CY-1253 NAD+/NADH Colorimetric Assay Kit
Contents
1. 0 10 20 30 40 50 60 Reaction time min Fig 2 NADH Standard curve Ow NADH Standard curve Reaction time 30min IO ee N A450 0 1 000 2 000 3 000 4 000 NADH conc nM C CY 1253 12 Version 130823 NAD NADH Colorimetric Assay Kit Pa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Specificity of CycLex NAD NADH Colorimetric Assay Kit As low as 62 5 nM of NADH ca detected with 60 min incubation time n 2 there is no response to NADPH 2 0 1 5 ra 2 1 0 0 5 kk A 0 0 0 1 000 2 000 3 000 4 000 NADH NADPH conc nM Fig 4 NAD and NADH concentration in the cell e fact of v and SW480 cells 250 E NAD NADH 200 150 r NAD or NADH conc pmol 10 7 cells 0 Q SW480 Jurkat C CY 1253 13 Version 130823 NAD NADH Colorimetric Assay Kit yy od ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 5 NAD and NADH concentration in the cell extracts of Jurkat cells that have been treated Nampt specific inhibitor FK866 at indicated concentrations NAD and NADH concentration OA O 200 f NADH 150 F NAD or NADH conc pmol 10 cells 100 gt 50 f 0 Es GS 0 3 3 10 33 FK866 conc nM Fig 6 Comparisons of NAD and NADH concent ai2. et n sensitivity and atlo Since it is very simple to measure and it can be performed at the measurement of NAD and NADH concentrations in most laboratories is possible if t are ipped with a microtiter plate reader Considering that the use of fully automatic apparat itor the absorbance has become widespread NAD and NADH nae measur now possible with the CycLex NAD NADH Colorimetric Assay Kit using the s This method of measurement should dramatically raise the efficiency of measuring NAD concentrations in mammalian cells and tissues Measuring Principle of The CycLex rA Colorimetric Assay Kit ADH A me EtOH NAD WST 1 formazan Read A450 Acetoaldehyde s NADH WST 1 C CY 1253 2 Version 130823 K NAD NADH Colorimetric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Provided Each kit contains 10X NAD NADH Assay Buffer 1 5mLx1 0 Y 400 uM NADH 200 uL x 1 70 0X WST 1 10X Standard Dilution Buffer 2mLx1 71 20 5 200 uLx1 50X ADH alcohol dehydrogenase 200 uLx1 9 50X Diaphorase I0X EIOH Solution A S Loe Instruction manual Sie uk Materials Required but not Provided e NAD Extraction Buffer See page 5 section Detailed Pr e NADH extraction Buffer See page 5 section Detaile e Neutralization Buffer for NAD extraction Se e Spsection Deta
3. for 5 min Note Each investigator should optimize the number of cells used Ka or For _non adherent cells harvest and transfer the cells 1 5 s to microcentrifuge tubes followed by centrifugation at 2 000 rpm for 5 min Note Each investigator should optimize the number of c er test 2 Wash the cells twice with cold PBS by centrifugati 0 for 5 min 3 Spin down the cells by microcentrifuge at 10 0 or min Aspirate the supernatant remove as much of the PBS as possible For NAD measurement 4 Vortex the cell pellet gently Extract the ce 100 uL of NAD Extraction Buffer See page 5 i by vortexing 3 4 times for 1 min eac same time intervals or by homogenization using standard techniques i e sonicate 4 times for 5 c each on ice Then stand for 30 min on ice 5 Add 100 pL of Neutralization r NAD extraction See page 5 to the acid extract and mix well by vortexing for neutralizat 6 Spin the neutralized cell ex ve at 15 000 rpm for 5 min at 4 C Transfer a supernatant to new microcentrifuge tube H of the supernatant should be 7 5 8 5 Please make sure that the pH is within this ran f not adjust pH 7 5 8 5 using either Neutralization Buffers for NAD ction see page 5 extraction or for N 7 Keep the K tract for NAD measurement refer to as an acid extract AcE on ice t which is ready o step 8 for NAD measurement 8 Measure p feid eoncentration for example using the BCA protein assay da
4. of NAD and NADH This assay kit isf use only and not for use in diagnostic or therapeutic procedures CyY 1253 Version 130823 NAD NADH Colorimetric Assay Kit 4 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Nicotinamide adenine dinucleotide NAD as well as nicotinamide adenine dinucleotide phos NADP is an important cofactor found in cells NADH is the reduced form of NAD and N the oxidized form of NADH It has been reported that NAD metabolism regulates importa effects including life span NAD through poly ADP ribosyl polymerase PARP ribosyltransferase ARTs and recently characterized sirtuin enzymes exerts potential bio These enzymes modify proteins to regulate their function via ADP ribosylation or deace presence of NAD These enzymes are involved in several pathways including apoptosis D repair senescence and endocrine signaling suggesting that either the enzymes could an important therapeutical target for cancer diabetes atherosclerosis and so on The traditional NAD NADH and NADP NADPH assays are done by moni f NADH or NADPH absorption at 340 nm This method suffers low sensitivity and high in rence since the assay is done in the UV range that requires expensive quartz microplate CycLex N DH Colorimetric Assay Kit employs an enzyme cycling reaction which significantly increa provides a convenient method for sensitive detection of NAD NADH a
5. sample wells The concentration o or NADH can be expressed in pmol 10 cells or pmol mg protein Q NAD concentration pmol 10 cells D concentration of AcE nM x 2 x 10 cell number NADH concentration pmol 10 NADH concentration of AIE nM x 2 x 10 cell number Total volume of AcE 20 Total volume of AIK e of AcE for assay 25 uL lume of AIE for assay 25 uL AD concentration pmol 10 cells NADENAD Q NADH concentration pmol 10 cells Note 1 The abso e background increases with time thus it is important to subtract the absorbance value zero standard Std 7 wells for each data point appropriate reaction time is from 60 to 90 min This value is variable depending on onditions and preparation of cell extract Decreasing the amount of cell extract in the ay may help to lengthen the time range CyY 1253 Version 130823 K NAD NADH Colorimetric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Cautions 1 Since this kit is based on enzyme cycling reaction it is not possible to measure NAD and N concentration in conventional crude cell extract without special extraction and treatment for D and NADH Please follow the section Preparation of Sample at page 7 8 2 Please avoid mixing of any reagents containing SH group like DTT or reduced gl ad Or alkyl amine in the sample that will interfere this assay WwW For
6. Use Only Not for use in diagnostic procedures 5 Prepare NAD NADH Reaction Buffer Quantity Required 75 uL assay e Mix following reagents and put in ice Please use within 30 min after prepared this NAD NADH Reaction Buffer Discard any se NAD NADH Reaction Buffer after use Assay reagents Volume 10X NAD NADH Assay Buffer 10 pL 50X WST 1 2 uL 50X ADH 2 pL 50X Diaphorase 2 uL 10X EtOH Solution 10 dH O Total C CY 1253 6 Version 130823 NAD NADH Colorimetric Assay Kit G ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 2 Preparation of Sample Numerous extraction methods can be used to isolate NAD and NADH The following protocol NAD and NADH have been shown to work with a number of different mammalian cell li provided as examples of suitable methods If desired you can employ other methods for tionyof NAD and NADH All extraction and neutralization buffers are not provided in this kit Determination of NAD and NADH requires two separate samples acid extract fo and alkaline extract for NADH utilizing the character of NAD resistant to acidic conditio heat labile and NADH resistant to alkaline condition and relatively heat stable Q Preparation of Cell Extract 1 For adherent cells after washing with PBS trypsinize harvest and une hA 1 5 x 10 cells to microcentrifuge tubes followed by centrifugation at 2 000 rpm
7. at 15 000 r extract should be clear of any sediments some loss of NAD NADH ultiple freeze thaw cycles After inutes at 4 C again since the cell matters However this may result in Note 2 Although this protocol has been successf should optimize the cell extraction procedure pplied to many mammalian cell lines users their own applications Note 3 Although we suggest to conduct rents as outlined above the optimal experimental conditions will vary dependin t meters being investigated and must be determined by the individual user Especially pl ase make sure the final preparations of cell extract are in neutral pH hopefully pH_7 sodium carbonate based buffer as a NAD NADH ction buffer and alkaline extraction buffer for NAD and se the extracts prepared by using sodium carbonate based D and NADH concentrations and ratio See Fig 6 and 7 at CycLex does NOT recommen extraction buffer instead of aci NADH extraction respectiv buffer give you an inappr page 14 and 15 C CY 1253 8 Version 130823 p NAD NADH Colorimetric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 3 NAD NADH Assay Procedures NADH Assay reagents Test sample Standard NAD NADH Reaction Buffer see page 6 75 uL 75 uL NADH Standards 25 uL Your sample Test sample 25 uL 1 Following the above table add 25 uL of NADH Standards Std 1 Std 7 and Blan ard Diluti
8. ay not be ld modified for resale or used to manufacture commercial such commercial use please us via email N products without prior sin al from CycLex Co Ltd To inquire about licensing for C CY 1253 17 Version 130823
9. e SW480 cell extract measured by extraction methods and two kits CycLex NAD NADH Assay kit and Company B s NAD NADH Quantitation Kit 4 250 E NAD 3 NADH NAD or NADH conc pmol 10 6 cells CycLex Kit CycLex Kit Sodium Company B Kit Conventional extraction carbonate extraction C CY 1253 14 Version 130823 NAD NADH Colorimetric Assay Kit Pa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 7 Comparisons of NAD NADH ratio in SW480 cell extract measured by extraction methods N two kits CycLex NAD NADH Assay kit and Company B s NAD NADH Quantitatio Kit N NAD NADH ratio 18 gt 16 2 14 ia 12 x A 10 z 8 eo Z 4 2 te 0 CycLex Kit CycLex Kit Company B Kit Conventional Sodium carbonate extraction extraction C CY 1253 15 Version 130823 p NAD NADH Colorimetric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures References 1 Ziegenhorn J Senn M Bucher T 1976 Molar absorptivities of beta NADH and beta NADPH Chem 22 151 2 Matsumura H and Miyachi S 1980 Cycling assay for nicotinamide adenine dinucleo Enzymol 69 465 470 3 Zerez CR Lee SJ Tanaka KR 1987 Spectrophotometric determination of oxidized a duced pyridine nucleotides in erythrocytes using a single extraction p
10. iled Protocol Neutralization Buffer for NADH extraction e 5 section Detailed Protocol e Microplate for ELISA e Water bath or heating block Qy e Plate reader capable of measuring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual wavelengths of 450 550 of 450 595 nm can also be used The plate can also be read at a single wavelength of 450 nm w ve a somewhat higher reading Pipettors 2 20 uL 20 200 uL and200 1000 uL precision pipettors with disposable tips e Multi channel pipette e Microplate shaker Deionized e efhighest quality e Reagent rese FK866 FK866 is available from Axon Medchem Cat Axon 1279 or Cayman Cat 1 mM stock solution in DMSO optional el 4 Stop solution optional C CY 1253 3 Version 130823 p NAD NADH Colorimetric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions e Please thaw all reagents in crushed ice before use e Please keep ADH alcohol dehydrogenase and diaphorase in this kit on ice and getuso immediately to 70 C after use There is a possibility that the enzyme activity may be i g Q e Please avoid mixing of any reagents containing SH group like DTT or reduced glu alkyl amine in the sample that will interfere this assay e Do not use kit components beyond the indicated kit expiration date Qy e Rinse all detergent residue from glassware e Use deionized water
11. of the highest quality A e Do not mix reagents from different kits QO Do not mouth pipette or ingest any of the reagents e Do not smoke eat or drink when performing the assay or ea ere samples or reagents are handled e Biological samples may be contaminated with i agents Do not ingest expose to open wounds or breathe aerosols Wear protective gl ose of biological samples properly CAUTION NAD Extraction Buffer and NAD a strong acid and alkaline respective when handling Stop Solution H SO Q N traction Buffer Not provided in this kit are ear disposable gloves and eye protection C CY 1253 4 Version 130823 NAD NADH Colorimetric Assay Kit Pa cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol CycLex NAD NADH Colorimetric Assay Kit can measure NAD and NADH concentratio enzyme cycling reaction using alcohol dehydrogenase ADH diaphorase and WST 1 Since the re tio is not stopped it is necessary to monitor absorbance of WST 1 formazan at 450 nm at reg int s after the reaction is initiated and to determine appropriate reaction time 1 Preparation of Assay Reagents NAD Extraction Buffer 0 5 M perchloric acid HC104 1 Prepare Extraction Buffers and Neutralization Buffers Not provided in this kit amp NADH Extraction Buffer 50 mM NaOH and 1mM EDTA e Neutralization Buffer for NAD extraction 0 55 M K CO Ne
12. ol 181 9 12 Pogrebniak A et al cas emopotentiating effects of a novel NAD biosynthesis inhibitor FK866 i 1 in combination O lastic agents Eur J Med Res 11 313 21 C CY 1253 16 Version 130823 p NAD NADH Colorimetric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Related Products CycLex NAMPT Colorimetric Assay Kit Cat CY 1251 CycLex NMNAT Colorimetric Assay kit Cat CY 1252 CycLex NAD NADH Colorimetric Assay Kit Cat CY 1253 NAMPT Nicotinamide Phosphoribosyltransferase Cat CY E1251 NMNAT1 Nicotinamide Mononucleotide Adenylyltransferase 1 Cat CY E1252 1 NMNAT2 Nicotinamide Mononucleotide Adenylyltransferase 1 Cat CY E1252 2 CycLex SIRT1 Sir2 Deacetylase Fluorometric Assay Kit Cat CY 1151 CycLex SIRT2 Deacetylase Fluorometric Assay Kit Cat CY 1152 CycLex SIRT3 Deacetylase Fluorometric Assay Kit Cat CY 1153 CycLex SIRT6 Deacetylase Fluorometric Assay Kit Cat CY 1156 NAD Dependent Deacetylase SIRT1 Cat CY E1151 NAD Dependent Deacetylase SIRT2 Cat CY E1152 NAD Dependent Deacetylase SIRT3 Cat CY E1153 NAD Dependent Deacetylase SIRT6 Cat CY E1156 QO PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co jp URL http www cyclex co jp CycLex CircuLex products are for research use only CycLex CircuLex products and components thereof m
13. on Buffer and Your sample to each well of microplate Next initiate reactii b ing 75 uL of NAD NADH Reaction Buffer to the each well and mix thoroughly Inc oom temperature ca 25 C 2 Monitor the absorbance at 450 nm for 30 to 90 minutes at 10 min inter microplate reader 2 Alternatively after 60 min or appropriate time incubation the rea uL of Stop Solution 1 N H SO not provided in this kit into with the absorbance at 450 nm an be stopped by adding 50 and mix well Take a reading Table Layout of NADH standards and test samples AcE a in 96 well microplate 1 2 3 4 8 9 10 11 12 A Blank Blank AcE 1 AcE 1 B Std 7 Std 7 AIE 1 AIE 1 C Std 6 Std 6 AcE 2 AcE D Std 5 Std 5 AIE 2 2 E Std 4 Std 4 AcE 3 F Std 3 Std 3 AIE 3 G Std 2 Std 2 cE 4 H Std 1 Std 1 eA AIE 4 AcE Acid Extract AIE Alkaline Exdpac Note The NA ndards and test samples should be run in duplicate C CY 1253 9 Version 130823 NAD NADH Colorimetric Assay Kit Pa cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 1 Run reactions as described in the Detailed Protocol 2 Subtract A450 at the 0 time from all reaction time points 3 Plot A450 versus reaction time 4 Determine the reaction time range in which the increase in A450 is linear 5 Fix an appropriate reaction time usually 60 min 6 Take a
14. p NAD NADH Colorimetric Assay Kit cLex User s Manual For Research Use Only Not for use in diagnostic procedures Quantitative test kit for NAD NADH CycLex NAD NADH Colorimetric cA Assay Kit Fo ays Cat CY 1253 Intended USe cccccccccessecceeeeseesseeeeeeeeeees 1 of NN EEE T T 1 Introduction cccccccsscessssessssssseneees 2 Principle of the Assay 2 Materials Provided ccccccceccceceeeeeeeeees 3 Materials Required but not Provided 3 PRECAUTIONS yae0523 24 esevecdeccscessesdeevereds i 4 Detailed Protocol ccccccecceeeessseeeeeeeseeeees 5 9 Evaluation of Results ccceceeeeeeeeeneeee 10 Cations aeien eani oin 11 Assay Characteristics ccccccsscsssceesenees 11 Trouble shia tin 8 53 5 cicacsessnnctaadasseaseaimmasvehaguce 11 Reagent StaDity s sjsvecacadssssacesecsnvwnssovaresecsveds Example of Test Results eee eeeeeeeeeeeeeee 1 References Related Products Intended Use The CycLex Research Product CyeLex4NAD NADH Colorimetric Assay Kit provides a convenient method for sensitive measur tof NAD NADH and their ratio This kit is designed for specific detection of NAD and NA nzyme cycling reaction alization buffers are NOT provided in this kit NAD and NADH extraction and n Applications for this kit inc 1 Measuring NAD and NA ncentrations in cell or tissue extracts 2 Measuring NAD NADEL ratio 3 Detecting effects ological agents on cellular level
15. reading with the absorbance at 450 nm standard Std 7 optical density Plot the optical density for the standards versus the ration of the Average the duplicate readings for each standard control and sample and subtr average zero standards and draw the best curve The data can be linearized by using log logep d regression analysis may be applied to the log transformation To determine the NAD o concentration of each sample first find the absorbance value on the y axis and extend a horiz e to the standard curve At the point of intersection extend a vertical line to the x axis and esponding NAD or NADH concentration If the samples have been diluted the concenffation tead from the standard curve must be multiplied by the dilution factor See Fig 2 NADH Standa gt at page 12 ker ogram having a 5 parameter adjustment to accommodate for 1 The results of unknown samples can be calculated with any cont logistic function It is important to make an appropriate mathemati the dilution factor 2 Most microplate readers perform automatic calculati o curve is constructed by plotting the absorbang concentration X of calibrators using the four p g alyte concentration The calibration ibrators versus log of the known funetion 3 The concentration of NAD or NADH in the sample can be calculated then divide the NAD or NADH concentration by the sample amount e g cell umber or extract protein amount you added into the
16. research use only not for use in diagnostic or therapeutic procedures Assay Characteristics The CycLex Research Product CycLex NAD NADH Assay kit has been and NADH concentrations or NAD NADH ratio crude cell extract using assay shows good linearity of sample response measure NAD cling reaction The Troubleshooting When test sample have not adjusted to neutral pH Enzyme cyeling ion might be inhibited so that NAD and NADH concentration cannot be measured 2 The test samples should be run in duplicate using the rotoco described in the Detailed Protocol Incubation times or temperatures significantly those specified may give erroneous results 3 Poor duplicates indicate inaccurate dispensing I instructions in the Detailed Protocol were followed accurately such results indicate a need for multi channel pipettor maintenance All of the reagents included in the C search Product CycLex NAD NADH Assay kit have been tested for stability Reagents sh used beyond the stated expiration date Upon receipt all kit reagents should be stored at 70 C amp Y N amp Y o C CY 1253 11 Version 130823 4 NAD NADH Colorimetric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Time course 3 0 p e 4000 nM 2000 nM a 1000 nM 500 nM 250 nM y gt 125 nM D 62 5 nM 0nM A450
17. rocedure Anal Bioc 164 367 73 4 Zhao Z Hu X and Ross CW 1987 Comparison of Tissue Preparation M or Assay of Nicotinamide Coenzymes Plant Physiol 84 987 988 5 Umemura K and Kimura H 2005 Determination of oxidized and red dinucleotide in cell monolayers using a single extraction procedure an Anal Biochem 338 131 5 tinamide adenine ectrophotometric assay 6 Hasmann M and Schemainda I 2003 FK866 a Highly Spe Nicotinamide Phosphoribosyltransferase Represents a Novel Apoptosis Cancer Research 63 7436 7442 2003 oncompetitive Inhibitor of for Induction of Tumor Cell 7 Vilcheze C et al 2005 Altered NADH NAD Ratio iates Coresistance to Isoniazid and Ethionamide in Mycobacteria Antimicrobial Agents an erapy 49 708 720 8 Kimura N Fukuwatari T Sasaki R Shibata K 06 omparison of metabolic fates of nicotinamide NAD and NADH administered orally and intraperitoneally characterization of oral NADH J Nutr Sci Vitaminol Tokyo 52 142 9 O Donnell JM et al 2004 Limited tran ytosolic NADH into mitochondria at high cardiac workload Am J Physiol Heart Circ Physiol 286 H2237 10 Richard A et al 2008 Characterization 6f NAD Uptake in Mammalian Cells J Biol Chem 283 6367 6374 11 Rongvaux A et al 2008 Nicotinamide Phosphoribosyl Transferase Pre B Cell Colony Enhancing Factor Visfatin Is Require phocyte Development and Cellular Resistance to Genotoxic Stress J Immun
18. ta can be normalized and expr as pmol NAD mg of protein CyY 1253 Version 130823 NAD NADH Colorimetric Assay Kit G ycLex User s Manual For Research Use Only Not for use in diagnostic procedures For NADH measurement 4 Vortex the cell pellet gently Extract the cells with 100 uL of NADH Extraction Buffer See p by vortexing 2 3 times for 1 min each with same time intervals or by homogenization using st d techniques i e sonicate 4 times for 5 sec each on ice Then incubate at 60 C for 30 O the viscosity of the samples 5 Add 100 uL of Neutralization Buffer for NADH extraction See page 5 to the alkalin t and mix well by vortexing for neutralization then stand on ice for at least 5 min 6 Spin the neutralized cell extract at 15 000 rpm for 5 min at 4 C Transfer Cen to new microcentrifuge tube The final pH of the supernatant should be 7 5 8 5 Please sure that the pH is within this range If not adjust pH 7 5 8 5 using either Neutralizati uffers for NAD extraction or for NADH extraction see page 5 T Keep the tube of the cell extract for NADH measurement refer to a aline extract AlE on ice which is ready to go to step 8 for NADH measurement 8 Measure protein concentration for example using the BCA pr data can be normalized and expressed as pmol NADH mg of protein Note 1 If necessary the cell extract can be stored at 70 C thaw the cell extract centrifuge
19. utralization Buffer for NADH extraction 0 3 M potassium pho r PH 7 4 2 Thaw 10X NAD NADH Assay Buffer at room temperature i bath Stand all other reagents on ice to thaw Use them after they thaw completely 3 Prepare a working solution of Standard Dilution Buffer 10X Standard Dilution Buffer 1 10 with distilled water Mix well Store at 4 C two ks or 20 C for long term storage 4 Prepare NADH Standards as follows Use 400 uM NADH to produce a serial dil es ow Mix each tube thoroughly before the next transfer The 4 000 nM standard Std es as the high standard The NADH Standard Dilution Buffer serves as the zero standard Blank Volume of Standard aera Concentration Std l 10 pL of 400 uM NAD 990 uL 4 000 nM Std 2 100 uL of Std 1 4 000 100 pL 2 000 nM Std 3 100 uL of Std 2 2 00 100 pL 1 000 nM Std 4 100 uL of Std 3 1 100 pL 500 nM Std 5 100 uL of Std 4 100 pL 250 nM Std 6 100 uL of Std 100 pL 125 nM Std 7 100 uL of St 100 pL 62 5 nM Blank 100 pL 0 ng mL Note 1 Do nof ts ting pipette Change tips for every dilution Wet tip with Standard before dispe portions of NADH Standards should be discarded Note 2 Si is converted to NADH in enzyme cycling reaction and relatively labile than cLex only provides NADH Standard CyY 1253 Version 130823 wn NAD NADH Colorimetric Assay Kit Car yCLex User s Manual For Research
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