dojindo · 2014-02-07 · chemicals. trinder’s reagents new trinder’s reagents, which are...

S t a t e o f t h e a r t L i f e S c i e n c e t e c h n o L o g i e S

D o j i n D oChemiCals

trinDer’S reagentS

New Trinder’s Reagents, which are highly water-soluble aniline derivatives, are applicable for the colorimetric determination of hydrogen peroxide. In the presence of a coupler such as 4-aminoantipyrine(4-AA), the Trinder’s reagents are oxidized with peroxidase and two molecules of hydrogen peroxide and are developing the color to purple or blue.

For more details of these products, please visit our web-site at www.dojindo.eu.com

TA b l e 1New Trinder’s Reagents developed by DOJINDO

2

OC08 HDAOS OC13 TOOS OC21 MADB

OC11 MAOS OC02 ADPS OC22 TODB

OC14 TOPS OC06 DAOS

R1

R2 SO3Na

H3C

H3C NH2

O+

+

-

NN

R3

N R4 R1

R2Trinder,s reagent 4-aminoantipyrine SO3

R3

N R4

H3CH2O2

POD

H3C N

ONN

R2

R1

R3

N N

NN+substrate

dehydrogenase

substrate(oxidized form)

NAD(P) tetrazolium salt

NAD(P)H

electron mediator (reduced form)

electron mediator

H3CO SO3Na

·H2OCH2CH3

N

H3CO

H3CO SO3Na

CH2CH3

N OH

H3C

H3C SO3Na

CH2CH3

N OH ·H2O

H3C

H3CSO3Na

SO3Na

N

H3CO

H3CSO3Na

SO3Na

N

H3C SO3Na

CH2CH3

N OH ·2H2O

H3CO SO3Na

HN OH

H3C SO3Na

CH2CH3

N ·H2O

I

Na

NO2

SO3

NN

NN

R3

R2

R1HNN

NNformazan dye

+

+

–O3S–

I

Na

NO2

O2NSO3

NN

NN+

+

–O3S–

O3SO

NH

–

NO2

Na

N

O3S

NN

N

NN+

+

–

SO3–

CO2H

H3CO

NN

N

NS

N+

O3SO

N

–

SO3–

H3CO

NN

N

NS

N+

SO3

O

N

–

SO3–

2Na+

OCH3

NN

N

NS

N+

-I

N+ N

NO

OO

O

-I

N+ N

NO

OO

O

O

O

NH2

NH2

NO2

F

NO

N

SO2NH2

F

NO

N

SO3 NH4

F

NO

N

- +

·2HCI

NH2

NH2

H3CO

H3CO

·2HCI

NBr

N O

CH3

H3CO

H3CO N COCI

N O

CH3

H3CO

H3COtetrazolium salt

1-Methoxy PMS 1-Methoxy PMS(reduced form)

NAD(P)H

formazan dye

electronOCH3

N

N

CH3

+-CH3SO4

OCH3

NH

N

CH3

R1

R2 SO3Na

H3C

H3C NH2

O+

+

-

NN

R3

N R4 R1


R3

N R4

H3CH2O2

POD

H3C N

ONN

R2

R1

R3

N N

NN+substrate

dehydrogenase



NAD(P)H


electron mediator

H3CO SO3Na

·H2OCH2CH3

N

H3CO

H3CO SO3Na

CH2CH3

N OH

H3C

H3C SO3Na

CH2CH3

N OH ·H2O

H3C

H3CSO3Na

SO3Na

N

H3CO

H3CSO3Na

SO3Na

N

H3C SO3Na

CH2CH3

N OH ·2H2O

H3CO SO3Na

HN OH

H3C SO3Na

CH2CH3

N ·H2O

I

Na

NO2

SO3

NN

NN

R3

R2

R1HNN

NNformazan dye

+

+

–O3S–

I

Na

NO2

O2NSO3

NN

NN+

+

–O3S–

O3SO

NH

–

NO2

Na

N

O3S

NN

N

NN+

+

–

SO3–

CO2H

H3CO

NN

N

NS

N+

O3SO

N

–

SO3–

H3CO

NN

N

NS

N+

SO3

O

N

–

SO3–

2Na+

OCH3

NN

N

NS

N+

-I

N+ N

NO

OO

O

-I

N+ N

NO

OO

O

O

O

NH2

NH2

NO2

F

NO

N

SO2NH2

F

NO

N

SO3 NH4

F

NO

N

- +

·2HCI

NH2

NH2

H3CO

H3CO

·2HCI

NBr

N O

CH3

H3CO

H3CO N COCI

N O

CH3

H3CO



NAD(P)H

formazan dye

electronOCH3

N

N

CH3

+-CH3SO4

OCH3

NH

N

CH3

Code# Description Cas# ⋋ max (nm) of oxidized ℇ condensation product, pH

OC08 HDAOS 82692-88-4 583, 5.5-9.5 1.73 x 104

OC13 TOOS 82692-93-1 555, 5.5-9.5 3.92 x 104

OC21 MADb 209518-16-1 630, 5.5-9.5 1.65 x 104

OC11 MAOS 82692-97-5 630, 5.5-9.5 2.25 x 104

OC02 ADPS 82611-88-9 540, 5.9-9.5 2.79 x 104

OC22 TODb 1044537-70-3 550, 5.5-9.5 3.80 x 104

OC14 TOPS 40567-80-4 550, 5.5-9.5 3.74 x 104

OC06 DAOS 83777-30-4 593, 5.5-9.5 1.75 x 104

tetrazoLium SaLtS

Tetrazolium salts have been developed to determine dehydrogenase activity which oxidizes its substrate and reduces its co-enzyme, NAD or NADP and produce NADH or NADPH. The colored formazan dyes are

produced upon reduction of NADH in the presence of an electron mediator.To perform the homogenious assay in aqueous condition, Dojindo developed Water-Soluble Tetrazolium salts (WST) which are widely

applied for the diagnostic analysis and lifescience research due to much improved water-solubility than water insoluble tetrazolium salts, such like NbT and INT.


W204 WST-5 W217 WST-9

TA b l e 2Water-Soluble-Tetrazolium Salts (WST) developed by DOJINDO

TA b l e 3Water Insoluble Tetrazolium Salts

TA b l e 4electron Carrier

3

R1

R2 SO3Na

H3C

H3C NH2

O+

+

-

NN

R3

N R4 R1


R3

N R4

H3CH2O2

POD

H3C N

ONN

R2

R1

R3

N N

NN+substrate

dehydrogenase



NAD(P)H


electron mediator

H3CO SO3Na

·H2OCH2CH3

N

H3CO

H3CO SO3Na

CH2CH3

N OH

H3C

H3C SO3Na

CH2CH3

N OH ·H2O

H3C

H3CSO3Na

SO3Na

N

H3CO

H3CSO3Na

SO3Na

N

H3C SO3Na

CH2CH3

N OH ·2H2O

H3CO SO3Na

HN OH

H3C SO3Na

CH2CH3

N ·H2O

I

Na

NO2

SO3

NN

NN

R3

R2

R1HNN

NNformazan dye

+

+

–O3S–

I

Na

NO2

O2NSO3

NN

NN+

+

–O3S–

O3SO

NH

–

NO2

Na

N

O3S

NN

N

NN+

+

–

SO3–

CO2H

H3CO

NN

N

NS

N+

O3SO

N

–

SO3–

H3CO

NN

N

NS

N+

SO3

O

N

–

SO3–

2Na+

OCH3

NN

N

NS

N+

-I

N+ N

NO

OO

O

-I

N+ N

NO

OO

O

O

O

NH2

NH2

NO2

F

NO

N

SO2NH2

F

NO

N

SO3 NH4

F

NO

N

- +

·2HCI

NH2

NH2

H3CO

H3CO

·2HCI

NBr

N O

CH3

H3CO

H3CO N COCI

N O

CH3

H3CO



NAD(P)H

formazan dye

electronOCH3

N

N

CH3

+-CH3SO4

OCH3

NH

N

CH3

R1

R2 SO3Na

H3C

H3C NH2

O+

+

-

NN

R3

N R4 R1


R3

N R4

H3CH2O2

POD

H3C N

ONN

R2

R1

R3

N N

NN+substrate

dehydrogenase



NAD(P)H


electron mediator

H3CO SO3Na

·H2OCH2CH3

N

H3CO

H3CO SO3Na

CH2CH3

N OH

H3C

H3C SO3Na

CH2CH3

N OH ·H2O

H3C

H3CSO3Na

SO3Na

N

H3CO

H3CSO3Na

SO3Na

N

H3C SO3Na

CH2CH3

N OH ·2H2O

H3CO SO3Na

HN OH

H3C SO3Na

CH2CH3

N ·H2O

I

Na

NO2

SO3

NN

NN

R3

R2

R1HNN

NNformazan dye

+

+

–O3S–

I

Na

NO2

O2NSO3

NN

NN+

+

–O3S–

O3SO

NH

–

NO2

Na

N

O3S

NN

N

NN+

+

–

SO3–

CO2H

H3CO

NN

N

NS

N+

O3SO

N

–

SO3–

H3CO

NN

N

NS

N+

SO3

O

N

–

SO3–

2Na+

OCH3

NN

N

NS

N+

-I

N+ N

NO

OO

O

-I

N+ N

NO

OO

O

O

O

NH2

NH2

NO2

F

NO

N

SO2NH2

F

NO

N

SO3 NH4

F

NO

N

- +

·2HCI

NH2

NH2

H3CO

H3CO

·2HCI

NBr

N O

CH3

H3CO

H3CO N COCI

N O

CH3

H3CO



NAD(P)H

formazan dye

electronOCH3

N

N

CH3

+-CH3SO4

OCH3

NH

N

CH3

Code# Description ℇ (as formazan dye) ⋋ max (as formazan dye) CAS#

N011 Nitro-Tb (NbT) 3.6 x 104 530 nm 298-83-9I003 INT 1.5 x 104 490 nm 146-68-9T012 Tb 2.6 x 104 525 nm 1871-22-3

Code# Description Standard CAS# Redox Potential

M003 1-Methoxy +0.063V 65162-13-2 PMS

Code# Description ℇ (as formazan dye) ⋋ max (as formazan dye) CAS#

W201 WST-1 3.7 x 104 438 nm 150849-52-8W202 WST-3 3.1 x 104 433 nm 515111-36-1W203 WST-4 1.0 x 104 530 nm 178925-54-7W204 WST-5 2.7 x 104 550 nm 178925-55-8W217 WST-9 1.6 x 104 490 nm NA

W201 WST-1 W202 WST-3 W203 WST-4

reagentS for ion Detection

Metal indicators, colorimetric metal chelators, and fluorometric metal chelators can be used to determine the metal ion concentration in solutions. In general, metal indicators are utilized for chelate titration, and colorimetric and fluorometric chelating agents are utilized for the determination of the metal concentration by spectrophotometry.


TA b l e 5Table for

Ion Detection

4

Target Description CAS Number Code Number

Ag o-Phenanthroline 5144-89-8 P007Al Calcein 1461-15-0 C001 Cu-PAN N/A C016 PR (Pyrogallol Red) 32638-88-3 P012 XO (Xylenol Orange) 1611-35-4 X003As Arsemate 1470-61-7 A012b Azomethine H 32266-60-7 (as free acid) A015ba Calcein 1461-15-0 C001 Chlorophosphonazo-III 1914-99-4 C010 HNb 63451-35-4 H007 PC (Phthalein complexone) 2411-89-4 P004Ca bAPTA 85233-19-8 (as free acid) b019 bT (eriochrome black T) 1787-61-7 b015 Calcein 1461-15-0 C001 Chlorophosphonazo-III 1914-99-4 C010 Cu-PAN N/A C016 eGTA (GeDTA) 67-42-5 G002 t-HDOPP-Ca N/A H014 HNb 63451-35-4 H007 Murexide 3051-09-0 M011 MX N/A M012 NN 3737-95-9 N013 NN diluted with potassium sulfate N/A N012 PC (Phthalein complexone) 2411-89-4 P004 Zincon 62625-22-3 Z002Cd bT (eriochrome black T) 1787-61-7 b015 TPPS 35218-75-8 (as anhydrous) T003 XO (Xylenol Orange) 1611-35-4 X003 Zincon 62625-22-3 Z002Ce o-Phenanthroline 5144-89-8 P007 PR (Pyrogallol Red) 32638-88-3 P012 XO (Xylenol Orange) 1611-35-4 X003Cl- bisthiourea-1 187404-67-7 b432CN- Cyanoline blue N/A C017Co bT (eriochrome black T) 1787-61-7 b015 5-br-PAPS 81608-06-2(as free acid) b026 5-br-PSAA 87035-60-7 b027 Cu-PAN N/A C016 Nitro-PAPS 143205-66-7 (as anhydrous) N031 Nitroso-PSAP 80459-15-0 N010 PAN 85-85-8 P002 PAR 1141-59-9 P003 PR (Pyrogallol Red) 32638-88-3 P012 TPPS 35218-75-8 (as anhydrous) T003Cr Calcein blue 54375-47-2 C002Cs Cesibor 25776-12-9 C007

5

Target Description CAS Number Code Number

Cu+ bathocuproine 4733-39-5 b001 bathocuproinedisulfonic acid, disodium salt 98645-85-3 b002 5-br-PSAA 87035-60-7 b027 Calcein 1461-15-0 C001 Cu-PAN N/A C016 3,5-Dibr-PAeSA 100743-65-5 D041 PR (Pyrogallol Red) 32638-88-3 P012 Sodium bicinchoninate 979-88-4 b037Cu2+ 5-br-PAPS 81608-06-2(as free acid) b026 Nitro-PAPS 143205-66-7 (as anhydrous) N031 Nitroso-PSAP 80459-15-0 N010 TMPyP 36951-72-1 T001 TPPS 35218-75-8 (as anhydrous) T003 Zincon 62625-22-3 Z002F- AlC 3952-78-1 A006Fe2+ bathophenanthroline 1662-01-7 b003 bathophenanthrolinedisulfonic acid, disodium salt 98645-86-4 b004 5-br-PAPS 81608-06-2(as free acid) b026 Nitro-PAPS 143205-66-7 (as anhydrous) N031 Nitroso-PSAP 80459-15-0 N010 TPTZ 3682-35-7 T028Fe3+ Diantipyrylmethane 1251-85-0 D008 XO (Xylenol Orange) 1611-35-4 X003Hg2+ bT (eriochrome black T) 1787-61-7 b015 PC (Phthalein complexone) 2411-89-4 P004K bis(benzo-15-crown-5) 69271-98-3 b020 Kalibor® 143-66-8 K003li Dibenzyl-14-crown-4 106868-21-7 D043 TTD-14-crown-4 151460-00-3 T302Mg bT (eriochrome black T) 1787-61-7 b015 Calcein 1461-15-0 C001 Chlorophosphonazo-III 1914-99-4 C010 C14-K22b5 N/A C391 Cu-PAN N/A C016 Xb-I (Xylidyl blue-I) 14936-97-1 X001Na bis(12-crown-4) 80403-59-4 b021NH3 Cyanoline blue N/A C017 TD19C6 N/A T402Ni 5-br-PSAA 87035-60-7 b027 Cu-PAN N/A C016 Nitro-PAPS 143205-66-7 (as anhydrous) N031 PAN 85-85-8 P002 PAR 1141-59-9 P003OCN- Cyanoline blue N/A C017Os PAN 85-85-8 P002Pb bT (eriochrome black T) 1787-61-7 b015 TPPS 35218-75-8 (as anhydrous) T003 Zincon 62625-22-3 Z002Pd 5-br-PAPS 81608-06-2 (as free acid) b026SCN- Cyanoline blue N/A C017Se DAb 7411-49-6 D006 DAN 771-97-1 D027Sn SATP 3449-05-6 S003Zn 5-br-PAPS 81608-06-2 (as free acid) b026 bT (eriochrome black T) 1787-61-7 b015 Calcein 1461-15-0 C001 Cu-PAN N/A C016 Nitro-PAPS 143205-66-7 (as anhydrous) N031 PAN 85-85-8 P002 PAR 1141-59-9 P003 Zincon 62625-22-3 Z002

TA b l e 6Table for Ion Detection

6

DetergentS

Detergents are normally used to isolate large insoluble molecules such as proteins. In the past, polyoxyethylene ether non-ionic detergents were widely used. These detergents, however, had several problems, such as denaturation of proteins and low CMC vale, which cannot be separated easily by dialysis. Most of the current detergents are non-ionic type and easily separated by dialysis. Since Dojindo detergents are highly purified and characteristically designed, these detergents are used as one of the materials in diagnostic research.


Successfully Crystalized bovine Heart Cytochrome C oxidase. Detergents used: n-Decyl-ß-D-maltoside + Trehalose C8. This data was kindly provided from Prof. Shinya Yoshikawa at University of Hyogo.

Successfully Crystalized Twelve-transmembrane Protein. Detergent used: Trehalose C12

TA b l e 7Physical Property

of Detergents

Description cmc M.W. Code#

NON-IONIC DeTeRGeNTSTrehalose-type Trehalose C8 5.6 mM 468,49 T459 Trehalose C10 3 mM 496,55 T460 Trehalose C12 0.15 mM 524,6 T461 Trehalose C14 0.012 mM 552,65 T464 Trehalose C16 0.0061 mM 580,71 T465Glucose-type n-Heptyl-ß-D-thioglucoside 30 mM 294,41 H015 n-Octyl-ß-D-glucoside 25 mM 292,37 O001 n-Octyl-ß-D-thioglucoside 9 mM 308,44 O003Mannose-type 3-Oxatridecyl-a-D-mannoside 0.63 mM 364,47 O401Maltose-type n-Dodecyl-ß-D-maltoside 0.17 mM 510,62 D316 n-Decyl-ß-D-maltoside 1.8 mM 482,57 D382 n-Nonyl-ß-D-thiomaltoside 2.4 mM 484,6 N373 n-Octyl-ß-D-maltoside 23.4 mM 454,51 O393Gluconamide-type bIGCHAP 2.9 mM 878,06 b043 deoxy-bIGCHAP 1.4 mM 862,06 D045Glucamine-type MeGA-8 55 - 67 mM 321,41 M014 MeGA-9 25 mM 335,44 M015 MeGA-10 7 mM 349,46 M016ANIONIC DeTeRGeNTSCholic acid-type Sodium cholate (purified) 14 mM 448,57 C321 Sodium deoxycholate 5 mM 432,57 D520ZWITTeR-IONIC DeTeRGeNTSCholamide-type CHAPS 8 mM 614,88 C008 CHAPSO 8 mM 630,88 C020

Detergent Micelle

Solubilized ProteinMembrane Protein

R1

R2 SO3Na

H3C

H3C NH2

O+

+

-

NN

R3

N R4 R1


R3

N R4

H3CH2O2

POD

H3C N

ONN

R2

R1

R3

N N

NN+substrate

dehydrogenase



NAD(P)H


electron mediator

H3CO SO3Na

·H2OCH2CH3

N

H3CO

H3CO SO3Na

CH2CH3

N OH

H3C

H3C SO3Na

CH2CH3

N OH ·H2O

H3C

H3CSO3Na

SO3Na

N

H3CO

H3CSO3Na

SO3Na

N

H3C SO3Na

CH2CH3

N OH ·2H2O

H3CO SO3Na

HN OH

H3C SO3Na

CH2CH3

N ·H2O

I

Na

NO2

SO3

NN

NN

R3

R2

R1HNN

NNformazan dye

+

+

–O3S–

I

Na

NO2

O2NSO3

NN

NN+

+

–O3S–

O3SO

NH

–

NO2

Na

N

O3S

NN

N

NN+

+

–

SO3–

CO2H

H3CO

NN

N

NS

N+

O3SO

N

–

SO3–

H3CO

NN

N

NS

N+

SO3

O

N

–

SO3–

2Na+

OCH3

NN

N

NS

N+

-I

N+ N

NO

OO

O

-I

N+ N

NO

OO

O

O

O

NH2

NH2

NO2

F

NO

N

SO2NH2

F

NO

N

SO3 NH4

F

NO

N

- +

·2HCI

NH2

NH2

H3CO

H3CO

·2HCI

NBr

N O

CH3

H3CO

H3CO N COCI

N O

CH3

H3CO



NAD(P)H

formazan dye

electronOCH3

N

N

CH3

+-CH3SO4

OCH3

NH

N

CH3

Application Target Code# Description ex em

PROTeIN lAbelING -NH2 I271 IC3-OSu special packaging 550 570 I272 IC5-OSu special packaging 640 660HPlC ANAlYSIS -NH2 N020 NbD-F 470 530 -SH A016 AbD-F 389 513 S013 SbD-F 385 515 -COOH b036 br-DMeQ 370 450 -OH D049 DMeQ-COCl 400 500 Ar-CHO D034 DDb 338 402 a-keto acid M021 MDb 367 445

7

LabeLing reagentS

Fluorometric derivatizing compounds enable higher sensitive determination of the biological samples such as proteins, nucleotides, peptides, and amino acids. The functional groups of amine, carboxylic acid, sulfhydryl,

hydroxyl, aromatic aldehyde in a sample are targeted to derivatized by each reagent. The detection and monitoring of fluorescent-labeled materials are performed by fluorescent microscopy, flow cytometry, or

HPlC analysis.


TA b l e 8labeling Reagents

I271 IC3-OSu I272 IC5-OSu

N020 NBD-F A016 ABD-F S013 SBD-F

D034 DDB M021 MDB

B036 Br-DMEQ D049 DMEQ-COCI

GeRMANY belGIUM FINlAND FRANCe

THe NeTHeRlANDSSWITZeRlAND

THe UNITeD KINGDOM

JAPAN

CHINA INDIA

MAlAYSIA SINGAPORe

SOUTH KOReA

AUSTRAlIA

NORTH AMeRICASOUTH AMeRICA

CANADA

eUROPeAN HeADQUARTeRSDOJINDO eU GMbH

leopoldstr. 254 D-80807 Munich

GeRMANYTel . +49 89 354048-05 Fax +49 89 354048-06

www.doj indo.eu.com

WorLDWiDe

dojindo · 2014-02-07 · chemicals. trinder’s reagents new trinder’s reagents, which are...

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