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Excellent substrate for alkaline phosphatase-based ELISA assays. Produces yellow soluble end product that can be read at 405 nm to 410 nm. Both kinetic and endpoint determinations can be performed. Supplied as ready-to-use. A ready-to-use diluent is available separately (Cat. No. 487664).
Functionality check performed on the final product in ELISA.
Suggested procedure for use of p-NPP in alkaline phosphatase-based ELISAs:
1. Prepare 1X p-NPP Substrate Solution: To 39 parts dH2O add 1 part 50X p-NPP Concentrate and 10 parts p-NPP Substrate Buffer (Cat. No. 487664). Mix well and store protected from light. This diluted reagent is suitable for up to 10 h after preparation.
Note: Cat. No. 487664 is an AMP (2-amino-2-methyl-1-propanol)-based buffer. Other buffers, e.g. diethanolamine (DEA)-based and 2-amino-2-methyl-1,3-propanediol (AMPD)-based, may also be employed.
2. Complete all required incubations with antibodies and alkaline phosphatase-labeled probes. 3. Wash plate wells at least 4 times with Tris-buffered saline (TBS) containing 0.1% Tween®-20 detergent. DO NOT USE PHOSPHATE BUFFERS; inorganic phosphate is a potent inhibitor of alkaline phosphatase. 4. After the final wash, shake and blot all residual buffer from the wells. 5. Add 100 µl of 1X p-NPP Substrate Solution (prepared in Step 2) to each well and incubate for 15-60 min. (If a kinetic study is desired, readings at 405 nm may be taken at intermediate intervals).
[Note: Optimal incubation times may vary depending on the amount of enzyme present. If color develops too quickly, dilution of the probe, antibody, or alkaline phosphatase-labeled reagent may be required.]
6. Stop the reaction by adding 100 µl 1 N NaOH. 7. Read absorbance at 405 nm.
Note: Variations in time, reagent volumes, and temperature may require further standardization by the user.
Biological Information
Primary Target
Substrate for alkaline phosphatase-based ELISA assays
Physicochemical Information
Cell permeable
N
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Storage and Shipping Information
Ship Code
Ambient Temperature Only
Toxicity
Standard Handling
Storage
+2°C to +8°C
Protect from Light
Protect from light
Do not freeze
Ok to freeze
Special Instructions
Discard if solution turns bright yellow or becomes turbid.
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
20-November-2009 JSW
Synonyms
pNPP
Application
ELISA
Description
p-Nitrophenyl phosphate supplied as a 50X concentrate (0.19 mol/l). For use with p-NPP Substrate Buffer, Cat. No. 487664.
Background
p-Nitrophenyl phosphate is the most common substrate for alkaline phosphatase-based ELISA assays. Hydrolysis of the phosphate ester liberates p-nitrophenol, a soluble, yellow chromogen that can be measured spectrophotometrically at 405 nm (ε = 1.83 x 104 M-1cm-1). As the reaction is linear, kinetic assays can be performed. Endpoint determinations can also be performed by stopping the reaction with strong base and measuring the absorbance at 405 nm. This p-NPP preparation is a stable, convenient, liquid concentrate in a proprietary solvent.
Form
Clear to slightly yellow tinged liquid
Formulation
Supplied as a 50X concentrate.
Preservative
None
Comments
Functionality check performed on the final product in ELISA.
Suggested procedure for use of p-NPP in alkaline phosphatase-based ELISAs:
1. Prepare 1X p-NPP Substrate Solution: To 39 parts dH2O add 1 part 50X p-NPP Concentrate and 10 parts p-NPP Substrate Buffer (Cat. No. 487664). Mix well and store protected from light. This diluted reagent is suitable for up to 10 h after preparation.
Note: Cat. No. 487664 is an AMP (2-amino-2-methyl-1-propanol)-based buffer. Other buffers, e.g. diethanolamine (DEA)-based and 2-amino-2-methyl-1,3-propanediol (AMPD)-based, may also be employed.
2. Complete all required incubations with antibodies and alkaline phosphatase-labeled probes. 3. Wash plate wells at least 4 times with Tris-buffered saline (TBS) containing 0.1% Tween®-20 detergent. DO NOT USE PHOSPHATE BUFFERS; inorganic phosphate is a potent inhibitor of alkaline phosphatase. 4. After the final wash, shake and blot all residual buffer from the wells. 5. Add 100 µl of 1X p-NPP Substrate Solution (prepared in Step 2) to each well and incubate for 15-60 min. (If a kinetic study is desired, readings at 405 nm may be taken at intermediate intervals).
[Note: Optimal incubation times may vary depending on the amount of enzyme present. If color develops too quickly, dilution of the probe, antibody, or alkaline phosphatase-labeled reagent may be required.]
6. Stop the reaction by adding 100 µl 1 N NaOH. 7. Read absorbance at 405 nm.
Note: Variations in time, reagent volumes, and temperature may require further standardization by the user.
Storage
Protect from light
+2°C to +8°C
Do Not Freeze
Ok to freeze
Special Instructions
Discard if solution turns bright yellow or becomes turbid.
