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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.
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 distributoror e-mail to infodesk@be.acros.com.
for packsizes and prices see http://www.acros.be or our catalogue on CD-Rom2
Appl iware : Substrates
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
Appl iware : Substrates
for packsizes and prices see http://www.acros.be or our catalogue on CD-Rom4
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
Substrates for GlycosidasesCode Product Synonym
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
Substrates for GlycosidasesCode 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-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
Substrates for EsterasesCode Product Synonym
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
Appl iware : Substrates
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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
Substrates for GlycosidasesCode 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-glucopyranoside34041 Salmon-beta-D-glcUA.CHX 6-Chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt
Substrates for EsterasesCode Product Synonym
34042 Salmon-butyrate 6-Chloro-3-indoxyl butyrate
34043 Salmon-caprylate 6-Chloro-3-indoxyl caprylate34044 Salmon-palmitate 6-Chloro-3-indoxyl palmitate
Substrates for PhosphatasesCode 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 AgentCode Product Synonym
41592 NBT Nitrotertazolium blue chloride
15833 TNBT Tetranitrobluetetrazolium chloride
Appl iware : Substrates
for packsizes and prices see http://www.acros.be or our catalogue on CD-Rom6
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.
Substrates for GlycosidasesCode 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-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
Appl iware : Substrates
7contact us for availability
Substrates for PhosphatasesCode Product Synonym
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.
Substrates for GlycosidasesCode 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-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
Appl iware : Substrates
for packsizes and prices see http://www.acros.be or our catalogue on CD-Rom8
Substrates for EsterasesCode Product Synonym
34071 4-MU-nonanoate 4-Methylumbelliferyl nonanoate
33698 4-MU-palmitate 4-Methylumbelliferyl palmitate
Substrates for PhosphatasesCode Product Synonym
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.
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 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
Appl iware : Substrates
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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-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
for packsizes and prices see http://www.acros.be or our catalogue on CD-Rom10
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
Appl iware : Substrates
Enzyme Code Substrate
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
Appl iware : Substrates
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Appl iware : Substrates
Esterases
Enzyme Code Substrate
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
Enzyme Code Substrate
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
for packsizes and prices see http://www.acros.be or our catalogue on CD-Rom12
Appl iware : Substrates
13contact us for availability
Peptidases
Enzyme Code Substrate
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
Appl iware : Substrates
for packsizes and prices see http://www.acros.be or our catalogue on CD-Rom14
Sulfatases
Enzyme Code Substrate
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.
Appl iware : Substrates
15contact us for availability
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.
Appl iware : Substrates
for packsizes and prices see http://www.acros.be or our catalogue on CD-Rom16
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.
Appl iware : Substrates
17contact us for availability
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.
Appl iware : Substrates
for packsizes and prices see http://www.acros.be or our catalogue on CD-Rom18
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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Janssen Pharmaceuticalaan 3aB-2440 Geel, Belgium
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