CY-1252 NMNAT Colorimetric Assay Kit
Contents
1. 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 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 supplied for research use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial products without prior written approval from CycLex Co Ltd To inquire about licensing for such commercial use please contact us via email Cat CY 1252 19 Version 1406042. 3 Monitor the absorbance at 450 nm for 30 reaction velocity remains constant Measure and calculate the rate of reaction while 9 Version 140604 NMNAT Colorimetric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Assay control 1 For Inhibitor screening The CycLex NMNAT Colorimetric Assay kit measures the NMNATI enzyme activity enzyme coupled reaction where three enzymes are involved NMNAT1 ADH and diaphorase If test chemicals have an inhibitory effect on one of these enzymes the signal will be reduce ne such a possibility please carry out the experiment of NAD NADH recycling assay i table to ascertain which enzyme is target of the test chemical Assay reagents Test chemical Vehicle con NAD 5 uM 10 pL 50X Inhibitor candidate 2 pL Vehicle for Inhibitor candidate dH20 68 uL 3 2 Step Assay Buffer II 20 uL 20 uL NAD Not provided in this kit See page 3 O and 50X Inhibitor candidate or nd mix well Next initiate reaction by ell and mix thoroughly Incubate at 30 C 1 Following the above table add 10 uL of NAD 5 Vehicle for Inhibitor candidate to each well of adding 20 uL of 3 2 Step Assay Buffer II t 2 Monitor the absorbance at 450 nm for 60 min at 5 min intervals using microtiter plate reader Measure and calculate the rate of reaction while the reaction velocity remains constant
3. NMNAT Colorimetric Assay Kit is d signed for the rapid and sensitive evaluation of NMNAT1 inhibitors or activators using recombiant NMNAT1 Since this kit is based on NAD detection system it is not possible to directly detect NMNAT activity in crude cell extract in which NAD concentration is relatively high Applications for this kit include 1 Screening inhibitors or activators of NMUNAT1 2 Detecting the effectssof pharmacological agents on NMNATI This assay kit is forresearch use only and not for use in diagnostic or therapeutic procedures Storage e Upon receipustore all components at 70 C e Don t exposeme agents to excessive light Cat CY 1252 1 Version 140604 oe NMNAT Colorimetric Assay Kit cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Nicotinamide mononucleotide adenylyltransferase 1 NMNAT1 EC 2 7 7 1 is a central enzymegin NAD biosynthesis transferring the adenylyl moiety of ATP to nicotinamide mononucleotide NMN or nicotinic acid mononucleotide NaMN resulting in the formation of NAD or NaAD and the release of pyrophosphate As this reaction is reversible the enzyme may in principle be used to form ATP and NMN from NAD and pyrophosphate This enzyme could be a potential target for therapeutical applications because its activity is rather low in tumor cells The deduced protein contains an N terminal nuclear localization signal Immunofluor
4. 70 C 10X ATP 1mLx1l 70 C i xK 50X WST 1 200 uLx1 70 C 200 pL x 18 370 C 200 R 70 C 10X EtOH 1 mLyx 1 70 C Instruction manual 1 Room temp Nicotinamide mononucleotide Human NMNAT1 nicotinamide mononucleotide adenylyltransferase I jexpressed in E coil Materials Required but not Provided e Microplate for ELISA e Plate reader capable of measuring absorbance in 963well plates at dual wavelengths of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used The plate can also be read at a single wavelength of 450 nm which will give somewhat higher reading e Pipettors 2 20 uL 20 200 uL and 2002000 ML precision pipettors with disposable tips e Multi channel pipette e Microplate shaker e Deionized water of the highest quality e 500 or 1000 mL graduated cylinder e Reagent reseryoirs e NAD NAD B Nicotinamide adenine dinucleotide hydrate is from Sigma Cat N7004 Prepare fleshly 5 uM solution in H2O from 1 mM stock solution in H20 Discard any unused 5 uM NAD Optional e Gallotannin fannic acid Gallotannin is from Sigma Cat T3437 Prepare 0 5 mM solution in H20 Optional Cat CY 1252 3 Version 140604 Pae NMNAT Colorimetric Assay Kit Ce ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions e Please thaw all reagents on crushed ice before use e Please avoid repeated freezi
5. Inhibitor candidate or Vehicle for Inhibitor candidate or Gallotannin 0 5 mM to each well of microplate and mix well Next initiate reaction by adding 50 ME of 1 1 Step Assay Buffer to each well and mix thoroughly Incubate at 30 C 2 Monitor the absorbance at 450 nm for 60 niingat 5 min intervals using a microtiter plate reader Measure and calculate the rate of reactionpwhilesthe reaction velocity remains constant Cat CY 1252 8 Version 140604 Py NMNAT Colorimetric Assay Kit ycLex eer eMail For Research Use Only Not for use in diagnostic procedures II 2 Step Method Assay reagents Test chemical Vehicle control Inhibitor control 1X recombinant NMNAT1 2 uL 2 uL 2 uL 50X Inhibitor candidate 2 uL Vehicle for Inhibitor candidate 2e o N Gallotannin 0 5 mm tit e 2nL dH20 66 uL 66 uL 2 2 Step Assay Buffer I 30 pL 30 pL pL Gallotannin 0 5 mM Not provided in this kit See page 3 20 and 50X Inhibitor mM to each well of the y Buffer I to each well and 1 Following the above table add 2 uL of 1X recombinant N candidate or Vehicle for Inhibitor candidate or Gallota microplate Next initiate reaction by adding 30 uL of 2 2 mix thoroughly to initiate reaction Incubate at 30 C for 6Qumi 2 Add 20 uL of 3 2 Step Assay Buffer II to each we microplate and mix thoroughly Incubate at 30 C min intervals using a microtiter plate reader
6. not stopped it is necessary to monitor absorbance of WST 1 formazan at 450 nm at regular intervals after the reaction is initiated and to determine reaction velocity The CycLex NMNAT Colorimetric Assay Kit can measure the enzyme activity of NMNAT1 with two different measuring methods the 1 Step Method and the 2 Step Method Mie 1 Step Method is accomplished by mixing with three enzymes i e NMNATI1 alcohol dehydtogenase and diaphorase Since two coupled reactions are promoted simultaneously with NMNAT1 enzyme reaction detection of this method is less sensitive than the 2 Step Method Conversely the 2 Step Method is begun by initiating reactions of NMNAT 1 within a set time period to produce NDA from NMN and ATP then in the second step followed b adding ADH diaphorase and WST 1 the resultant WST 1 formazan is formed by NAD NADH cycling enzyme reaction Preparation Method for Assay Reagents Place all reagents in ice to thaw Use them after they thaw completely 1 1 Step Assay Buffer Quantity Required 50 pL assay e Mix following reagents and put on ice This 1 Step Assay Buffer should be used within 30 min after prepared Discard any unused 1 Step Assay Buffer after use Reagents Volume 10X NMNAT Assay Buffer 10 pL 10X NMN 10 pL 10X ATP 10 pL 50X WSTI 2 pL gt 50X ADH 2 uL M 50X Diaphorase 2 uL 10X EtOH 10 uL dH20 4 uL Total 50 uL Cat CY 1252 5 Version 14060
7. page 11 or 12 3 Duplicate measurement is strongly recommefided for accurate measurement of NMNAT1 activity 4 Although we suggest to conduct experiments as outlined in Protocol for immunoprecipitation on page 15 the optimal experimental conditions will vary depending on the parameters being investigated and must be detesmined by the individual user For research use only not for use in diagnostic or therapeutic procedures Cat CY 1252 13 Version 140604 oy NMNAT Colorimetric Assay Kit 2 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Characteristics The CycLex Research Product CycLex NMNAT Colorimetric Assay Kit has been shown to detect the activity of nicotinamide phosphoribosyltransferase activity in recombinant NMNATI or an immunoprecipitate using the specific antibody against NMNAT1 The assay shows good linearity of sample response The assay may be used to follow the purification of NMNATI1 Troubleshooting 1 When test chemicals have an inhibitory effect on ADH or diaphorase precise inhibitory effect on NMNATI1 enzyme activity cannot be measured 2 The recombinant NMNAT1 should be run in duplicate using the protocol d scribed in the Detailed Protocol Incubation times or temperatures significantly different from those specified may give erroneous results 3 Poor duplicates indicate inaccurate dispensing If all instructions in the Detailed Protocol were fol
8. y reag sample control control Your enzyme sample Test sample 10 uL 1X recombinant NMNAT1 2 uL dH20 40 uL 48 uL 50 uD 1 1 Step Assay Buffer see page 5 50 uL 50 uL 50 pL Following the above table add 10 uL of Your enzyme sample or_2 uL of 1X recombinant NMNAT1 and dH20 to the well of microplate and mix well Next initiate r action by adding 50 uL of 1 1 Step Assay Buffer to each well and mix thoroughly Incubate at30 C Monitor the absorbance at 450 nm for 60 min at 5 min interyals using a microtiter plate reader Measure and calculate the rate of reaction while the reaction velocitysremains constant 2 2 Step Method 1st reaction Conversion of nicotinamide to NAD Assay reagents Test Positive No enzyme sample control control Your enzyme sample Test sample 10 pL 1X recombinant NMNATI l7 Loo ee eee dH20 60 uL 68 uL 70 uL 2 2 Step Assay Buffer I see page 6 30 uL 30 uL 30 uL 1 Following the above table add Your enzyme sample or 2 uL of 1X recombinant NMNAT1 and dH20 to each well of the mi foplate Finally initiate reaction by adding 30 uL of 2 2 Step Assay Buffer I to each well and mix thofoughly to initiate reaction Incubate at 30 C for 60 min 2nd reaction Measurementof NAD 2 Add 20 uL of 3 2 StepsAssay Buffer II see page 6 to each well of the microplate and mixing thoroughly Incubate at30 C 3 Monitor thefaBbsor
9. 4 NMNAT Colorimetric Assay Kit Ce NycLex User s Manual For Research Use Only Not for use in diagnostic procedures 2 2 Step Assay Buffer I Quantity Required 30 uL assay Mix following reagents and put on ice This 2 Step Assay Buffer I should be used within 30 min after prepared Discard any unused 2 Assay Buffer I after use Assay reagents Volume 10X NMNAT Assay Buffer 10 pL 10X NMN 10 pL 10X ATP 10 uL Total 30 uL D 3 2 Step Assay Buffer II Quantity Required 20 uL assay e Mix following reagents and put in ice This 2 Step Assay Buffer II should be used within 30 min after p Discard any unused 2 Step Assay Buffer II after use Assay reagents e 50X WST 1 L 50X ADH uL 50X Diaphorase 2 uL 10X EtOH 10 uL dH 0 Q 4 uL O 20 uL 4 Prepare 1X NMNAT Assay by diluting the 10X NMNAT Assay Buffer 10 fold with deionized distilled water at th e Of assay 5 Prepare 1X recombinant 1 by diluting the 100X recombinant NMNATI 100 fold with 1X NMNAT Assay Buffer ce of assay Prepare C e for your assay Discard any unused 1X recombinant NMNATI after diluted C CY 1252 6 Version 140604 oy NMNAT Colorimetric Assay Kit 2 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures NMNATI1 Assay Procedures 1 1 Step Method Per Test Positive No enzyme
10. ILVNAN oseroydeig References 1 Emanuelli M Rajaelli N Amici A Balducci E Natalini P Ruggieri S and Magni G 1995 Biochem Pharmacol 49 575 579 2 Jayaram H N Cooney D A and Grusc Curr Med Chem 6 561 574 3 Schweiger M Hennig K Lerner F M Hirsch Kauffmann M Specht T Weise C Oei S L Ziegler M 2001 FEBS Left 95 100 4 Zhou T Kurnasov O Tom L Zhang H 2002 J Bio R Binns D D Grishin N V Marquez V E Osterman A 7 13148 13154 5 Felicitas Berger Corin au Mathias Ziegler 2007 Proc Natl Acad Sci 104 3765 37706 Araki T Sasaki Y Milbrandt 004 Science 305 1010 1013 M Frizzell M J Gamble M E DuMond R Krishnakumar T Yang A 2009 J Biol Chem 284 20408 20417 mann M Ziegler M 2005 J Biol Chem 280 36334 36341 7 Berger F Cj 8 Y Vohra R H Baloh and J Milbrandt 2009 J Neurosci 29 6526 6534 18 Version 140604 oy NMNAT Colorimetric Assay Kit 2 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 NAMPT Nicotinamide Phosphoribosyltransferase Cat CY E1251 NMNATI Nicotinamide Mononucleotide Adenylyltransferase 1 Cat CY E1252 CycLex SIRT1 Sir2 Deacetylase Fluorometric Assay Kit Cat CY 1151
11. MNAT using 1 Step Method e 0 ng 3 5 r aang amp 8 ng a 16 ng 2 5 31 ng A 63 ng 2 0 125 ng 2 250 ng 1 5 1 0 0 5 oo ss 0 10 20 30 40 50 60 Reaction time min ww Fig 2 Dose dependent curve of recombinant NUNA ivity using 1 Step Method 0 20 0 18 fF 0 16 0 14 0 12 0 10 0 08 Activity A450 min 0 06 0 04 0 02 0 00 0 50 100 150 200 250 Recombinant NMNAT1 ng C CY 1252 16 Version 140604 NMNAT Colorimetric Assay Kit g ycLex User s Manual For Research Use Only Not for use in diagnostic procedures o Fig 3 Effect of Gallotannin on recombinant NMNAT1 activity 120 peer retreat of control 0 0 10 20 Gallotannin uM Fig 4 Measurement of endogenous NMNATI a in mmunoprecipitate using anti Human NMNAT1 Rabbit Polyclonal Antibody from of Raji cells 0 07 0 06 fF 0 05 t A g L 0 04 wn vt 2 gt 0 03 lt 0 02 0 01 7 0 00 L Anti NMNAT1 pAb Nomal Rabbit IgG control C CY 1252 17 Version 140604 gry NMNAT Colorimetric Assay Kit 3 cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 5 Requirement of each assay component for measurement of NMNAT1 activity 120 100 f 80 F 40 F 20 of control a O13 D Ly aya dur0s HOV Hav C
12. When there is an inhibitory effect of te emical on NAD NADH recycling reaction A450 will not increase C CY 1252 10 Version 140604 NMNAT Colorimetric Assay Kit A yCLex User s Manual For Research Use Only Not for use in diagnostic procedures II For NMNAT1 activity assay in an immunoprecipitate Since NAD level in cells is relatively high around several hundreds micromolar on NAD might mix easily with purified NMNATI1 from various cells or an immunoprecipitate us the specific antibody against NMNAT1 Such contaminated NAD in the test sample causes a positive result by initiating NAD NADH cycling enzyme reaction If there is such a possibility please carry out the experiment of NAD NADH recycling assay in the following table Assay reagents Test sample NAD control Your enzyme sample Test sample 10 pL INAD 5uM o a Ud 10 p e2 dH20 70 pL L 3 2 Step Assay Buffer II 20 uL NAD Not provided in this kit See page 3 1 Following the above table add 10 uL of Your enzyme samp 0 uL of NAD 5 uM and 2 Monitor the absorbance at 450 nm A450 for 60 intervals using a microtiter plate reader Measure and calculate the rate of reactio i tion velocity remains constant When there is contaminated NAD in the ple A450 will increase compared to NAD control Version 140604 y AA ycLex User s Manual NMNAT Colorimetric Assay Kit For R
13. bance at 450 nm for 30 min at 5 min intervals using a microtiter plate reader Measure and galctilate the rate of reaction while the reaction velocity remains constant Cat CY 1252 T Version 140604 oy NMNAT Colorimetric Assay Kit P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 3 Special considerations when screening inhibitors or activators Please consider the following in order to correctly estimate the inhibitory effect on NMNAT enzymatic activity It is necessary to conduct 1 Vehicle Control at least once for the first experiment 2 Inhibitor Control at least once for the first experiment and 3 Test Chemical as indicated on the following table When test chemicals cause an inhibitory effect on NMNAT1 enzyme activity the level of A450 is weakened as compared with Vehicle control The high level of A450 is not obsetyed in Inhibitor control usually A450 lt 0 3 at 30 min reaction point I 1 Step Method Assay reagents Test chemical Vehicle control Inhibitor control 1X recombinant NMUNAT1 2 uL 2 uL 2 uL 50X Inhibitor candidate 2 uL Vehicle for Inhibitor candidate uA Gallotannin 0 5 mM o aA 2p dH 20 46 uL 46 nL 46 uL 1 1 Step Assay Buffer 50 uL 50 uL 50 pL Gallotannin 0 5 mM Not provided in this kit See page 3 1 Following the above table add 2 uL of 1X recombinant NMNAT1 dH20 and 50X
14. escence microscopy localized endogenous NMNATI to the nucleus in human fibroblasts and in a hepatoma cell line It was demonstrated that NMNAT1 inhibited recombinant human poly ADP ribose polymerase 1 by about 35 and it compl telypprevented the formation of branched ADP ribose polymers Principle of the Assay Since it is very simple to measure and it can be performed at a low price the measurement of NMNAT1 activity in most laboratories is possible if they are equipped With a microtiter plate reader Considering that the use of fully automatic apparatus to monitor the abSOrbance has become widespread NMNAT1 activity measurement which could not be made by the conventional method is now possible with the CycLex NMNAT Colorimetric Assay Kit using the same equipment This new method of measurement shall dramatically raise the efficiency of inhibitof Screening and biochemical analysis of this enzyme Measuring Principle of The CycLex NMNAT Golorimetric Assay Kit NMN nicotinamide mononucleotide ATP NMNAT nicotinamide mononucleotide adenylyltransferase NAD nicotinamide adenine dinucleotide ADH Alcohol dehydrogenase t Diaphorase NADH WJ NADH WST 1 WSF formazan l Measure OD at 450 nm Cat CY 1252 2 Version 140604 ry NMNAT Colorimetric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Provided Each kit contains i2mLx1 0G 10X NMN 1mLx1
15. esearch Use Only Not for use in diagnostic procedures II For NMNAT1 activity assay in an immunoprecipitate Alternative method In order to measure the activity of NMNAT1 correctly it is necessary to conduct the control experiments for No enzyme control and No NMN control at least once in addition to Your enzyme sample as indicated in the below table of assay method Although A450 increases in Test sample when NMNAT enzyme activity is in the sample the increase in A450 is not obsefvedjin No enzyme control and No NMN control Assay reagents Test sample Positive No enzyme No NMN control control control 10X NMNAT Assay Buffer 10 uL 10 pL 10 pL 10 uL 10X NMN 10 pL 10 pL 10 uk 10X ATP 10 pL 10 pL 10 ple 10 pL 3 2 Step Assay Buffer II see page 6 20 uL 20 uL 20 uL 20 uL dH20 40 pL 48 pL 50 pL 50 uL Your enzyme sample Test sample 10 pL 10 pL 1X recombinant NMNAT1 2 nD 1 Following the above table add all reagents Of 10X NMNAT Assay Buffer to dH20 to each well of microplate and mix well Next initiate reaction by adding 10 uL of Your enzyme sample or 2 uL of 1X recombinant NMNAT1 to each well andimix thoroughly Incubate at 30 C 2 Monitor the absorbance at 450 nm for 30 min at 5 min intervals using a microtiter plate reader Measure and calculate the rate of reaction whil the reaction velocity remains constant The differe
16. lowed accurately such results indicate a need for multi channel pipettor maintenance Reagent Stability All of the reagents included in the CycLex Research Produes Cychex NMNAT Colorimetric Assay Kit have been tested for stability Reagents should not be ased b yond the stated expiration date Upon receipt all kit reagents should be stored at 70 C Cat CY 1252 14 Version 140604 oe NMNAT Colorimetric Assay Kit cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Protocol for immunoprecipitation Solutions and Reagents Note Prepare solutions with Milli Q or equivalently purified water Cell Lysis Buffer 1X 20 mM Tris pH 8 0 250 mM NaCl 1 mM EDTA 1 mM EGTA 1 Triton X 100 1 mM DTT and protease inhibitor cocktail Protein A Agarose Beads Add 5 mL of 1X PBS to 1 5 g of Protein A Agarose Beads Shake 2 hours at 4 C spin down Wash the pellet twice with PBS Resuspend beads in 1 volume of PBS Can be stored for 2 weeks at 4 C 10X TBS Tris buffered saline For 1 liter of 10X TBS use 24 2 g Tris b se a d 80 g NaCl Adjust pH to 7 6 with HCI use at 1X Wash Buffer TBS T 1X TBS 0 1 Tween 20 Preparing Cell Lysates 1 Aspirate media Treat cells by adding fresh media containing test compoundfor desired time 2 To harvest cells under nondenaturing conditions remove mediagamd rmse cells once with ice cold PBS 3 Remove PBS and add 0 5 mL 1X ice cold Cell Lysis Buffer
17. nce in the reaction velocity between Test sample and No NMN control indicates the NMNAT activity Note Duplicate measurements strongly recommended Cat CY 1252 12 Version 140604 oY NMNAT Colorimetric Assay Kit aa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Evaluation of Results Analysis of Kinetics Time course curve 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 Calculate activity A450 of Test Sample Activity reaction velocity Reaction time min Note Usually the linear range is from 10 to 30 min This value dS Wariable depending on reaction conditions and storage handling of the recombinant NMNATYI Decreasing the amount of recombinant NMNAT1 in the assay may help to lengthen fhe timerange Cautions p Since this kit is based on NAD detection system it s NOtpossible to detect NMNATI1 activity in crude cell extract in which NAD concentration s relatively high Please use an immunoprecipitate as a test sample using the specific antibody against NMNAT1 2 Contaminated NAD in the test sample causes a false positive result by initiating NAD NADH cycling enzyme reaction Please confirm no NAD in the test sample according to 4 Assay control
18. ng and thawing of the recombinant NMNATI in this kit a possibility that the enzyme activity may be inactivated Aliquot to 25 50 uL and store at e Please avoid mixing of any reagents containing group like DTT or reduced g or 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 of the highest quality G e Do not mix reagents from different kits QO e Do not mouth pipette or ingest any of the reagents e Do not smoke eat or drink when performing the assay or im are ere samples or reagents are handled e Biological samples may be contaminated with infec ts Do not ingest expose to open wounds or breathe aerosols Wear protective co se of biological samples properly C CY 1252 4 Version 140604 oe NMNAT Colorimetric Assay Kit aa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol Description of assay system The CycLex NMNAT Colorimetric Assay Kit can measure the enzyme activity of nicotinamide mononucleotide adenylyltransferase NMNA by an enzyme coupled reaction as shown if Measuring Principle of The CycLex NMNAT Colorimetric Assay Kit at page 2 In this method NMUNAT1 converts NMN to NAD Resultant NAD can be measured by enzyme cycling reaction using alcohol dehydrogenase ADH diaphorase and WST 1 Since the reaction is
19. oe NMNAT Colorimetric Assay Kit aa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Quantitative test kit for nicotinamide mononucleotide adenylyltransferase activity CycLex NMNAT Colorimetric Assay Kit For 100 assays Cat CY 1252 Intended Use cisssiesalstatsntcssaasaaaianabalenaaeidacas 1 BOS A ETT 1 TCEOOU CTO a epscascsnceadatacatantanatabotecianac vateens 2 Principle of the Assay 2 Materials Provided jiscisccccssssccceccessnsdxctccassavens 3 Materials Required but not Provided 3 PC ANIONS iccoecsceudtonssaantsatauanrananrasetactinnraes 4 Detailed Protocol ivi sassceigessdeamanssassesiacriqinals 5 12 Evaluation Of RESUS ccsiscisassssasssanecasscaeseee 13 CAUCUS ssc cuscatc uacpeweeeaagccaccinends quaniesatatedeences 13 Assay Characteristics seee 14 TrOUbleSHO OUI sas veauscaststanincensaiscasseaspouunnns 14 Reagent Sia DILLY x iuusiinasssniacuesagniadeateansanne 14 Protocol for immunoprecipitation 15 Example of Test RESUS issiisivsssccsecsosossieanns 16 18 RELCTSM CES sscctecssanszrasace taryeuntvationevensouartanatias 18 Related Products vecscsscaanccssassteatearancessenteaasaias 19 Intended Use The CycLex Research Product CycLex NMNAT Colorimetric Assay Kit detects nicotinamide mononucleotide adenylyltransferase NMNAT activity in recombinant NMNATI1 or endogenous NMNATI1 immunoprecipitated from cell extract Primarily the CycLex Research Product CycLex
20. to each plate 10 cm dish and incubate the plate on ice for 5 minutes 4 Scrape cells off the plate and transfer to microcentrifugeitubes Keep on ice 5 Sonicate 4 times for 5 seconds each on ice 6 Microcentrifuge for 10 minutes at 4 C and transfer the supernatant to a new tube The supernatant is the cell lysate If necessary the lysate canbe stored at 70 C Immunoprecipitation 1 Take 250 ul cell lysate and add protein A agarose beads 40 ul of 50 bead slurry Incubate with gentle rocking for 1 3 hours at 4 C for pr clearance 2 Microcentrifuge for 30 seconds at 496 Take the supernatant and transfer to a new tube W Add and add 1 2 ug of aft NMNAT1 antibody which can be used for immunoprecipitation incubate with gentle rockingg og hours or overnight at 4 C 4 Add protein A agarose beads 20 ul of 50 bead slurry Incubate with gentle rocking for 1 3 hours at 4 C Nn Microcentrifge for 10 seconds at 4 C Wash the beads twice with 500 ul of 1X Cell Lysis Buffer subsequently twice with NMNAT Assay Buffer Keep on ice during washes 6 Resuspend the beads with 20 ul NAMPT Assay Buffer and measure NMNAT1 activity according to NMINAT Assay Procedures page 7 Cat CY 1252 15 Version 140604 4 NMNAT 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 of NMNAT activity in recombinant N
Download Pdf Manuals
Related Search