NAD + /NADH Assay [Colorimetric]

G-Biosciences 1-800-628-7730 1-314-991-6034 technical@gbiosciences.com A Geno Technology, Inc. (USA) brand name NAD + /NADH Assay [Colorimetric] (Cat. #786 1539, 786 1540) think proteins! think G-Biosciences www.gbiosciences.com

INTRODUCTION... 3 ITEM(S) SUPPLIED... 3 STORAGE CONDITIONS... 4 ADDITIONAL ITEMS REQUIRED... 4 IMPORTANT INFORMATION... 4 PREPARATION BEFORE USE... 4 PROTOCOL... 5 PREPARATION OF SAMPLE AND DEPROTEINATION WITH SPINOUT GT 100 COLUMNS... 5 WORKING ASSAY MIXTURE PREPARATION... 6 WST 1 DEVELOPER [1X] PREPARATION... 6 ASSAY PROTOCOL... 6 INTERFERENCES... 8 TROUBLESHOOTING... 8 REFERENCES... 9 RELATED PRODUCTS... 9 Page 2 of 10

INTRODUCTION Nicotinamide adenine dinucleotide (NAD) found in all living cells plays an important role in oxidation reduction reactions and energy metabolism. It exists in oxidized (NAD + ) and reduced forms (NADH). NAD serves as a cofactor in large number of cellular redox reactions, carrying reducing equivalents from one reaction to other. Thus, maintaining proper levels of NAD is essential for normal cellular respiratory function. In addition, NAD plays critical roles in ADP ribosylation reactions and is a substrate for sirtuins. There are studies that show that the cytosolic NAD + concentrations range from 300 nm in mammalian cells to 2 mm in yeast. Depletion of NAD in cells is a major cause of cell death. Thus levels of NAD +, NADH and their ratio are critical indicator of cell health. NAD + /NADH Assay [Colorimetric] is designed for sensitive detection of intracellular nucleotides, NADH, NAD and their ratio. This colorimetric assay is based on NAD cycling enzyme mix which increases the sensitivity of detection (Fig.1). In this assay, NAD + present in the samples is reduced to NADH by alcohol dehydrogenase which oxidizes ethanol to acetaldehyde. The newly formed NADH and NADH present in samples are oxidized by diaphorase in presence of WST 1 which in turn is reduced to highly colored formazan. Formazan absorbs at 450 nm and amount of formazan made is directly proportional to total NAD in samples and thus serves as indicator of NAD in cell samples. ITEM(S) SUPPLIED Description Fig1: NAD+/NADH Assay reaction Cat. # 786 1539 100 assays Cat. # 786 1540 200 assays NAD + /NADH Extraction Buffer [10X] 10 ml 2 x 10 ml NADH Standard [1 mm] 1 vial 2 vials NAD + /NADH Assay Buffer [5X] 5 ml 2 x 5 ml NAD + Cycling Enzyme Mix 1 vial 2 vials WST 1 Developer [10X] 1 vial 2 vials Page 3 of 10

STORAGE CONDITIONS The kit is shipped on blue ice. Store the kit at components as indicated on label. The kit when stored as directed without reconstitution is stable for 1 year. Reconstituted component are stable for 2 months. ADDITIONAL ITEMS REQUIRED Microtiter plate with clear flat bottom. Microplate reader that can measure absorbance at 450 nm SpinOUT GT 100, 1ml (Cat. # 786 866) or SpinOUT GT 100, 5ml (Cat. # 786 868) column for deproteinization step Bicinchoninic Acid (BCA) Protein Assay (Cat. # 786 570) IMPORTANT INFORMATION 1. It is recommended that fresh samples are used for assay. If the assay cannot be performed at same time, then make sure that sample preparation step is complete before storing the samples. Samples should be stored in one time used aliquot at 80 C. Alternatively, samples extracted can be snap freezed in liquid nitrogen and then stored immediately at 80 C for processing later. 2. Prepared or unprepared samples should be thawed on ice for assay or sample preparation respectively. 3. Use 2 3 dilutions of sample to ensure that the readings fall within standard graph 4. Always prepare fresh standard for assaying the samples for NAD +, NADH and total NAD. PREPARATION BEFORE USE 1. Bring reagents to room temperature before performing assay and spin down the vials before reconstitution. 2. Dilute NAD + /NADH Extraction Buffer [10X] to [1X] by adding 1 ml of NAD + /NADH Extraction Buffer [10X] to 9 ml of deionized water. NOTE: NAD + /NADH Extraction Buffer [1X] is used for sample preparation and for making working dilutions of NADH Standard 3. Add 200 µl of deionized water to one vial of NADH Standard [1 mm] and dissolve the lyophilized NADH by mixing several times with a pipette. Make small one time use aliquots and store them at 20 C in dark. NOTE: NADH is light sensitive. Prepare NADH avoiding direct light and store the NADH in dark or protected from light. 4. Dilute NAD + /NADH Assay Buffer [5X] to [1X] by adding 1 ml of NAD + /NADH Assay Buffer [5X] to 4 ml of deionized water. 5. Add 120 µl of deionized water to one vial of NAD + Cycling Enzyme Mix and mix well. Make small one time use aliquots and store them at 20 C. 6. Add 120 µl of deionized water to one vial of WST 1 Developer [10X] to make 10X stock solution of WST 1. Make one time use aliquots and store them in dark at 20 C. Page 4 of 10

PROTOCOL NOTE: WST 1 is light sensitive. Avoid direct contact with light and store the vials in dark or protected from light. Preparation of sample and Deproteination with SpinOUT GT 100 columns Cell culture (adherent and suspension) samples 1. Culture cells in appropriate flasks and at appropriate density in incubator at 37 C and 5% CO 2. 2. Trypsinize (for adherent cells) and harvest the number of cells required for the assay NOTE: Initial recommended numbers for assay is 2 x 10 5 cells 3. Wash the cells once with cold PBS. 4. Pellet cells at 2000 rpm for 5 minutes at 4 C. Remove the supernatant. 5. Add 400 µl of ice cold 1 x NAD + /NADH Extraction Buffer to the pellet and resuspend the cells. 6. Lyse the cells either by homogenization on ice or freeze/thaw for 2 cycles of 20 minutes at 20 C with thawing in between for 10 minutes at room temperature. 7. Vortex the sample for 10 seconds and the centrifuge at 13000 g for 10 minutes at 4 C. 8. Collect the supernatant. 9. Filter the supernatant by passing through SpinOUT GT 100 spin column. Add the supernatant collected to SpinOUT GT 100 spin column and centrifuge at 1000 x g for 10 minutes at 4 C. Collect the filtrate. NOTE: This deproteination step is employed to remove enzymes that may use NAD + and NADH and interfere with NAD quantification. NOTE: Use of deproteination reagents such as TCA and PCA is avoided NADH is degraded in acidic ph. 10. The filtrate is the sample and should be either used immediately for assay or store as one time use aliquots at 80 C. Tissue samples 1. Remove the tissue and wash several times with cold isotonic saline (150 mm) or PBS. Weight 20 mg of tissue. NOTE: 20 mg is initial recommended amount. It needs to be standardized by end user. 2. Wash the weight tissue once with cold PBS. 3. Add 400 µl of ice cold 1 x NAD + /NADH Extraction Buffer to the tissue sample and homogenize using douncer homogenizer for 30 50 passages on ice. 4. Centrifuge the sample at 13000 g for 10 minutes at 4 C. 5. Collect the supernatant. Page 5 of 10

6. Filter the supernatant by passing through SpinOUT GT 100 spin column. Add the supernatant collected to SpinOUT GT 100 spin column and centrifuge at 1000 x g for 10 minutes at 4 C. Collect the filtrate. NOTE: This deproteination step is employed to remove enzymes that may use NAD + and NADH and interfere with NAD quantification. NOTE: Use of deproteination reagents such as TCA and PCA is avoided NADH is degraded in acidic ph. 7. The filtrate is the sample and should be either used immediately for assay or store as one time use aliquots at 80 C. Working Assay Mixture preparation Before starting the assay prepare Working Assay Mixture as listed in table1. Table 1 preparation is suitable for 45 reactions Reagents Volume NAD + /NADH Assay Buffer [1X] 4.45 ml NAD + Cycling Enzyme Mix 50 µl Table 1: Working Assay Mixture preparation WST 1 Developer [1X] Preparation Prepare 1 X WST 1 Developer by adding 50 µl of WST 1 Developer [10X] to 450 µl of NAD + /NADH Assay Buffer [1X] and mix well. NOTE: WST 1 is light sensitive. Prepare WST 1 protected from light. Assay protocol Total NADH detection 1. NADH standard: Prepare 10 µm working stock of NADH by adding 10 µl of 1mM NADH stock solution to 990 µl of 1X NAD + /NADH Extraction Buffer. The standards prepared must be protected from light. Fresh standard should be made for every assay. Tube NADH [10µM] 1 X NAD + /NADH Extraction Buffer Concentration of NADH (µm ) Final Concentration of NADH (µm ) in assay A 300 0 0 B 6 294 0.2 0.1 C 12 288 0.4 0.2 D 24 276 0.8 0.4 E 36 264 1.2 0.6 F 48 252 1.6 0.8 G 60 240 2 1 NOTE: The linear detection range is 0.1 to 8µM, so standards with different concentrations can be made. NOTE: The final concentrations of the standards prepared would be half in the assay as the standards are diluted 1:2 in the assay. Page 6 of 10

NOTE: The 10µM NADH solution is unstable and must be used within 4 hrs. 2. Add 100 µl sample or standard per well on clear flat bottom microtiter plate. NOTE: Assay sample and each standard in duplicate set 3. Add 90 µl of Working Assay Mixture, mix well and incubate the plate for 5 minutes at room temperature on a shaker with gentle speed. 4. Add 10 µl of 1 X WST 1 Developer per well and mix well. 5. Incubate the plate for 60 minutes on shaker at room temperature in dark. Read OD at 450 nm. NOTE: Depending upon the standard and sample NADH concentration incubate plates at 30 minutes to 2 hrs and take reading at a point where the color development is good enough to read. One can read plate at multiple time points while color is developing to find suitable time point. Calculation: Plot the Standard curve (Fig.1) Total NADH= NADH detected by standards plot x dilution factor x 2* µm * The concentration of sample is multiplied by 2 as the sample is 1:2 diluted in the assay. Total NADH is also calculated related to cell number or protein content. Protein concentration can be detected after sample extraction and before filtration of sample using GT 100 spin columns by BCA method (Cat. #786 570). Thus total NADH amount is calculated per mg of protein. Page 7 of 10

NADH detection only 1. To detect NADH only in samples, remove NAD + by decomposing. Aliquot 500 µl of extracted samples in a microfuge tube and heat to 60 C for 30 minutes in a water bath or a heating block. 2. Cool the samples on ice and spin the samples briefly to remove any precipitate 3. Add 100 µl sample per well NOTE: For unknown samples different dilution of samples can be tested to bring the sample concentration with NADH standard range. 4. Add 90 µl of Working Assay Mixture, mix well and incubate the plate for 5 minutes at room temperature on a shaker with gentle speed. 5. Add 10 µl of 1 X WST 1 Developer per well and mix well. 6. Incubate the plate for 60 minutes on shaker at room temperature in dark. Read OD at 450 nm. NOTE: Depending upon the standard and sample NADH concentration incubate plates at 30 minutes to 2 hrs and take reading at a point where the color development is good enough to read. One can read plate at multiple time points while color is developing to find suitable time point. Calculation: Plot the Standard curve NADH= NADH detected by standards plot x dilution factor x 2* µm * The concentration of sample is multiplied by 2 as the sample is 1:2 diluted in the assay. NADH is also calculated related to cell number or protein content. Protein concentration can be detected after sample extraction and before filtration of sample using GT 100 spin columns by BCA method (Cat. #786 570). Thus total NADH amount be calculated per mg of protein. NAD + = Total NADH NADH NAD + /NADH (Ratio)= Total NADH NADH NADH INTERFERENCES 5% detergents such as Triton X 100, NP 40 and ascorbic acid interfere with the assay. TROUBLESHOOTING Issue Suggested reason Possible solution NAD + Bring assay buffer to room temperature /NADH Assay buffer is cold Assay not working Plate reader is not set at correct wave length 96 well plate used in not correct Check the filter setting of instrument and set to appropriate filter (450 nm read) Use clear well plates with flat Page 8 of 10

Samples with erratic reading Non linear standard Steps not performed per protocol or missed a step Bubbles in the wells Samples were not deproteinized Presence of interfering substance in samples Pipetting error in preparing standards or reaction mix Air bubbles formed in well Use of reagents from old kits bottom Check the protocol and follow steps as mentioned in protocol Mix the reagents gently in the well by pipetting gently against the wall to avoid bubble formation. Use SpinOUT GT 100 columns to filter the sample to remove proteins Dilute the samples Avoid preparing small volume of standards. Prepare master mix (Working Assay Mix) and avoid adding single component directly to the well Pipette gently against the wall of the well Use reagents from the same kit REFERENCES 1. Bernofsky, C. and Swan, M. (1973).An improved cycling Assay for Nicotinamide Adenine dinucleotide. Analytical Biochemistry 53, 452 458. 2. Umemura, K. and Kimura, H. (2005). Determination of oxidized and reduced nicotinamide adenine dinucleotide in cell monolayers using a single extraction procedure and a spectrophotometric assay. Analytical Biochemistry 338, 131 135. RELATED PRODUCTS Download our Bioassays Handbook. http://info2.gbiosciences.com/complete bioassay handbook For other related products, visit our website at www.gbiosciences.com or contact us. Page 9 of 10

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