CY-1251 NAMPT Colorimetric Assay Kit
Contents
1. Ognjanovic S Bao S Yamamoto SY Garibay Tupas J Samal B et al 20 Endocrinol 26 107 117 7 Max Hasmann and Isabel Schemainda 2003 Cancer Res 63 7 2 M Hasmann and I Schemainda 2003 Cancer Res 63 7436 7442 Q Kathryn Moynihan Ramsey et al 2009 Science 324 651 Yasukazu Nakahata et al 2009 Science 324 654 CY 1251 21 Version 121015 H9 NAMPT Colorimetric Assay Kit e Vclex 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 SIRTI 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 PRODUCED BY CycLex Co Ltd 1063 103 Tera sawaoka 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 onl2. NMNA 50x ADH and 9 50x Diaphorase in this kit There is a possibility that the enz inactivated Aliquot to 25 50 uL and store at 70 C e Please avoid mixing of any reagents containing SH group like DTT or reduced gluta alkyl amine in the sample that will interfere this assay Do not use kit components beyond the indicated kit expiration date Q Rinse all detergent residue from glassware G Use deionized water of the highest quality Q Do not mix reagents from different kits Do not mouth pipette or ingest any of the reagents Do not smoke eat or drink when performing the assay or i as Where samples or reagents are handled Biological samples may be contaminated with Anfectio ents Do not ingest expose to open wounds or breathe aerosols Wear protective g d dispose of biological samples properly C CY 1251 4 Version 121015 oe NAMPT Colorimetric Assay Kit e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol Description of assay system The CycLex NAMPT Colorimetric Assay Kit can measure the enzyme activity of nicotinamide phosphoribosyltransferase NAMPT by an enzyme coupled reaction as shown in Measufing Principle of The CycLex NAMPT Colorimetric Assay Kit at page 2 In this method NAMPT converts nicotinamide to nicotinamide mononucleotide NMN subsequently nicotinamide mononucleotide adenylyltransferase 1 NMNATI converts NMN to NAD
3. Resultant NAD can be measured by enzyme cycling reaction using alcohol dehydrogenase ADH diaphorase and WST 1 Since the reaction is 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 NAMPT Colorimetric Assay Kit can measure the enzyme activity of NAMPT with two kinds of measuring methods the 1 Step Method and the 2 Step Method The 1 Step Method is accomplished by mixing with four enzymes i e NAMPT NMNATI alcohol dehydrogenase and diaphorase Since three coupled reactions are promoted simultaneously with NAMPT enzyme reaction detection sensitivity of this method is less than that of 2 Step Method Conversely the 2 Step Method is begun by initiating reactions of two enzymes NAMPT NMNATI within a set time period to produce NDA from nicotinamide and PRPP then in the second step followed by adding ADH diaphorase and WST 1 the resultant WST 1 formazan is formed by NAD NADH cycling enzyme reaction Preparation Method for Assay Reagents Stand all reagents in ice to thaw Use them after they thaw completely 1 1 Step Assay Buffer Quantity Required 60 uL assay e Mix following reagents and put in 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 D 10X NAMPT assay buffer 10 u
4. each well and mix thoroughly Incubate at 30 C 2 Monitor the absorbance at 450 nm for 6 min at 5 min intervals using microtiter plate reader Measure and calculate the rate of reaefion while the reaction velocity remains constant The difference in the reaction velocitysbetween Test sample and No Nicotinamide control indicates the NAMPT activity Cat CY 1251 12 Version 121015 oe NAMPT Colorimetric Assay Kit e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Evaluation of Results Analysis of Kinetics Time course curve 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 min4 NOTE Usually the linear range is from 20 to 40 min This value is variable depending on reaction conditions and storage handling of the Recombinant NAMPT Decreasing the amount of Recombinant NAMPT in the assay may help to lengthenjthe time range Cautions Since this kit is based on NAD detection system it is not possible to detect NAMPT activity in crude cell extract in which NAD concentration is relatively high Please use an immunoprecipitate using the specific antibody against NAMPT as a test sample CycLex
5. 450 will increase in Test sample C CY 1251 11 Version 121015 H9 NAMPT Colorimetric Assay Kit 5 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures III For NAMPT activity assay in an immunoprecipitate Alternative method In order to measure the activity of NAMPT correctly it is necessary to conduct the control experiments for No enzyme control and No Nicotinamide control at least once in addition_to Your enzyme sample as indicated in the below table of assay method Although A450 increases i Test sample when NAMPT enzyme activity is in the sample the increase in A450 is not obsefyed in No enzyme control and No Nicotinamide control Test Positive Ne Ko 4 Assay reagents sample otitro l enzyme Nicotinamide control control D 10X NAMPT assay buffer 10 uL 10 uL 10 uL 10 uL Q 10X Nicotinamide 10 uL 10 uL 10 uL 3 10X PRPP 10 uL 10 uL 10 uL 10 uL D 10X ATP 10 pL 10 pL 10 mL 10 uL recombinant NMNATI 2 uL 2 uL 2uL 2 uL 3 2 Step Assay Buffer II see page 6 20 uL 20 uL 20 pL 20 uL dH20 28 pL 36 uL 38 uL 38 uL Your enzyme sample Test sample 10 uL 10 uL recombinant NAMPT 2 uh 1 Following the above table add all reagents D TOX NAMPT Assay Buffer to dH2O to each well of microplate and mix well Next initiate reaction byadding 10 uL of Your enzyme sample or 2 uL of recombinant NAMPT to
6. AMPT Assay Procedures page 7 Cat CY 1251 15 Version 121015 4 NAMPT Colorimetric Assay Kit v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results 8 E N wt 5 p o 4 0 500 1000 1500 2000 2500 Nampt ng Fig 2 Time course of NAMPT activity in rooming ur Ming 1 Step Method 3 5 S 0 ng 78 ng S gm amp 313 ng E 1250 ng 2 5 2500 ng 2 0 wn 15 1 0 0 5 0 0 0 10 20 30 40 50 60 Reaction time min C CY 1251 16 Version 121015 NAMPT Colorimetric Assay Kit v yclex User s Manual AN For Research Use Only Not for use in diagnostic procedures Fig 3 Effect of FK866 on recombinant NAMPT activity 120 LLL LLL 100 80 60 of control 40 20 0 ii iiri Lo irrt Lo pILILI Lo a a 0 1 10 100 1000 FK866 nM V Fig 4 Measurement of endogenous NAMPT activi mmunoprecipitate using anti Human NAMPT Mouse Monoclonal Antibody CY M1035 fro racts of three cell lines 0 006 Raji K562 HL60 0 005 0 004 0 003 0 002 Activity A450 min 0 001 SIR SSAAAAAg SSSAAAAAg SSAAAAg SRA SSE RRR RH SSSeeeee eS ERI KERRI KERRI KER Rh 0 000 Isotypic Anti Isotypic Anti Isotypic Anti mouse Nampt mouse Nampt mo
7. L 10X Nicotinamide 10 uL 3 I0X PRPP 10 pL QD 10X ATP 10 pL 8 recombinant NMNATI 2 uL 50X WST 1 2 uL 50X ADH 2 uL 9 50X Diaphorase 2 uL 10X EtOH 10 pL dH20 2 uL Total 60 uL Cat CY 1251 5 Version 121015 NAMPT Colorimetric Assay Kit P es yclex User s Manual For Research Use Only Not for use in diagnostic procedures 2 2 Steps Assay Buffer I Quantity Required 90 uL assay Mix following reagents and put in 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 D 10X NAMPT assay buffer 10 uL 10X Nicotinamide 10 uL 8 10X PRPP 10 uL Qy 10X ATP 10 pL recombinant NMNATI 2 uL dH20 48 u Total 3 2 Step Assay Buffer II Quantity Required 20 uL assa Mix following reagents and put in ice This 2 Step Assay Buffer II should be used within 30 after prepared Discard any unused 2 Step Assay Buffer II after use Assay reagents Volume 50X WST 1 2 uL 50X ADH Q 2 uL 9 50X Diaphor 2 uL 10X 10 uL d 4 uL Q otal 20 uL Q C CY 1251 6 Version 121015 H9 NAMPT Colorimetric Assay Kit e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures NAMPT Assay Procedures 1 1 Step Method Assay reagents Test Positive No enzyme sample control control
8. Your enzyme sample Test sample 10 pL recombinant NAMPT 2u 4 dH20 30 uL 38 pL 40 uL 1 1 Step Assay Buffer see page 5 60 pL 60 uL 60 uL 1 Following the above table add 10 uL of Your enzyme sample or 2 uL of recombinant NAMPT and dH5O to the well of microplate and mix well Next initiate r action by adding 60 uL 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 min at 5 min intervals using microtiter plate reader Measure and calculate the rate of reaction while the reaction velocity remains constant 2 2 Step Method 1st reaction Conversion of nicotinamide to NAD Assay pagent Test Positive No enzyme sample control control Your enzyme sample Test sample 1QuL recombinant NAMPT fP L o Asb d dH20 8 pL 10 uL 2 2 Step Assay Buffer I see page 6 90 uL 90 uL 90 uL 1 Following the above table add 20 pW of Your enzyme sample or 2 uL of recombinant NAMPT and dEH5O to eachewell of the microplate Finally initiate reaction by adding 90 uL of 2 2 Step Assay Buffer I to each well and mix thoroughly to initiate reaction Incubate at 30 C for 60 min 2nd reaction Measurement of NAD 2 Add 20 uL of 43 2 Step Assay Buffer II see page 6 to each well of the microplate and mix thoroughly In ubate at 30 C 3 Monitor th absorbance at 450 nm for 30 min at 5 min i
9. ble to directly detect NAMP T activity in crude cell extract in which NAD concentration is relatively high Applications for this kit include 1 Screening inhibitors r activators of NAMPT 2 Detecting the effectssof pharmacological agents on NAMPT This assay kit is foryresearch use only and not for use in diagnostic or therapeutic procedures Storage Upon rec ipttote all components at 70 C e Don t exposeme agents to excessive light Cat CY 1251 1 Version 121015 NAMPT Colorimetric Assay Kit jde Drs Mi GA y gt X ser s Manu For Research Use Only Not for use in diagnostic procedures Introduction Nicotinamide phosphoribosyltransferase NAMPT also known as pre B cell colony enhancing faetor is the rate limiting enzyme that converts nicotinamide to nicotinamide mononucleotide NMN from nicotinamide in the salvage pathway of NAD biosynthesis in mammals Nicotinamide mononucleotide adenylyltransferase 1 NMNATI converts NMN to NAD The expression of NAMPT is upregulated during activation of immune cells such as monocytes macrophages dendritic cells T and B cells as well as in amniotic epithelial cells upon stimulation with several inflammatory cytokines NAMPT specific inhibitor FK866 was found to deplete intracellular NAD content resulting in apoptotic cell death in many cancer cell lines without any DNA damaging effect Recently Nakahata K et al demonstrated that NAMPT is required to modulate circad
10. control 9 FK866 20uM A450 QR wa Reaction time min C CY 1251 19 Version 121015 NAMPT Colorimetric Assay Kit es yclex User s Manual A For Research Use Only Not for use in diagnostic procedures Fig 9 Typical data of 2 Step Method according to section 3 Special considerations when screeni inhibitors or activators at page 9 2 Step method Rc MM ee ee C Positive control 9 No enzyme control Vehicle control 2 5 FK866 20uM lt Reaction time min Fig 10 Requirement of each assay component for ent of NAMPT activity 120 100 r 80 r 60 2 b l e 40 20 r 0 HEL a om S gt zZz 7 2 Z 9 gt z SEE TERFZS 25 lor BOSE z 2 B 7 cav T g T C CY 1251 20 Version 121015 NAMPT Colorimetric Assay Kit t ycLex User s Manual For Research Use Only Not for use in diagnostic procedures o CA oo Revollo J R Grimm A A Imai S 2004 J Biol Chem 279 50764 50763 2004 References Rongvaux A Shea RJ Mulks MH Gigot D Urbain J et al 2002 Eur J Immunol 32 3225 32 Iqbal J Zaidi M 2006 Biochem Biophys Res Commun 342 1312 1318 Nau GJ Richmond JF Schlesinger A Jennings EG Lander ES et al 2002 Proc Natl i 99 1503 1508 Huang Q Liu D Majewski P Schulte LC Korn JM et al 2001 Science 294 87
11. ian gene expression and circadian oscillation of NAD Principle of the Assay Since it is very simple to measure and it can be performed at a low price the measurement of NAMPT 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 ias pbecome widespread NAMPT activity measurement which could not be made by the conventional method is now possible with the CycLex NAMPT Colorimetric Assay Kit using the same equipm nt This new method of measurement shall dramatically raise the efficiency of inhibitor screening and biochemical analysis of this enzyme Measuring Principle of The CycLex NAMPT Colorimetric Assay Kit Nicotinamide PRPP phosphoribosyl pyrophosphate NAMPT nicotinamide phospheribosyltransferase NMN nicotinamide mononucleotide NMNAT nicotinami mononucleotide adenylyltransferase NAD nicotinamide adenine dinucleotide ADH Alcohol d hyd egenase I Diaphorase NADH c 7 NADH WST 1 WST 1 formazan Measure OD at 450 nm Cat CY 1251 2 Version 121015 A NAMPT Colorimetric Assay Kit Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Provided Each kit contains moix Lak 200 uLx ig IPC 50X ADH alcohol dehydrogenase 200 uEX1 70 C 9 50X Diaphorase 200 uL x 1 70 C 10X E
12. 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 followed accurately such results indicate a need for multi channel pipettor maintenance Reagent Stability All of the reagents included in the CycLex Research Product GycLex NAMPT Assay kit have been tested for stability Reagents should not be used beyofid the stated expiration date All kit components should be stored at 70 C Cat CY 1251 14 Versionft 121015 oe NAMPT Colorimetric Assay Kit e Vclex 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 and 80 g NaCl Adjust pH to 7 6 with HCl 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 conta
13. ining test compound for desired time 2 To harvest cells under non denaturing conditions remove media and finse cells once with ice cold PBS 3 Remove PBS and add 0 5 mL 1X ice cold Cell Lysis Buffer to each plate 10 cm dish and incubate the plate on ice for 5 minutes 4 Scrape cells off the plate and transfer to microcehtrifuge tubes 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 lysate can be 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 pre clearance 2 Microcentrifuge for 30 secofids at 4C Take the supernatant and transfer to a new tube o2 Add and add 1 2 ug of anticNAMPT antibody which can be used for immunoprecipitation incubate with gentle focking for 2 hrs or overnight at 4 C CycLex recommend Anti Human NAMPT Mouse Monoclonal Antibody Cat CY M1035 4 Add protein Aagarose beads 20 ul of 50 bead slurry Incubate with gentle rocking for 1 3 hours at 4 C Nn Microcentrifugesfor 10 seconds at 4 C Wash the beads twice with 500 ul of 1X Cell Lysis Buffer subSequently twice with NAMPT assay buffer Keep on ice during washes 6 Resuspend the beads with 20 ul NAMPT Assay Buffer and measure NAMPT activity according to N
14. ity remains constant When there is an inhibitory effe A450 will not increase chemical on NAD NADH recycling reaction C CY 1251 10 Version 121015 NAMPT Colorimetric Assay Kit A yclex User s Manual For Research Use Only Not for use in diagnostic procedures II For NAMPT activity assay in an immunoprecipitate Since NAD level in cells is relatively high around several hundreds micromolar concentrati NAD might mix easily in purified NAMPT from various cells or an immunoprecipitate using the specific antibody against NAMPT Such contaminated NAD in the test sample causes false po ou result by initiating NAD NADH cycling enzyme reaction If there is such a possibility please c the experiment of NAD NADH recycling assay in the following Table Assay reagents Test sample NAD contro Your enzyme sample Test sample 10 uL E N mew O dH O 70 uL u 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 n 10 uL of NAD 5 uM and dH20 to each well of microplate and mix well Next initiate reaction by adding 20 uL of 3 2 Step Assay Buffer II to each well and mix thoroughly 2 Monitor the absorbance at 450 nm for 60 min at 5 als using microtiter plate reader Measure and calculate the rate of reaction while the reaction velocity remains constant When there is contaminated NAD in the test A
15. ntervals using microtiter plate reader Measure and e lculate the rate of reaction while the reaction velocity remains constant Cat CY 1251 T Version 121015 Pat YCLEX NAMPT Colorimetric Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures 3 Special considerations when screening inhibitors or activators In order to estimate the inhibitory effect on NAMPT enzyme activity in the test chemicals correctly it is necessary to conduct the control experiment of Vehicle control at least once for every experiment and Inhibitor control at least once for the first experiment in addition to Test chemical as indicated in the following table When test chemicals cause an inhibitory effect on NAMPT enzyme activity the level of A450 is weakened as compared with Vehicle control The high level of A450 is not observed in Inhibitor control usually A450 0 2 I 1 Step Method Assay reagents an al Vehicle control or recombinant NAMPT 2 uL 2 uL 2 uL 50X Inhibitor candidate 2 uL E Vehicle for Inhibitor candidate 2mby Jh iE FKS660mM 44 9 cw dH20 36 uL 36 uL 36 uL 1 1 Step Assay Buffer 60 uL 60 uL 60 uL FK866 1 mM Not provided in this kit See pag 3 1 Following the above table add 2 uL of recombinant NAMPT dH20O and 50X Inhibitor candidate or Vehicle for Inhibitor candidate or FK866 1 mM
16. oe NAMPT Colorimetric Assay Kit aA Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Quantitative test kit for nicotinamide phosphoribosyltransferase activity CycLex NAMPT Colorimetric Assay Kit For 100 assays Cat CY 1251 Intended T T 1 s lez 1 Lor rge raliter NN ME 2 Principle of the Assay epe 2 Materials Provided ieceee ont 3 Materials Required but not Provided 3 Dio ANIONS iat niboI ain rai ti PIDE eU E biRpEQU 4 Detailed Protocol sassceigessdeamanssassesiacriqinals 5 12 Evaluation of Results ere rena 13 CAUCUS ssc cuscatc Eu are tre t assce rU Da diac ERU RE U IUD 13 Assay Characteristics seee 14 TroubleshOOBBp assess stir or bla atia petit 14 Reagent Sia DILLY cs jusiinasssnincuesagniadeatcansaane 14 Protocol for immunoprecipitation 15 Example of Test Results 16 20 imn 21 Related Products vecsssscaacccssasstaatearancesssanysasates 22 Intended Use The CycLex Research Product CycLex NAMPT Colorimetric Assay Kit detects nicotinamide phosphoribosyltransferase NAMPT activity in recombinant NAMPT or endogenous NAMPT immunoprecipitated from cell extract Primarily the CycLex Research Product CycLex NAMPT Colorimetric Assay Kit is designedefor the rapid and sensitive evaluation of NAMPT inhibitors or activators using recombinant NAMPT Since this kit is based on NAD detection system it is not possi
17. rate of reaction while the reaction velocity remains c o 9 Versions 121015 Py NAMPT Colorimetric Assay Kit t ycLex User s Manual For Research Use Only Not for use in diagnostic procedures 4 Assay control I For Inhibitor screening Since the CycLex NAMPT Colorimetric Assay Kit can measure the NAMPT enzyme activity enzyme coupled reaction in which four enzymes involved including NAMPT NMNATI A diaphorase when test chemicals that have an inhibitory effect on one of these enzymes be reduced If there is such a possibility please carry out the experiment of NAD N assay in the following Table and NMNATI assay using CycLex NMNATI Colorimet CY 1252 to ascertain which enzyme is target of the test chemical Test i Assay reagents diameal Vehicle Qu NAD 5 uM 10 pL 1 50X Inhibitor candidate 2uL Vehicle for Inhibitor n _ candidate B 000000 LR dH20 68 pL pL 3 2 Step Assay Buffer II 20 uL 20 uL NAD Not provided in this kit See page 20 and 50X Inhibitor candidate or p and mix well Next initiate reaction by ell and mix thoroughly Incubate at 30 C 1 Following the above table add 10 uL of NA Vehicle for Inhibitor candidate to each wel adding 20 uL of 3 2 Step Assay Buffer II t 2 Monitor the absorbance at 450 nm for 60 min at min intervals using microtiter plate reader Measure and calculate the rate of O reaction veloc
18. recommends Anti Human NAMPT Mouse Monoclonal Antibody Cat CY M1035 2 Contaminated NAD in the test sample causespfalse positive result by initiating NAD NADH cycling enzyme reaction Please confirm no NAD in the test sample according to 4 Assay control page 11 or 12 3 Duplicate measurement is strongly recommended for accurate measurement of NAMPT activity 4 Although we suggest to conduct experiments as outlined in Protocol for immunoprecipitation at page 15 the optimal experimental conditions will vary depending on the parameters being investigated and must besdetermined by the individual user For research use only not for use in diagnostic or therapeutic procedures Cat CY 1251 13 Version 121015 H9 NAMPT Colorimetric Assay Kit e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Characteristics The CycLex Research Product CycLex NAMPT Assay kit has been shown to detect the activitysof nicotinamide phosphoribosyltransferase activity in recombinant NAMPT or an immunoprecipitate using the specific antibody against NAMPT The assay shows good linearity of sample response The assay may be used to follow the purification of NAMPT Troubleshooting 1 When test chemicals have an inhibitory effect on NMNATI ADH or diaphorase precise inhibitory effect on NAMPT enzyme activity cannot be measured 2 The recombinant NAMPT should be run in duplicate using the protocol des ribed
19. tOH ImLx1 70 C Instruction manual T room temp Phosphoribosyl pyrophosphate Human NAMPT nicotinamide phosphoribosyltransferase expressed in F coil x x Human NMNATI nicotinamide mononucleotide adenylyltransferase 1 expressed in E coil Materials Required but not Provided e Microplate for ELISA Plate reader capable of measuring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 mm can also be used The plate can also be read at a single wavelength of 450 nm which will giv a Somewhat higher reading Pipettors 2 20 uL 20 200 uL and 2001000 pF precision pipettors with disposable tips Multi channel pipette Microplate shaker Deionized water of the highest quality 500 or 1000 mL graduated cylinder Reagent reservoirs FK866 APO866 FK866 is available from Axon Medchem Cat Axon 1279 or Cayman Cat 13287 Make 1 mM stock solution in DMSO Optional NAD NAD B Ni otinamide adenine dinucleotide hydrate is from Sigma Cat N7004 Prepare fleshly 5 uM solution in H20 from 1 mM stock solution in H20 Discard any unused 5 uM NAD Optional Cat CY 1251 3 Version 121015 Fg NAMPT Colorimetric Assay Kit es yclex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions Please thaw all reagents in crushed ice before use e Please avoid freezing and thawing cycle of recombinant NAMPT recombinant
20. to each well of microplate and mix well Next initiate reaction by adding 60 uL of 1 1 Step Assay Buffer to each well and mix thoroughly Incubate at 30 C Measure and calculate the rate of reactionfwhile the reaction velocity remains constant II 2 Step Method Monitor the absorbance at 450 nmgfor 60 min at 5 min intervals using microtiter plate reader Cati Assay reagents Test Vehicle control Inpipitor chemical control recombinant NAMPT 2 uL 2 uL 2 uL 50X Inhibitor candidate 2 uL m V ehidefgFInhibito t jq Lo candidate 70 TES FK866 1 mM 2uL dILO 6 uL 6 uL 6 uL 2 2 Step Assay Buffer I 90 uL 90 uL 90 uL CY 1251 8 Version 121015 Fae NAMPT Colorimetric Assay Kit yclex User s Manual For Research Use Only Not for use in diagnostic procedures FK866 1 mM Not provided in this kit See page 3 1 Following the above table add 2 uL of recombinant NAMPT dH O and 50X Inhibi candidate or Vehicle for Inhibitor candidate or FK866 1 mM to each well of the micro Next initiate reaction by adding 90 uL of 2 2 Step Assay Buffer I to each well a thoroughly to initiate reaction Incubate at 30 C for 60 min phy 2 Add 20 uL of 3 2 Step Assay Buffer II to each well of the microplate and Incubate at 30 C 3 Monitor the absorbance at 450 nm for 30 min at 5 min intervals using mi plate reader Measure and calculate the
21. use Nampt IgG mAb IgG mAb IgG mAb control control control C CY 1251 17 Version 121015 NAMPT Colorimetric Assay Kit 1 Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 5 Measurement of NAMPT activity in the immunoprecipitate from cell extract of 293T cells whi had been transfected with NAMPT expression plasmid DNA 0 020 a 0 010 0 005 0 000 Anti Flag tag Anti Nampt mAb Activity A450 min Fig 6 Effect of FK866 on NAMPT activity in th anti Human NAMPT Mouse Monoclonal Antibo unoprecipitate from Raji cells extract using Y M1035 120 100 80 E 9 60 8 L 40 I i 0 500 FK866 nM C CY 1251 18 Versionft 121015 NAMPT Colorimetric Assay Kit g ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 7 Effect of FK866 on NAMPT activity in activity in the immunoprecipitate using anti Flag antib from cell extract of 293T cells which had been transfected with Flag NAMPT expression plasmi DNA 120 i Q 80 Qj 6 7 z 2 L 40 20 0 0 5 50 500 FK866 nM Fig 8 Typical data of 1 Step Method according n 3 Special considerations when screening inhibitors or activators at page 8 1 Step method 3 PRR RSA BE SE ea ee ee ee aie e Positive control e No enzyme control 25 L Vehicle
22. y 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 1251 22 Version 121015
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