Product Name:	NADP/NADPH Assay Kit (BA0238)
SKU:	BA0238
Detection Method:	Fluorimetric (FL530/585 nm)
Detection Range:	0.01 µM to 1 µM NADP+/NADPH (96-well plate)
Sample Type:	['Cell extracts', 'Tissue extracts']
Species Reactivity:	All
Assay Time:	30 min
Kit Size:	100 Assays
Equipment Required:	Microplate reader
Storage:	-20°C
Shelf Life:	12 months
Shipping:	Gel Pack

A fluorimetric assay kit for the quantitative determination of NADP+, NADPH and their ratio in cell and tissue extracts. The kit uses a glucose dehydrogenase cycling reaction that generates a highly fluorescent product measured at λex/em = 530/585 nm, with a detection limit of 0.01 µM and linearity up to 1 µM in a 96-well plate format.

Sensitive and accurate. Detection limit of 0.01 µM and linearity up to 1 µM NADP+/NADPH in a 96-well plate assay.
Convenient. The procedure involves adding a single working reagent and reading fluorescence at time zero and 30 min. Room temperature assay.
High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.

Direct assays of NADP+/NADPH concentrations and ratios in cell or tissue extracts.
Research into energy transformation and the redox state of cells and tissue.

Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1	Sample Preparation. For tissues, weigh ~20 mg tissue per sample and wash with cold PBS. For cells, wash with cold PBS and pellet ~10^5 cells per sample. Homogenise samples in a 1.5 mL tube with either 100 µL NADP+ extraction buffer (for NADP+) or 100 µL NADPH extraction buffer (for NADPH). Heat extracts at 60°C for 5 min, then add 20 µL Assay Buffer and 100 µL of the opposite extraction buffer to neutralise. Briefly vortex and spin down at 14,000 rpm for 5 min; use the supernatant for the assay. Determination of both NADP+ and NADPH requires extractions from two separate samples.
2	Calibration Curve. Prepare 5000 µL of 1 µM NADP+ Premix by mixing 5 µL of 1 mM Standard with 4995 µL distilled water. Dilute the standard to give 1.0, 0.6, 0.3 and 0 µM points (see Standard Dilution table). Transfer 50 µL standards into wells of a black flat-bottom 96-well plate.
3	Samples. Add 50 µL sample per well in separate wells.
4	Reagent Preparation. For each reaction well, prepare Working Reagent by mixing 45 µL Assay Buffer, 1 µL Enzyme A, 1 µL Enzyme B, 1 µL G6P and 5 µL Probe. Fresh reconstitution is recommended.
5	Reaction. Add 50 µL Working Reagent per well quickly. Tap plate to mix.
6	Read fluorescence at λex/em = 530/585 nm for time zero (F0) and again (F30) after a 30-min incubation at room temperature. Protect the plate from light during incubation.

Compute the ΔF for each standard and sample by subtracting F0 from F30. Plot the standard ΔF values and determine the slope. [NADP(H)] = (ΔF_SAMPLE – ΔF_BLANK) / Slope (µM^-1) × n (µM), where ΔF_SAMPLE and ΔF_BLANK are the changes in fluorescence intensity of the Sample and Blank (STD 4) respectively, Slope is the slope of the standard curve and n is the dilution factor. If sample ΔF values exceed that of the 1 µM standard, dilute the sample in distilled water and repeat, multiplying the result by the dilution factor.

Component	Quantity	Storage
Assay Buffer	10 mL	-20°C
Enzyme A	120 µL	-20°C
G6P	120 µL	-20°C
Enzyme B	120 µL	-20°C
Probe	750 µL	-20°C
NADP Standard	0.5 mL	-20°C
NADP Extraction Buffer	12 mL	-20°C
NADPH Extraction Buffer	12 mL	-20°C

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NADP/NADPH Assay Kit (BA0238)

Description