Description

Pyridine nucleotides play an important role in metabolism and, thus, there is continual interest in monitoring their concentration levels. Quantitative determination of NADP⁺/NADPH has applications in research pertaining to energy transformation and redox state of cells or tissue.

Simple, direct and automation-ready procedures for measuring NADP⁺/NADPH concentration are very desirable. BioAssay Systems EnzyChrom^TM NADP⁺/NADPH assay kit is based on a glucose dehydrogenase cycling reaction, in which the formed NADPH reduces a formazan (MTT) reagent. The intensity of the reduced product color, measured at 565 nm, is proportionate to the NADP⁺/NADPH concentration in the sample. This assay is highly specific for NADP⁺/NADPH and is not interfered by NAD⁺/NADH. Our assay is a convenient method to measure NADP, NADPH and their ratio.

Applications

Direct Assays: NADP⁺/NADPH concentrations and ratios in cell or tissue extracts.

Key features

Sensitive and accurate. Detection limit 0.1 mM, linearity up to 10 mM NADP⁺/NADPH in 96-well plate assay.

Convenient. The procedure involves adding a single working reagent, and reading the optical density at time zero and 30 min at room temperature. No 37°C heater is required.

High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.

Kit Contents (100 tests in 96-well plates)

   Assay Buffer:       10 mL                Glucose (1 M): 1.5 mL

   MTT Solution:     1.5 mL                Enzyme Mix:   120 mL  

   NADP Standard: 0.5 mL 1 mM   

   NADP/NADPH Extraction Buffers:  each 12 mL    

Storage conditions. Store all reagents at -20°C. Shelf life: 6 months after receipt.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

ProcedureS

1. Sample Preparation. For tissues weigh ~20 mg tissue for each sample, wash with cold PBS.  For cell samples, wash cells with cold PBS and pellet ~10⁵ cells for each sample. Homogenize samples (either tissue or cells) in a 1.5 mL eppindorf tube with either 100 mL NADP extraction buffer for NADP determination or 100 mL NADPH extraction buffer for NADPH determination. Heat extracts at 60°C for 5 min and then add 20 mL Assay Buffer and 100 mL of the opposite extraction buffer to neutralize the extracts. Briefly vortex and spin the samples down at 14,000 rpm for 5 min. Use supernatant for NADP/NADPH assays. Determination of both NADP and NADPH concentrations requires extractions from two separate samples.

2. Calibration Curve. Prepare 500 mL 10 mM NADP Premix by mixing  5 mL 1 mM Standard and 495 mL distilled water.

Dilute standard as shown in the Table. Transfer 40 mL standards into wells of a clear bottom 96-well plate.

Samples:  add 40 mL sample per well in separate wells.

3. Reagent Preparation. For best results allow Enzyme to come to RT (15-30 min) before preparing the Working Reagent.  For each well of reaction, prepare Working Reagent by mixing 60 mL Assay Buffer, 1 mL Enzyme Mix, 10 mL Glucose and 14 mL MTT. Fresh reconstitution is recommended.

4. Reaction. Add 80 mL Working Reagent per well quickly. Tap plate to mix briefly and thoroughly.

5. Read optical density (OD0) for time “zero” at 565 nm (520-600nm) and OD30 after a 30-min incubation at room temperature.

6. Calculation. Subtract OD0 from OD30 for the standard and sample wells. Use the DOD values to determine sample NADP/NADPH concentration from the standard curve.

Note: If the sample DOD values are higher than the DOD value for the 10 mM standard, dilute sample in distilled water and repeat this assay. Multiply the results by the dilution factor.

Materials Required, but not provided

Pipetting (multi-channel) devices. Clear-bottom 96-well plates (e.g. Corning Costar) and plate reader.

General Considerations

1.  At these concentrations, the standard curves for NADP and NADPH are identical. Since NADPH in solution is unstable, we provide only NADP as the standard.  

2.  This assay is based on an enzyme-catalyzed kinetic reaction. Addition of Working Reagent should be quick and mixing should be brief but thorough. Use of multi-channel pipettor is recommended.

3.  The following substances interfere and should be avoided in sample preparation. EDTA (>0.5 mM), ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%) and Tween-20 (>1%).

Standard Curve in 96-well plate assay

Publications

1. Ding X et al (2009). Enhanced HtrA2/Omi expression in oxidative injury to retinal pigment epithelial cells and murine models of neurodegeneration. Invest Ophthalmol Vis Sci. 50(10):4957-66. 

2. Tseng HC