate

Phosphatase (misc.) 34072 Bis(4-methylumbelliferyl) phosphate34054 5-Bromo-4-chloro-3-indoxyl phosphate, diAMPD salt

33723 5-Bromo-4-chloro-3-indoxyl phosphate, p-tol salt34121 1-Naphthyl phosphate, calcium salt

22689 1-Naphthyl phosphate, sodium salt34122 2-Naphthyl phosphate, monosodium salt

22799 4-Nitrophenyl phosphate, dicyclohexylammonium salt12886 4-Nitrophenyl phosphate, disodium salt

34123 Phenolphtalein diphosphate, tetrasodium salt




Peptidases


Cathepsin 33730 L-Arginine 7-amido-4-methylcoumarin hydrochloride

Dipeptidyl Aminopeptidase II 34124 L-Lysyl-L-alanine-4-methoxy-beta-napthylamide hydrobromideg-Glutamyl Transpeptidase 33732 gamma-L-Glutamic acid 7-amido-4-methylcoumarin

22871 L-gamma-glutamyl-4-nitroanilide monohydratePyroglutamyl Peptidase 33726 L-Pyroglutamic acid 7-amido-4-methylcoumarin

34125 L-Pyroglutamic acid 2-naphthylamidePeptidase (misc.) 34077 N-Acetyl-L-lysine 7-amido-4-methylcoumarin, acetate salt

33737 L-Alanine 7-amido-methylcoumarin, trifluoroacetate salt33730 L-Arginine 7-amido-4-methylcoumarin hydrochloride

34078 L-Arginyl-L-arginine 7-amido-4-methylcoumarin hydrochloride33692 L-Aspartic acid 7-amido-4-methylcoumarin

33731 N-alpha-CBZ-L-arginine 7-amido-4-methylcoumarin HCl34079 N-CBZ-glycyl-L-proline 7-amido-4-methylcoumarin

34080 L-Citrulline 7-amido-4-methylcoumarin hydrobromide34081 L-Citrulline 7-amido-4-methylcoumarin, trifluoroacetate salt

33732 gamma-L-glutamic acid 7-amido-4-methylcoumarin33677 L-Glutaryl-glycyl-arginine 7-amido-4-methylcoumarin

33681 L-Glycine 7-amido-4-methylcoumarin hydrobromide34082 Glycyl-glycine 7-amido-4-methylcoumarin hydrochloride

34083 Glycyl-L-phenylalanine 7-amido-4-methylcoumarin34084 Glycyl-L-proline 7-amido-4-methylcoumarin hydrobromide

34085 L-Histidine 7-amido-4-methylcoumarin34086 L-Hydroxyproline 7-amido-4-methylcoumarin

34087 L-Isoleucine 7-amido-4-methylcoumarin, trifluoroacetate salt30310 L-Leucine 7-amido-4-methylcoumarin

33728 L-Leucine 7-amido-4-methylcoumarin hydrochloride34088 L-Lysine 7-amido-4-methylcoumarin, acetate salt

34089 L-Methionine 7-amido-4-methylcoumarin, acetate salt33736 L-Methionine 7-amido-4-methylcoumarin, trifluoroacetate salt

34090 L-Ornithine 7-amido-4-methylcoumarin, carbonate salt34091 L-Phenylalanine 7-amido-4-methylcoumarin, TFA salt

33682 L-Proline 7-amido-4-methylcoumarin hydrobromide33726 L-Pyroglutamic acid 7-amido-4-methylcoumarin

33683 L-Serine 7-amido-4-methylcoumarin hydrochloride34092 L-Seryl-L-tyrosine 7-amido-4-methylcoumarin

34093 L-Tryptophan 7-amido-4-methylcoumarin34094 L-Tyrosine 7-amido-4-methylcoumarin

34095 L-Valine 7-amido-4-methylcoumarin34096 L-Valine 7-amido-4-methylcoumarin trifluoroacetate salt



Sulfatases


Arylsulfatase 33723 5-Bromo-4-chloro-3-indoxyl sulfate, potassium salt

33724 5-Bromo-6-chloro-3-indoxyl sulfate, potassium salt33706 3-Indoxyl sulfate, potassium salt

34074 4-Methylumbelliferylsulfate, potassium saltSulfatases (misc.) 34126 Phenolphthalein disulfate, tripotassium salt

5 How to use selected Enzyme Substrates

Substrate Reaction Product ColorX-gal or X-glcUA BlueMagenta-gal or Magenta-glcUA MagentaSalmon-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 dilutioninto the final media. There is no need for sterilization of the stock solutions when they aremade in DMF or DMSO.

Concentration of substrate in agar: The recommended starting concentrations of thesesubstrates are listed below, but the concentration should be optimized for each application.

Substrate Final Concentration in MediaX-gal or X-glcUA 50 µg/mlMagenta-gal or Magenta-glcUA 100 µg/mlSalmon-gal or Salmon-glcUA 150 µg/ml

Types of agar media: Minimal media are preferable, but other media will work. Due tocatabolite 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 hascooled down to 80ºC. The X- and Magenta-substrates, but not the Salmon-substrates, canalso be added to the media prior to autoclaving.

Stability of substrates in prepared agar plates: Prepared plates with these substrateshave been found to be stable when stored for up to several months at 4ºC. In general theX-substrates are the most stable (< 6 months), followed by the Magenta-substrates (up to3 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 mediacontaining either Magenta- or Salmon-substrates can show a diffuse halo of the coloredproduct 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 6.0resulted in even greater diffusion, whereas raising the pH to pH 7.0 or pH 7.5 resulted inthe color being localized within the colonies.



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 possessthe corresponding permease. The function of a specific permease is to transport the enzy-me substrates across the bacterial membrane. For example, beta-Glucuronidase substratesare 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 thatthey do contain the gus enzyme. However, almost all wild isolates of E. coli do have bothan activity beta-Glucuronidase and an active glucuronide permease, and hence showstrong 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 forthe 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. Thefirst is in environmental microbiology, where possession of beta-Galactosidase and/orbeta-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, 11-184. 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-MicrobeInteracitons", 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 andthe occurence and use of gus in other bacteria. In: "Gus Protocols: Using the gus Gene as a Reporterof Gene Expression". Ed. S. Gallagher. Academic Press, New York. pp 7-23.



6 Immunological substrates

Acros Organics offers a high quality range of substrates as ready to use tablets andliquids for the immunological detection of HRP and AP. Quality of water is essential inimmunological detection. Acros Organics offers high grade water and a revolutionarysupport for water free of heavy metals (for more information, ask the AffinityChromatography brochure).

Enzyme: HRP (Horse Radish Peroxidase)

Product Formulation Code Product Packsize Suggested Colourapplication

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 Colourapplication

FAST RED tablet (without buffer) 34207 FAST RED on request HISTOCHEMISTRY REDBLOTTING

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 ahigher 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.



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) forimmunohistochemistry when dissolved in water.Short term storage at 4°C, long term storage at -20°C. Keep the lid tightly closedduring 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 Whatmanno.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 ELISAOPD EASY (ortho-phylenediamine) tablets for ELISA contain also the citrate buffer giving you theproper 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 thetablets 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 EASYtabletsAEC 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 thetablets 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.



5. UREA PEROXIDE TabletsUREA PEROXIDE tablets are a simple and convenient method of adding peroxide. To ease the usetablets are fizzing and dissolve in 5 minutes.Each tablet contains 5.68 mg of urea-peroxide. When dissolved in 10 ml substrate solution of substrateof 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 thetablets and do not remove the drying capsule from the container.

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

DO NOT USE METAL FORCEPS DURING HANDLING OF THE TABLETS !

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