• Detection of NADP/NADPH activity in cells or tissue extracts
• Detection of NADP/NADPH levels in antioxidation and oxidative stress
• Detection of NADP/NADPH levels in cell death, energy metabolism, and mitochondria function
• Species Independent – NADP/NADPH detection in Bacterial, fungal and plant cells
• Highly Sensitive – Detects up to 4nM NADP and NADPH
• Highly Specific – No Cross reactivity with NAD/NADH
• Easy to Use – 96 well Fluorescent Plate reader readout
   

Nicotinamide adenine dinucleotide phosphate (NADP+) is used in anabolic reactions, such as lipid and nucleic acid synthesis, which require NADPH as a reducing agent. NADPH is the reduced form of NADP+, and NADP+ is the oxidized form of NADPH. In cells, NADPH plays the role of a carrier of reducing power and is primarily involved in maintaining optimal redox metabolism. A simplified assay for the measurement of NAD and NADP is critical to understanding the roles of these pyridine nucleotides in normal and abnormal cells.

NADPH is produced in the oxidative phase of the pentose phosphate pathway in cells, a multifunctional pathway whose primary purpose is to generate reducing power in the form of NADPH. NADPH is a cofactor for enzymes that synthesize energy-rich molecules and provide the reducing equivalents for the oxidation-reduction involved in protecting the cell from the toxicity of reactive oxygen species (ROS) and NADPH oxidase-dependent ROS generation. Both NAD and NADP have been shown to influence hemoglobin affinity for oxygen in erythrocytes. In plant cells, NADPH is used as the reducing power for the biosynthetic reactions in the Calvin cycle of photosynthesis (1-2). 

Cell Technology’s Fluoro NADP/NADPH provides a highly reliable, sensitive fluorometric assay for the quantification of NADP, NADPH and their ratio in biological samples.

Reaction:

1. NADPH + non-fluorescent detection reagent + electron coupler → fluorescent analog + NADP

2. NADP + enzyme coupled reaction →  NADPH (then proceeds to reaction 1).

   Excitation: 530-570nm and Emission at 590-600nm
 

2. Bedard K and Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology.Physiol Rev 87:245 –313,2007.

3. Lowry, Oliver H., Passonneau, Janet V. and Rock, Martha K. The Stability of Pyridine Nucleotides. The Journal of Biological Chemistry, 236, #10, 1961.

2. NADP/NADPH Lysis Solution 12.5mL   Part# 3050 ……………………….4-8C

3. NADP Extraction Buffer 22 mL           Part# 3046 (Green label) ……….4-8C

4. NADPH Extraction Buffer 22 mL         Part# 3047 (Magenta label)……..4-8C

5. Reaction Buffer 11 mL.                    Part# 3049 ……………………….4-8C 

6. Standard curve diluent 15 ml.            Part # 3052……………………….4-8C

7. NADPH Detection reagent 110 μL       Part # 4019………………………-20C 

8. 3X Substrate Mix 6mL                     Part # 3048……………………… -20C 

9. NADPH Standard- 3 vials dried          Part# 7014………………………..-20C