NADP+/NADPH Assay Kit (Fluorometric)

1. Fluorometric Probe (40X) : One 150 μL amber tube
2. NADP Cycling Substrate : One 50 μL tube
3. Assay Buffer : One 25 mL bottle
4. Extraction Buffer (10X) : One 10 mL bottle
5. Electron Mediator (500X) : One 10 μL amber tube

Box 2 (shipped on blue ice packs)

• Detects NADP+, NADPH, or total NADP+/NADPH
• Compatible with cell or tissue lysates
• NADP+ standard included

1X Extraction Buffer: Dilute the stock 10X Extraction Buffer 1:10 with deionized water for a 1X solution. Stir or vortex to homogeneity. NADP Cycling Reagent: Prepare an NADP Cycling Reagent for the number of assays being tested and just before use. Prepare by diluting the NADP Cycling Substrate 1:100, NADP Cycling Enzyme 1:400, Electron Mediator 1:500, and Fluorometric Probe 1:40 in 1X Assay Buffer. (eg. For 100 assays, combine 50 μL NADP Cycling Substrate, 12.5 μL NADP Cycling Enzyme, 10 μL Electron Mediator, and 125 μL Fluorometric Probe to 4.803 mL of 1X Assay Buffer for a 5 mL total solution). When preparing, combine the NADP Cycling Substrate, NADP Cycling Enzyme, and Electron Mediator together first and mix prior to adding the Fluorometric Probe. Upon adding the probe, protect the solution from light to prevent the probe from oxidizing. Use the NADP Cycling Reagent immediately. Preparation of Samples These preparation protocols are intended as a guide for preparing unknown samples. The user may need to adjust the sample treatment accordingly. It is highly recommended that all samples should be assayed immediately upon preparation or stored for up to 1 month at -80 °C. A trial assay with a representative test sample should be performed to determine the sample compatibility with the dynamic range of the standard curve. High levels of interfering substances may cause variations in results. Samples may be diluted in deionized water as necessary before testing. Run proper controls and account for any sample dilutions. Always run a standard curve with samples. Tissue homogenates: Sonicate or homogenize 100 mg tissue sample in 0.5 mL cold 1X Extraction Buffer. Centrifuge at 14,000 rpm for 5 minutes at 4 °C to remove insoluble material. Filter the solution with a 10 kDa spin filter to deproteinate the sample. Collect flow through. Perform + dilutions in cold deionized water. Sample may be tested immediately for total NADP /NADPH quantification or extracted with acid or base to separate the cofactors. Store unused samples at - 80 °C for up to 1 month. Cell lysates: Culture cells until confluent and harvest. Centrifuge and wash cell pellet with 1X PBS. 6 Centrifuge to pellet cells and remove wash. Resuspend cells at 1-5 x 10 cells/mL in 0.5 mL 1X Extraction Buffer. Homogenize or sonicate the cells on ice. Centrifuge at 14,000 rpm for 5 minutes 4 °C to remove debris. Filter the solution with a 10 kDa spin filter to deproteinate the sample. Collect flow through. Perform dilutions in cold deionized water. Sample may be tested + immediately for total NADP /NADPH quantification or extracted with acid or base to separate the cofactors. Store unused samples at -80 °C for up to 1 month. 4 Note: Enzymes in tissue and cell sample lysates may deplete NADPH rapidly and affect results. Samples should be deproteinized before extracting the cofactors or using within the assay. A spin filter with a 10 kDa cutoff is recommended for efficient and clean separation. NADPH Extraction Procedure:

• To measure NADPH and destroy NADP , add 25 μL of sample to a microcentrifuge tube. Add 5 μL of 0.1 N NaOH and mix thoroughly. Incubate the tube at 80 °C for 60 minutes and protected from light. Centrifuge the tube to pool all sample solution. Add 20 μL of 1X Assay Buffer to shift the pH of the sample back to neutral. Vortex to mix and centrifuge to pool sample. Keep sample on ice until assaying.
• NADP Extraction Procedure:
• To measure NADP and destroy NADPH, add 25 μL of sample to a microcentrifuge tube. Add 5 μL of 0.1 N HCl and mix thoroughly. Incubate the tube at 80 °C for 60 minutes and protected from light. Centrifuge the tube to pool all sample solution. Add 20 μL of 1X Assay Buffer to shift the pH of the sample back to neutral. Vortex to mix and centrifuge to pool sample. Keep sample on ice until assaying. Notes: + +If testing both total NADP /NADPH and individual cofactors, dilute the total NADP /NADPH samples 1:2 with Assay buffer to maintain sample dilution consistency. Avoid samples containing SH groups like DTT, β-mercaptoethanol, or reduced gluathione. Samples should be close to neutral pH before harvesting. Samples with extremely high or low pH values could fail to yield reliable results.

1. Prepare and mix all reagents thoroughly before use. Each sample, including unknowns and standards, should be assayed in duplicate or triplicate. +
2. Add 50 μL of each NADP standard or unknown sample into wells of a 96-well microtiter plate.
3. Add 50 μL of NADP Cycling Reagent to each well. Mix the well contents thoroughly and incubate for 1-2 hours at room temperature protected from light. Note: This assay is continuous (not terminated) and therefore may be measured at multiple time points to follow the reaction kinetics.
4. Fluorescence microplate reader capable of reading excitation in the 530-570 nm range and emission in the 590-600 nm range. +4. Calculate the concentration of NADP /NADPH within samples by comparing the sample RFU to the standard curve.

1. Calculate the average fluorescence values for every standard, control, and sample. Subtract the average zero standard value from itself and all standard and sample values. This is the corrected background fluorescence. If sample background control value is high, subtract the sample background control value from the sample reading. +
2. Plot the corrected fluorescence for the NADP standards against the final concentration of the -1 standards from Table 1 to determine the best slope (μM ). See Figure 2 for an example standard curve.
3. + Since all NADP is converted to NADPH by the Cycling Reagent, use the standard curve to + determine the total NADP /NADPH concentration in pmoles within the sample. Determine the total concentration of the samples with the equation obtained from the linear regression analysis of the standard curve. Substitute the corrected fluorescence values for each sample. Remember to account for dilution factors. + Total NADP /NADPH = Sample corrected fluorescence x Sample dilution Slope