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Contains 50 tablets. Chromogenic substrate for horseradish peroxidase in ELISA procedures.
Catalogue Number
523121
Brand Family
Calbiochem®
Synonyms
OPD, o-PD, HCl
References
Product Information
CAS number
615-28-1
Form
Off-white tablets
Formulation
White tablets (10 mg each).
Hill Formula
C₆H₈N₂ . 2HCl
Chemical formula
C₆H₈N₂ . 2HCl
Preservative
None
Applications
Application Comments
The tablets are inspected visually and are examined spectrophotometrically at 492 nm for the background level in 0.1 M sodium phosphate/citric acid, pH 5.0, containing 6 mM H2O2. Tablets dissolve in the substrate buffer within 5 min (with shaking).
Suggested Procedure for Use of OPD tablets for HRP-based ELISAs:
REAGENTS REQUIRED BUT NOT PROVIDED: 1. Substrate buffer: 50 mM citric acid/sodium phosphate buffer, pH 5.0. 2. Hydrogen peroxide (H2O2) 30%. 3. Stop solution: 0.1 M H2SO4.
PROTOCOL: 1. Complete all required incubations with antibodies and HRP-labeled probes. 2. Wash plate wells at least 4 times with phosphate-buffered saline (PBS) or Tris-buffered saline, containing 0.1% TWEEN®-20 detergent. 3. After the final wash, shake and blot all residual buffer from the wells. 4. Within 30 min of completion of the above steps, prepare OPD Substrate Solution (the following recipe provides sufficient OPD Substrate Solution for one 96-well plate (100 µl per well): a. Remove the OPD tablet container from the refrigerator about 15 min before use. b. Dissolve 1 tablet (10 mg total) in 12.5 ml of Substrate Buffer at room temperature with stirring. Note: DO NOT use metal forceps during handling of the tablets, as metals catalyze the oxidation process. Protect the solution from direct sunlight. c. Add 12.5 µl of 30% H2O2, mix, and use within 30 min. [Final H2O2 concentration = 0.03% (v/v)] 5. Add 100 µl of OPD Substrate Solution to each well, and incubate 5-30 min. Note: Optimal incubation times may vary depending on the amount of HRP present. If color develops too quickly, dilution of the probe, antibody, or HRP-labeled reagent may be required. 6. Stop reaction by adding 100 µl of 0.1 M H2SO4. 7. Read the absorbance at 492 nm.
Note: Variations in incubation times and reagent concentrations may require further standardization by the user.
Biological Information
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
RTECS
ST0175000
Safety Information
R Phrase
R: 23/24/25-43-50/53
Toxic by inhalation, in contact with skin and if swallowed. May cause sensitization by skin contact. Very toxic to aquatic organisms; may cause long-term adverse effects in the aquatic environment.
S Phrase
S: 28-36/37-45-60-61
Wear suitable protective clothing and gloves. In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible). This material and its container must be disposed of as hazardous waste. Avoid release to the environment. Refer to special instructions/safety data sheet.
Product Usage Statements
Storage and Shipping Information
Ship Code
Blue Ice Only
Toxicity
Toxic & Carcinogenic / Teratogenic
Hazardous Materials Attention:
Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
Storage
-20°C
Protect from Light
Protect from light
Do not freeze
Ok to freeze
Special Instructions
Close the lid of the container tightly for storage, and do not remove the enclosed drying capsule.
Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.
Revision
10-August-2007 RFH
Synonyms
OPD, o-PD, HCl
Description
OPD (ortho-phenylenediamine) tablets for preparation of soluble substrate.
Background
OPD (ortho-phenylenediamine; o-PD) is a sensitive, chromogenic substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assays (ELISA). In the presence of HRP and hydrogen peroxide (H2O2), OPD is oxidized to a water-soluble, yellow-orange product that is measured spectrophotometrically at 492 nm, allowing a quantitative analysis of HRP-based systems.
Form
Off-white tablets
Formulation
White tablets (10 mg each).
CAS number
615-28-1
RTECS
ST0175000
Chemical formula
C₆H₈N₂ . 2HCl
Preservative
None
Comments
The tablets are inspected visually and are examined spectrophotometrically at 492 nm for the background level in 0.1 M sodium phosphate/citric acid, pH 5.0, containing 6 mM H2O2. Tablets dissolve in the substrate buffer within 5 min (with shaking).
Suggested Procedure for Use of OPD tablets for HRP-based ELISAs:
REAGENTS REQUIRED BUT NOT PROVIDED: 1. Substrate buffer: 50 mM citric acid/sodium phosphate buffer, pH 5.0. 2. Hydrogen peroxide (H2O2) 30%. 3. Stop solution: 0.1 M H2SO4.
PROTOCOL: 1. Complete all required incubations with antibodies and HRP-labeled probes. 2. Wash plate wells at least 4 times with phosphate-buffered saline (PBS) or Tris-buffered saline, containing 0.1% TWEEN®-20 detergent. 3. After the final wash, shake and blot all residual buffer from the wells. 4. Within 30 min of completion of the above steps, prepare OPD Substrate Solution (the following recipe provides sufficient OPD Substrate Solution for one 96-well plate (100 µl per well): a. Remove the OPD tablet container from the refrigerator about 15 min before use. b. Dissolve 1 tablet (10 mg total) in 12.5 ml of Substrate Buffer at room temperature with stirring. Note: DO NOT use metal forceps during handling of the tablets, as metals catalyze the oxidation process. Protect the solution from direct sunlight. c. Add 12.5 µl of 30% H2O2, mix, and use within 30 min. [Final H2O2 concentration = 0.03% (v/v)] 5. Add 100 µl of OPD Substrate Solution to each well, and incubate 5-30 min. Note: Optimal incubation times may vary depending on the amount of HRP present. If color develops too quickly, dilution of the probe, antibody, or HRP-labeled reagent may be required. 6. Stop reaction by adding 100 µl of 0.1 M H2SO4. 7. Read the absorbance at 492 nm.
Note: Variations in incubation times and reagent concentrations may require further standardization by the user.
Storage
Protect from light
-20°C
Do Not Freeze
Ok to freeze
Special Instructions
Close the lid of the container tightly for storage, and do not remove the enclosed drying capsule.
