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1. User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay CycLex SIRTI Sir2 Deacetylase Fluorometric Assay Kit measures the activity of SIRTI Sir2 b basic principle of changing a SIRTI Sir2 reaction into the activity of the protease In order to the enzyme activity of SIRTI Sir2 which is the NAD dependent Histone deacetylase and this kit is designed so that the activity of NAD dependent Histone deacetylase can be existence of Trichostatin A which is the powerful inhibitor of HDACs In this kit fluorophore and quencher are coupled to amino terminal and carboxyl termina peptide respectively and before reaction of deacetylase the fluorescence cannot be emitted However if SIRT1 Sir2 performs deacetylation substrate peptide will become cut by the actio rotease added simultaneously quencher will separate from fluorophore and fluorescence will be Deacetylase enzyme activity is measured by measuring this fluorescence intensity Since it is very simple to measure and it can be performed at a low pri e th Measurement of SIRTI Sir2 activity in most laboratories is possible if they are equipped wit escent reader for microtiter plates Considering that the use of fully automatic apparatus to has become widespread SIRTI Sir2 activity measurement which conventional method is now possible with CycLex SIRT1 Sir2 Deace using the same equipment This new method of measurement should2. 6 Poor duplicates ing s included in the CycLex Research Product CycLex SIRTI Sir2 Deacetylase ay Kit have been tested for stability Reagents should not be used beyond the stated Jpon receipt store the Recombinant SIRT1 at 70 C all other kit reagents should Version 130130 CY 1151 4 SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit t User s Manual For Research Use Only Not for use in diagnostic procedures Sample Preparation Numerous extraction and purification methods can be used to isolate SIRTI The following prot have been shown to work with a number of different cells and enzyme sources and are provi examples of suitable methods Crude samples can frequently be used without dilution whi concentrated or highly purified SIRT1 should be diluted It is strongly advised that t perform an initial experiment to determine the proper dilution to be used in subsequent This need not be any more than a single time point assay using serial dilutions of the crude lysate or sample fraction taken prior to a purification step All sample preparation should be performed at 4 C and recovered fractions should be kept at 70 C to prevent loss of enzymatic activi Buffers J Lysis Buffer Sucrose cushion Extraction buffer 10 mM Tris HCl pH7 5 30 Sucrose 50 mM Hepes KOH pH 10 mM NaCl 10 mM Tris HCl pH7 5 7 5 15 mM MgCl 10 mM NaCl 420 mM NaCl 250 mM Sucrose 3 mM MgCl 0 5 mM EDTA Na 0 5 NP 4
3. Troubleshooting j When chemicals that have an inhibitory effect on lysylendopeptida mixed in a crude SIRTI Sir2 fraction purified from various cells or the immunopreci us specific antibody against SIRTI1 Sir2 or other proteins precise SIRTI Sir2 enzy cannot be measured Since the protease inhibitors used in the usual protein purificati s inhibit lysylendopeptidase activity strongly please avoid the use of any protease inhibi e protein purification process test chemicals have an inhibitory effect on on lysylendopeptidase 2 Final fluorescence intensity will not increase b SIRTI Sir2 and also when there is an inhibitory e 3 If the test reagents themselves emit fl ence at excitation wavelength 340 360 nm and fluorescence wavelength 440 460 nm t tory effect of the test assay cannot be evaluated correctly 4 The recombinant SIRT1 should be run duplicate using the protocol described in the Detailed Protocol Incubation times or t S significantly different from those specified may give erroneous results 5 The reaction curve is nearly i ine if the kinetics of the assay is of the first order Variations in the protocol can lead to y of the curve as can assay kinetics that are other than first order For a non linear curve point nt or quadratic curve fit methods should be used curate dispensing If all instructions in the Detailed Protocol were esults indicate a need for multi channel pipettor maintenance
4. 5 000 4 000 3 000 2 000 1 000 F355 F460 x 10 counts 10 000 Sir2 substrate CycLex Sir2 Assay kit e crude HDAC e Recombinant SIRT1 bh F355 F460 x 10 counts 8 crude HDAC e Recombinant SIRTI 20 30 40 50 60 Time min Fig 6 Stability of Fluorescence Inten op the Reaction fili F355 F460 x 10 counts SIRTI 250ng 8 SIRTI 93ng k SIRTI 46 5ng SIRTI 15 625ng Time min C CY 1151 15 Versions 130130 SIRTI Sir2 Deacetylase Fluorometric Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures Fig 7 Measurement of 293T cell endogenous SIRTI activity in an immunoprecipitate using anti SI antibody CY P1016 400 350 x B9 gt em 259r S scm gt F 150 a 100 sm 0 Q _ BE Bl ET BE EB 35 B 222 22 RE Rz 5 2 2 z C CY 1151 16 Version 130130 FS SIRTI Sir2 Deacetylase Fluorometric Assay Kit es User s Manual For Research Use Only Not for use in diagnostic procedures References Imai S et al Nature 403 795 800 2000 2 Landry J et al Proc Natl Acad Sci U S A 97 5807 5811 2000 Smith JS et al Proc Natl Acad Sci U S 97
5. inhibitor screening and biochemical analysis of these enzymes easure rescence intensity ot be made by the luorometric Assay Kit ally raise the efficiency of Measuring Principle of The CycLex SIRT1 Sir2 Deacetylase Fluorometric Assay Kit fluorophore X X X Lys Ac X X cher lt Deacetylase fluorophore X X X Lys X er Lysylendopeptidase fluorophore X X XJLy quencher Measur fluorescence intensity Note This me iple and kit are covered under CycLex s patents U S Pa 778 and No 7256013 Europea ent No 1243658 Japa No 4267043 Can tent No 2392711 CY 1151 3 Versions 130130 4 SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit t User s Manual For Research Use Only Not for use diagnostic procedures Materials Provided Each kit contains Materials Quantity Storage Al D 10X SIRTI assay buffer 1mlx2 Below 20 50X Fluoro Substrate Peptide 1 mM 100u 1x1 Below 20 50X Fluoro Deacetylated Peptide 1 mM 50ulx1 Below 20 Lysylendopeptidase 100 mA U ml 50u 1x1 Belows20 C 100X NAD 20 mM 100u 1x1 Bel w 20 200X Trichostatin A 0 2 mM 100u 1x1 Below220 C Recombinant SIRT1 200n 1x1 L 70 100X Stop solution 100 u 1x Below 20 C 9 Instruction manual 1 e oom temp Materials Required but not Provided Microplate for fluorometer Microplate rea
6. 0 0 1 mM EGTA 0 1 mM EGTA 10 glycerol Procedure Isolation of Nuclei Suspend 1x 10 cells 100 mm dish sub conflu into 1ml of Lysis buffer Vortex for 10 second Keep on ice for 15 min Spin the cells through 4 ml of sucrose 1 300 g for 10 min at 4 0 T Discard the supernatant Wash the nuclei pellet once with c ris HCI pH7 5 10 mM NaCl Extraction of Nuclei Suspend the isolated nuclei i uL of extraction buffer Sonicate for 30 seconds Stand on ice for 30 min c f g at 20 000 x g for Take supernatant t Clear extract by Bradford method or equivalent tract at 70 C until use Determine protein kind of protease inhibitor SD OP Dp dor Store the crude C CY 1151 11 Version 130130 SIRTI Sir2 Deacetylase Fluorometric Assay Kit e User s Manual For Research Use Only Not for use in diagnostic procedures SIRTI activity inan immunoprecipitate Immunoprecipitation Followed by Measuring SITR1 Activity Protocol Solutions and Reagents Note Prepare solutions with Milli Q or equivalently purified water Cell Lysis Buffer 1X 20 mM Tris HCl pH 7 5 250 mM NaCl 1 mM EDTA 1 Triton X 100 1 mM DTT hours at 4 C spin down Wash the pellet twice with PBS Resuspend bea Can be stored for 2 weeks at 4 C Protein A Agarose Beads Add 5 mL of 1X PBS to 1 5 g of Protein A Preparing Cell Lysates 1 Aspirate
7. 6658 6663 2000 4 Vaziri H et al Cell 107 149 159 2001 5 Luo J et al Cell 107 137 148 2001 Q 6 Langley E et al EMBO J 21 2383 2396 2002 7 Smith J Trends Cell Biol 12 404 2002 2 8 Grozinger CM and Schreiber SL Chem Biol 9 3 16 2002 9 Sereno et al Antimicrob Agents Chemother 49 808 812 200 10 Nicola Ferrara et al ReJuvenation Research 11 139 150 2 11 Rameshwar U et al Chemical Biology amp Drug Design 71 506 2008 12 Fan Lan et al J Biol Chem 283 27628 2008 13 Joana TAVARES et al Biochemical Journal 415 86 2008 14 Yue Zhao et al Mol Cell Biol Dec 2008 10 1128 MCB 02123 07 Q S 1151 17 Version 130130 4 SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit t User s Manual For Research Use Only Not for use in diagnostic procedures Related Products CycLex Cellular Histone Acetylation Assay Kit Cat CY 1140 CycLex HDACS Deacetylase Fluorometric Assay Kit Cat CY 1150 CycLex HDACS Deacetylase Fluorometric Assay Kit Cat CY 1158 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 Anti Acetylated Histone p53 K382 Mouse Monoclonal Antibody Cat CY M1 Anti Histone Deacetylase 1 HDAC1 Rabbit Polyclonal Antibody C
8. SIRTI Sir2 Deacetylase Fluorometric Assay Kit e User s Manual For Research Use Only Not for use in diagnostic procedures Quantitative test kit for NAD dependent histone deacetylase activity CycLex SIRTI Sir2 Deacetylase Fluorometric Assay Kit Q Cat CY 1151 Intended Use n aaa 1 Inr M 1 ICE M UN 2 Principle of the Assay 3 Materials Provided 4 Materials Required but not Provided 4 Pr c80t00608S ET Detailed Protocol Caufi nS anan a Troubleshooting Reagent Stability Sample Preparation SIRTI activity in an immunoprecipitate Example of Test Results References ioi t ete ete Hooded Related Products Intended Use Qi CycLex Research Product ir2 Deacetylase Fluorometric Assay Kit detects SIRTI Sir2 activity in lysates Primarily he CycLex Research Product CycLex SIRT1 Sir2 Deacetylase Fluorometric Assay Kit is ed for the rapid and sensitive evaluation of SIRTI Sir2 inhibitors or activators using crud ir2 fraction or purified SIRTI Sir2 Additionally any cultured primary cell cell line or tissu mogenate can be assay
9. SIRTI assay buffer mixed with 9 ml of dH O and store SIRT1 assay buffer at 4 C 2 X20 diluted Lysylendopeptidase 5 mAU ml Quantity required 2 5 uL assay Dilute the Lysylendopeptidase 1 20 with 1 1 T1 assay buffer 3 10X NAD 2 mM NAD Quantity required 5 uL assay Dilute the 100 NAD 1 10 with 1 1X ssay buffer 4 10X TSA 10 uM Quantity required 5 uL assay Dilute the 200 Trichostatin ith 1 1X SIRTI assay buffer 5 10X Test sample 10X fin Quantity Required 5 uL assa Dilute Test sample to 10X fi 6 X5 diluted recom Quantity Required 1 Dilute the SIRTI 1 5 with 1 1X SIRTI assay buffer Note Use 6 iluted recombinant SIRT1 within the same day they are prepared tion e g a candidate of inhibitor or activator ired concentration with 1 1X SIRTI assay buffer 7 2X Stop s Quantity requi uL assay Dilute thes 100X Stop solution 1 50 with dH O CY 1151 6 Version 130130 Fg SIRTI Sir2 Deacetylase Fluorometric Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures 8 SIRTI reaction buffer Final 50 mM Tris HCl pH 8 8 0 5 mM DTT 0 25 mAU Lysylendopeptidase 1 uM Trichostatin A and 20 uM Fluoro Substrate Peptide in 50 uL of reactio mixture Quantity Required 30 uL assay in case of adding 10 uL of 6 X5 diluted recombinant SIRT1 uL of 5 10X Test sample M
10. ant appropriate time to stop the reaction e reader with excitation at 340 nm and emissi uL of 7 2X Stop solution to each well at re fluorescence intensity in a microplate fluorescence at 440 nm 3 The difference in fluorescence jin control indicates the SIRT1 Sir 1 ity between Test sample control and No enzyme Note 1 It is possible to c Q of assay reagents and sample as far as it sets up the final concentration of reagents in a reaction mixture as indicated as below Note 2 15 strongly recommended Version 130130 C CY 1151 8 SIRTI Sir2 Deacetylase Fluorometric Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures Vclex 2 Assay control 1 When the chemicals that have an inhibitory effect on lysylendopeptidase come to be mixe SIRTI Sir2 fraction purified from various cells or the immunoprecipitate using the specific anti against SIRTI Sir2 or other proteins precise SIRT1 Sir2 enzyme activity cannot be measure the protease inhibitors used in the usual protein purification process str lysylendopeptidase activity please avoid using any protease inhibitors during the proces purification If there is such a possibility please carry out the experiment of Positive control Assay control 1 in the following table using Fluoro Deacetylated Peptide to reference When Fluoro Deacetylated Peptide is used fluorescence intensity should inc
11. at CY P Anti Histone Deacetylase 2 HDAC2 Rabbit Polyclonal Antibody Cat CY P Anti Human SIRT1 Rabbit Polyclonal Antibody Cat CY P1016 NAD Dependent Deacetylase SIRT1 Cat CY E1151 NAD Dependent Deacetylase SIRT2 Cati CY E1152 NAD Dependent Deacetylase SIRT3 Cati CY E1153 NAMPT Nicotinamide Phosphoribosyltransferase Cat CY E125 NMNATI Nicotinamide Mononucleotide Adenylyltransferase E1252 Note This product is covered under CycLex s patents U S Patent No 7 033 778 and No 7256013 European Patent No 1243658 Japanese Patent No 4267043 Canadian Patent No 2392711 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex PRODUCED BY Q CycLex Co Ltd e ucts are supplied for research use only CycLex CircuLex products and y not be resold modified for resale or used to manufacture commercial components t ior written approval from CycLex Co Ltd To inquire about licensing for products wit please contact us via email CY 1151 Versions 130130
12. ay be contaminated wit jo ents Do not ingest expose to open wounds or breathe aerosols Wear protective d dispose of biological samples properly C CY 1151 5 Version 130130 4 SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit t User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol Description of assay system CycLex SIRTI Sir2 Deacetylase Fluorometric Assay Kit can measure the enzyme SIRTI Sir2 with a homogeneous method In this method the reaction is initiated and th intensity is measured by mixing simultaneously fluorescence labeled acetylated pept substrate SIRT1 Sir2 trichostatin A NAD and lysylendopeptidase Since the reaction is is necessary to measure fluorescence intensity at regular intervals after the reaction is initiated and to determine reaction velocity Alternatively within a time in which the reaction velocitydsskept constant it is also possible to stop the reaction by adding 2X stop solution and to measure fluorescenceantensity Preparation Method for Assay Reagents Thaw 250X Fluoro Substrate Peptide and 3 50X Fluoro Deacetylated 1 room temperature Stand other reagents in ice to thaw Use them after they thaw completely 1 1X SIRTI assay buffer 50 mM Tris HCl pH 8 8 0 5 mM DT Quantity Required 50 uL assay Dilute the 10X SIRTI assay buffer 1 10 with distilled wate Since this is the base buffer for the assay prepare vial f X
13. ction of p53 is made to strengthen powerfully by using together with DNA damaging reagent it is expected that inhibitor of SIRT1 becomes an effec e icancer drug However the conventional method for measuring SIRTI Sir2 activity is ve 4 plicated and laborious In order to measure SIRTI Sir2 enzyme activity it is necessar pare radioactive acetylated histone as a substrate First cells have to be labeled metabolic radioactivity by adding radioactive acetic acid to the culture medium Second radioactive acet histone has to be purified from the cells Following the reaction it is necessary to extrag eparate the radioactive acetyl group which has been released from acetylated histone using ethy to measure the activity of the enzyme based on the radioactivity Although a method for measuring the activity of deacetylase wi was reported in recent years owing to the use of fluorescent labe ted lysine as a substrate the reaction product must be separated from the intact substrate a escent intensity measured by reverse phase HPLC As mentioned above these measurgme stems are difficult to adapt for processing many samples under a variety of conditions beca eir complicated operation Thus a simple system for biochemical analysis as well as for nhibiter ser ening without the use of radioactive substances is preferred se of radioactive substances C CY 1151 2 Version 130130 4 SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit t
14. ding fluorometer capable of excitation at awavelength in the range 340 360 nm and detection of emitted light in the range 440 460 nm Pipettors 2 20 uL 20 200 uL and 200 1000 uL 1 pipettors with disposable tips multi channel pipette Microplate shaker Deionized water of the highest quality 500 or 1000 mL graduated cylinder Reagent reservoirs C CY 1151 4 Version 130130 FS SIRTI Sir2 Deacetylase Fluorometric Assay Kit es User s Manual For Research Use Only Not for use in diagnostic procedures Precautions e Please thaw 2 50 Fluoro Substrate Peptide and 3 50X Fluoro Deacetylated Peptide at temperature before use Then thaw the other reagents in ice and use after they are completely thawed en Please avoid mixing of protease inhibitors such as PMSF or alkyl amine in the sample that will be measured SIRTI Sir2 activity e Please avoid repeated freezing and thawing of the Recombinant SIRT1 in this possibility that the enzyme activity may be inactivated Aliquot to 10 20 uL and store at Do not use kit components beyond the indicated kit expiration date Qy e Rinse all detergent residue from glassware 2 Use deionized water of the highest quality Q Do not mix reagents from different kits Do not mouth pipet or ingest any of the reagents Do not smoke eat or drink when performing the assay handled S ere samples or reagents are Biological samples m
15. ecombinant SIRTI activity F355 F460 x10 counts SIRT1 ng Fig 2 Time course of SIRT 1 substrate A recombinant SIRT1 10 000 p 8 000 2 2 6 000 4 SIRT full 7 8125ng e 4 000 c SIRTIfull 15 625ng SIRTI full 31 25ng 2 000 D SIRTIfull 62 5ng SIRTIfull 125ng 0 0 10 20 30 Time min C CY 1151 13 Version 130130 Not for use in diagnostic procedures SIRTI Sir2 Deacetylase Fluorometric Assay Kit User s Manual For Research Use Only Fig 3 Effect of Trichostatin A and NAD on recombinant SIRTI activity yclex a lt 4 E Z E 2 e a 4 SRE fon 55 gt c Q I Bu 1 p D L 2 8 I 2 5 lt E 5 3 _ E E gt 2 22 J q S w lt w w lt w t en e a N 2 S e e e a Ste c om 2 2 2 2 92 s E co co o co o o lt o o c sjunoo x 5 01 O9PA SSCA A S o C 3 S gt bn 130130 Version 14 CY 1151 User s Manual Fig 5 Substrate Preference of HDAC and 51 1 SIRTI Sir2 Deacetylase Fluorometric Assay Kit For Research Use Only Not for use in diagnostic procedures 9 000 8 000 7 000 6 000
16. ed for SIRT1 Sir2 activity with the CycLex Research Product 1 Sir2 Deacetylase Fluorometric Assay Kit if the appropriate istuse antibody direct against SIRTI d for immunoprecipitation Applications for this kit in lude 1 Monitoring the purific 2 Screening inhibit 3 Detecting t earch use only and not for use in diagnostic or therapeutic procedures of SIRT1 Sir2 ators of SIRTI Sir2 harmacological agents on SIRT1 Sir2 ore recombinant SIRTI at 70 C and all other components below 20 C reagents to excessive light CY 1151 Versions 130130 4 SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures Introduction Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in bu yeast and nematode In 2000 it was reported that the yeast Sir2 protein is a NAD dependent histon deacetylase that plays a critical role in transcriptional silencing genome stability and longevi homologue of Sir2 SIRTI also functions as a NAD dependent p53 deacetylase NAD dependent histone deacetylase SIRT1 was shown to regulate the activity of the mor suppressor and inhibits apoptosis These results have significant implications regarding an ant role for in modulating the sensitivity of cells in p53 dependent apoptotic response and the possible effect in cancer therapy Since the fun
17. ix following reagents 30 uL 1 assay Q D10X SIRTI assay buffer 5uL 1 2 50 Fluoro Substrate Peptide l uL 3 2 X20 diluted Lysylendopeptidase 2 5uL 4 4 10X TSA 5 Q 5 dH O 16 Total 3 C CY 1151 7 Version 130130 SIRTI Sir2 Assay Procedures SIRTI Sir2 Deacetylase Fluorometric Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures 1 Assay method No enzyme Test Assay reagents Test sample sample control control 8 SIRTI reaction buffer 30 uL 30 uL 30 uL 3 10X NAD 5uL 5uL 5 5 10 Test sample 5 dH O 15 pL 5uL 10 uL 6 X5 diluted recombinant SIRT1 10 pL _ 10 uL or Your enzyme sample he microplate Finally 1 Following the above table add Reagent 3 5 and 8 or dH5O to each your enzyme sample to ervals using microtiter plate Measure and calculate the 2 Read fluorescence intensity for 30 to 60 minutes at 1 to inute fluorometer with excitation at 340 360 nm and emission rate of reaction while the reaction velocity remains constant Alternate procedure 1 Following the above table add Reagent 3 5 an or dH5O to each well of the microplate Finally initiate reaction by adding 10 uL of 6 X5 dilute ombinant SIRTI or your enzyme to each well and mixing thoroughly Incubate at roo erature Ca 25 C 2 While the reaction rate is kept const
18. media Treat cells by adding fresh media containing test co 2 To harvest cells under nondenaturing conditions remove media PBS 3 Remove PBS and add 0 5 mL 1X ice cold Cell Lysis Buffer t the plate on ice for 5 minutes 4 Scrape cells off the plate and transfer to microcentrifuge tu Sonicate 4 times for 5 seconds each on ice nn is the cell lysate If necessary lysate can be stor Immunoprecipitation 1 ds Shake 2 me of PBS Microcentrifuge for 10 minutes at 4 C and transfer the su atant to a new tube The supernatant 1 Take 200 uL cell lysate and add anti SIRT1 a dy CY P1016 1 2 ug incubate with gentle rocking for 2 hrs or overnight at 4 C 2 Add protein A agarose beads 20 uL o bead slurry Incubate with gentle rocking for 1 3 hours at 4 C 3 Microcentrifuge for 30 seconds at 4 ellet 3 times with 500 uL of 1X Cell Lysis Buffer and with SIRTI assay buffer 50 is HCl pH 8 8 0 5 mM DTT Keep on ice during washes 4 After immunoprecipitation ad mixture containing Fluoro Substrate peptide solution to protein A agarose beads easure NAD dependent deacetylase activity according to the procedure in CycLex SIRT Q C CY 1151 12 eacetylase Fluorometric Assay Kit Cat CY 1151 Version 130130 4 SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit t yclex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency curve of r
19. rease w there is no SIRTI Sir2 activity in your enzyme sample When there is an inhibitory effect o dopeptidase activity even if there is SIRT1 Sir2 activity in a sample fluorescence intensi t increase 2 Not only when an inhibitory effect on SIRTI Sir2 is in test chemicals b inhibitory effect on lysylendopeptidase final fluorescence intensity Fluoro Deacetylated Peptide instead of Fluoro Substrate Peptide and ple of Positive control and Assay control 2 that does not add Although fluorescence intensity increases when Fluoro Deacety inhibitory effect on lysylendopeptidase activity occurs in a testi not increase when there is an Increase Please use ury out the experiment in the following Table Peptide is used when an fluorescence intensity does Assay reagents Assay control ssay control 2 Positive control D 10X SIRTI assay buffer 5 5 5 8 50X Fluoro Deacetylated Peptide 1 1 3 10X NAD SuL 5 uL 4 10X TSA Su 5 5 5 10X Test sample 5 uL I 6 X5 diluted recombinant SIRT1 or Your enzyme sample dH O pL 26 5 uL 31 5 uL 2 X20 diluted Lysylendopeptidase 2 5 uL 2 5 uL 2 5 uL 1 Following the table above add gent 1 2 5 uL of 2 X20 diluted 2 Incubate for 10 min or desi 3 Add 50 uL of 7 2X h of time at room temperature Ca 25 C olution to each well 3 4 5 or 6 and dH5O to each well Finall
20. y add to each well and mix thoroughly to initiate reaction 4 Read using microtiter plate fluorometer with excitation at 340 nm and emission at 440 nm Q C CY 1151 130130 Version SIRTI Sir2 Deacetylase Fluorometric Assay Kit 4 yclex User s Manual For Research Use Only Not for use in diagnostic procedures Cautions In order to measure the activity of SIRTI Sir2 correctly it is necessary to conduct the co experiments for No enzyme control and No NAD control at least once in addition to es sample control as indicated in the above table of Assay method Although fluoresceneemmt increases in No Test sample control when SIRTI Sir2 enzyme activity is in t increase in fluorescence intensity is not observed in No enzyme control and No NA 2 In order to estimate the inhibitory effect on SIRTI Sir2 activity in the Test sample correctly it is necessary to conduct the control experiment of No Test sample control at once for every experiment and No NAD control at least once for the first experiment in additi Fest sample as indicated in the above table of Assay method When test chemicals cause an ry effect on SIRTI Sir2 activity the level of increase of fluorescence intensity is weaken ared with No Test sample control The increase in fluorescence intensity is not observed AD control For research use only not for use in diagnostic or therapeutic proced
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