p-Nitrophenyl Phosphate Substrate Buffer, 1 X 500 mL (487664-500ML)

Alkaline phosphatase substrate buffer supplied as ready-to-use. A diluent for Cat. No. 487663. ELISA (see comments) Product tested for stability at 4°C, 18-26°C, and 37°C. Product performance is tested on the final product by ELISA. Suggested procedure for use of p-NPP in alkaline phosphatase-based ELISAs: 1. To 39 parts distilled water add 1 part 50X p-NPP Concentrate (Cat. No. 487663) and 10 parts p-NPP Substrate Buffer. Mix well and store protected from light. This diluted reagent is suitable for up to 10 h after preparation. 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 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 phophatase-labeled reagent may be required.] 6. Stop the reaction by adding 100 µl of 1 N NaOH. 7. Read absorbance at 405 nm. Note: Variations in time, reagent volumes, and temperature may require further standardization by the user. Primary Target Alkaline phosphatase substrate buffer Toxicity: Standard Handling (A)

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