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1. CY 1140 CycLex HDACs Deacetylase Fluorometric Assay Kit Cat CY 1150 CycLex HDAC8 Deacetylase Fluorometric Assay Kit Cat CY 1158 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 Anti Acetylated Histone p53 K382 Mouse Monoclonal Antibody Cat CY M1 Anti Histone Deacetylase 1 HDAC1 Rabbit Polyclonal Antibody Cat 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 Cat CY E1152 NAD Dependent Deacetylase SIRT3 Cat CY E1153 NAMPT Nicotinamide Phosphoribosyltransferase Cat CY E125 NMNAT1 Nicotinamide Mononucleotide Adenylyltransferase E1252 Japanese Patent No 4267043 Canadian Patent No 2392711 amp PRODUCED BY cA Note This product is covered under CycLex s paten U S Patent No 7 033 778 and No 7256013 European Patent No 1243658 CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 7 e mail info c CycLex Cire oducts are supplied for research use only CycLex CircuLex products and compo may not be resold modified for resale or used to manufacture commercial pro hout prior writt2. Version 130130 p SIRT2 Deacetylase Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Cautions In order to measure the activity of SIRT2 correctly it is necessary to conduct the control experi for No enzyme control and No NAD control at least once in addition to No Test control as indicated in the above table of Assay method Although fluorescence intensity in No Test sample control when SIRT2 enzyme activity is in the sample t fluorescence intensity is not observed in No enzyme control and No NAD control 2 In order to estimate the inhibitory effect on SIRT2 activity in the Test sample correctly it 1 essary to conduct the control experiment of No Test sample control at least once for every experiment and No NAD control at least once for the first experiment in addition t sample as indicated in the above table of Assay method When test chemicals cause an itory effect on SIRT2 activity the level of increase of fluorescence intensity is weakened as with No Test sample control The increase in fluorescence intensity is not observed in control For research use only not for use in diagnostic or therapeutic proced Troubleshooting e mixed in a crude SIRT2 ific antibody against SIRT2 red Since the protease inhibitors tidase activity strongly please avoid When chemicals that have an
3. and its homologs are Proc Natl Acad Sci U S A 97 5807 5811 2000 pied s Lorraine Pillus and Rolf dependent protein deacetylases 7 Jeffrey S Smith Carrie Baker Brachmann Ivana Celi Vincent J Starai Jose L Avalos Jorge C Escala e Wolberger and Jef D Boeke A phylogenetica e activity in the Sir2 protein family Proc Natl Ac et A Kenna Shabazz Muhammad ena Charles Grubmeyer Cynthia AD dependent protein deacetylase A 97 6658 6663 2000 oo H Vaziri SK Dessain E Ng Eaton SI Imai RA TK Pandita L Guarente and RA Weinberg hSIR2 SIRT1 functions as an NAD depend 53 deacetylase Cell 107 149 159 2001 9 J Luo AY Nikolaev S Imai D Chen F Su h L Guarente and W Gu Negative control of p53 by Sir2alpha promotes cell survival un tress Cell 107 137 148 2001 10 E Langley M Pearson M Farett er RA Frye S Minucci PG Pelicci and T Kouzarides Human SIR2 deacetylates p53 gonizes PML p53 induced cellular senescence EMBO J 21 2383 2396 2002 11 J Smith Human Sir2 and Q of p53 activity Trends Cell Biol 12 404 2002 S 12 CM Grozinger and chreiber Deacetylase enzymes biological functions and the use of small molecule inhibi hem Biol 9 3 16 2002 C CY 1152 14 Version 130130 SIRT2 Deacetylase Fluorometric Assay Kit Pa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Related Products CycLex Cellular Histone Acetylation Assay Kit Cat
4. become widespread SIRT2 activity measurement which could not be ma conventional method is now possible with the CycLex SIRT2 Deacetylase Fluorometric Assay g the same equipment This new method of measurement should dramatically raise the e inhibitor screening and biochemical analysis of these enzymes Measuring Principle of The CycLex SIRT2 Deacetylas Bluorometric Assay Kit fluorophore X X X Lys Ac Deacetylase fluorophore X X X Lys X Ei l tM TMT Lysylendopeptidase oe X X ys X X X quencher act escence intensity Note This measuring pri Qi are covered under CycLex s patents U S Patent No 778 and No 7256013 European Pa 43658 Japanes 67043 Canadi 2392711 C CY 1152 3 Version 130130 SIRT2 Deacetylase Fluorometric Assay Kit yclex Materials Provided User s Manual For Research Use Only Not for use in diagnostic procedures Each kit contains Materials Quantity Storage Al 10X SIRT2 assay buffer 1mlx2 Below 20 50X Fluoro Substrate Peptide 1 mM 100u1x1 Below 20 50X Fluoro Deacetylated Peptide 50un1x1 Below 20 Lysylendopeptidase 100 mA U ml 50u 1x1 Beloyge20 C 100X NAD 80 mM 100n 1x1 Bely 20 200X Trichostatin A 0 2 mM 100n1x1 Below220 C Recombinant SIRT2 200n 1x1 EFC 100X Stop solution 100 u 1 x Jeg Below 20 C Instruction manual 1 oom temp Materials R
5. prepare 1 vial f X SIRT2 assay buffer mixed with 9 ml of dH O and store SIRT2 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 2 assay buffer 3 10X NAD 8 mM NAD Quantity required 5 uL assay Dilute the 100X NAD 1 10 with 1 1X ssay buffer 4 10X TSA 10 uM Quantity required 5 uL assay Dilute the 200X Trichostatin ith 1 1X SIRT2 assay buffer 5 10X Test sample 10X fin Quantity Required 5 uL assa Dilute Test sample to 10X fi tion e g a candidate of inhibitor or activator ired concentration with 1 1X SIRT2 assay buffer 6 X5 diluted recom Quantity Required Dilute the IRT2 1 5 with 1 1X SIRT2 assay buffer Note Use 6 iluted recombinant SIRT2 within the same day they are prepared 7 2X Stop s 0 Quantity requi uL assay Dilute thes 100X Stop solution 1 50 with dH O Cat i CY 1152 6 Version 130130 LA SIRT2 Deacetylase Fluorometric Assay Kit ANyCLex User s Manual For Research Use Only Not for use in diagnostic procedures 8 SIRT2 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 S 5 uL of 5 10X Test sampl
6. welljas for inhibitor screening without the use of radioactive substances is preferred c o measure the activity of the enzyme based on C CY 1152 2 Version 130130 4 SIRT2 Deacetylase Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay CycLex SIRT2 Deacetylase Fluorometric Assay Kit measures the activity of SIRT2 by the principle of changing a SIRT2 reaction into the activity of the protease In order to measure the e activity of SIRT2 which is the NAD dependent histone deacetylase this kit is designed activity of NAD dependent histone deacetylase can be measured under existence of 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 Howe SIRT2 performs deacetylation substrate peptide will become cut by the action 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 price t eas ent of SIRT2 activity in most laboratories is possible if they are equipped with a fluores er for microtiter plates Considering that the use of fully automatic apparatus to measur nce intensity has
7. 4 SIRT2 Deacetylase Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Quantitative test kit for NAD dependent histone deacetylase activity CycLex SIRT2 Deacetylase Fluoromettig N Assay Kit Cat CY 1152 Intended WSC soc auenadenscaccceesesivanceateceeasecteecenes 1 Qy Storage 1 ays TntroductiOn ccccccsscccccessesseeceeeeseesteeees 2 Principle of the Assay 3 Materials Provided ccccccsccccceeeesseeeeees 4 Materials Required but not Provided 4 PLECAULIONS cccsescececeeceesssnseececeesessseeeeees 5 Detailed Protocol cccccccccccssssseeeceeeeseees 6 9 CautiOMs cccccccccsssssscecceessesseceeeceesessenseeeeees 10 TTOUDISSG QUIN os sscanantnacreinanedicrneunteseeactiacs 10 Reagent SUADIILY eccixsosnadveneanearavdsics ieduneeaseeees 10 Example of Test Results cee eeeeeeeeeneeee 11 References ccccscsseccccecessssseeeecesssssseeeeees Related Products ccccccccccsssscccceeeessseeeeees 1 Intended Use The CycLex Research Product CycLex deacetylase activity of recombinant SIRT2 Deacetylase Fluorometric Assay Kit i inhibitors or activators using recombinant SIR Deacetylase Fluorometric Assay Kit detects y the CycLex Research Product CycLex SIRT2 for the rapid and sensitive evaluation of SIRT2 or purified SIRT2 Applications for this kit include 1 Screening inhibitors or activat f T2 2 Detect
8. RNA and protein expression are sever educed in a large fraction of human glioma cell lines 4 Ectopic expression of SIRT2 e cell lines suppressed colony formation and modified the microtubule network These results in at SIRT2 may act as a tumor suppressor and may function to control the cell cycle by ag on Of alpha tubulin It was reported that SIRT2 inhibitor rescued alpha synuclein toxicity and d inclusion morphology in a cellular model of Parkinson s disease however the exact mechani uncertain However the conventional method for measuring SIRT2 acti i complicated and laborious In order to measure SIRT2 enzyme activity it is necessary to pr ioact substrate First cells have to be labeled metabolically with radioactivity by adding radioactive acetic acid to the culture medium Second radioactive ace i Following the reaction it is necessary to extract ar been released from acetylated histone using ethyl the radioactivity Although a method for measuring the activity of dea ase without the use of radioactive substances was reported in recent years owing to the use uorescent labeled acetylated lysine as a substrate the reaction product must be separated from the ubstrate and the fluorescent intensity measured by reverse phase HPLC As mentioned above these measurement systems are difficult to adapt for processing many samples under a variet co ons because of their complicated operation Thus a simple system for biochemical analysis aS
9. ate fluorescence reader with excitation at 490 10 nm emission at 530 10 nm 3 The difference in fluorescence etween No Test sample control and No enzyme control indicates the SIRT2 activity Note 1 It is possible to ch e yolume of assay reagents and sample as far as it sets up the final concentration of ts in a reaction mixture as indicated as below r Note 2 Duplicate AS is strongly recommended C CY 1152 8 Version 130130 SIRT2 Deacetylase Fluorometric Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures yclex 2 Assay control 1 When the chemicals that have an inhibitory effect on lysylendopeptidase come to be mixed in S fraction purified from various cells or the immunoprecipitate using the specific antibody SIRT2 or other proteins precise SIRT2 enzyme activity cannot be measured Since the inhibitors used in the usual protein purification process strongly inhibit lysylendopepti please avoid using any protease inhibitors during the process of protein purification If there is such a possibility please carry out the experiment of Positive control control 1 in the following table using Fluoro Deacetylated Peptide to referene Fluoro Deacetylated Peptide is used fluorescence intensity should increase whenever there is no SIRT2 activity in your enzyme sample When there is an inhibitory effect o dopeptidase activity even if there is SIRT2 activity in a sample fluor
10. e e Mix following reagents 30 uL 1 assay 1 D10X SIRT2 assay buffer 5 pL 2 50X Fluoro Substrate Peptide l uL 3 2 X20 diluted Lysylendopeptidase 2 5 uL 4 4 10X TSA 5 dH O 16 Total C CY 1152 7 Version 130130 SIRT2 Deacetylase Fluorometric Assay Kit 4 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures SIRT2 Assay Procedures 1 Assay method No enzyme Na Test Assay reagents Test sample sample control control 8 SIRT2 reaction buffer 30 uL 30 uL 30 uL 3 10X NAD 5uL 5uL 5 uL 5 10X Test sample 5uL dH 0 15 pL 5 pL 10 pL 6 X5 diluted recombinant SIRT2 10 pL _ 10 pL or Your enzyme sample he microplate Finally your enzyme sample to ervals using microtiter plate fluorometer with excitation at 490 10 nm and emission at m Measure and calculate the rate of reaction while the reaction velocity remains constan Alternate procedure 1 Following the above table add Reagent 3 5 r dH O to each well of the microplate Finally initiate reaction by adding 10 uL of 6 X5 dilut combinant SIRT2 or your enzyme to each well and mixing thoroughly Incubate at room tempera Ca 25 C 2 While the reaction rate is kept constant uL of 7 2X Stop solution to each well at appropriate time to stop the reaction an fluorescence intensity in a micropl
11. en approval from CycLex Co Ltd To inquire about licensing for such use please contact us via email C CY 1152 15 Version 130130
12. equired but not Provided w e Microplate for fluorometer e Microplate reading fluorometer capable of excitation at ength in the range 490 10 nm and detection of emitted light in the range 530 10 nm Pipettors 2 20 uL 20 200 uL and 200 1000 uL iS e Multi channel pipette e Microplate shaker e Deionized water of the highest quality e 500 or 1000 mL graduated cylinder e Reagent reservoirs tors with disposable tips C CY 1152 4 Version 130130 wn SIRT2 Deacetylase Fluorometric Assay Kit C ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions e Please thaw 50X Fluoro Substrate Peptide and 50X Fluoro Deacetylated Peptide at temperature before use Then thaw the other reagents in ice and use after they are completely thawed e Please avoid repeated freezing and thawing of the recombinant SIRT2 in this kit There i that the enzyme activity may be inactivated Aliquot to 10 20 uL and store at 70 C e Please avoid mixing of protease inhibitors such as PMSF or alkyl amine in the sample tha measured SIRT2 activity e Do not use kit components beyond the indicated kit expiration date Qy e Rinse all detergent residue from glassware A e Use deionized water of the highest quality O e Do not mix reagents from different kits Do not mouth pipet or ingest any of the reagents e Do not smoke eat or drink when performing the assay fin atea
13. escence intensity should ase e is an inhibitory ase Please use out the experiment the following Table ide is used when an orescence intensity does 2 Not only when an inhibitory effect on SIRT2 is in test chemicals but also effect on lysylendopeptidase final fluorescence intensity will Fluoro Deacetylated Peptide instead of Fluoro Substrate Peptide and of Positive control and Assay control 2 that does not add S Although fluorescence intensity increases when Fluoro Deacet inhibitory effect on lysylendopeptidase activity occurs in a test ch not increase Assay reagents Assay control 1 control 2 Positive control 10X SIRT2 assay buffer 5uL 5 uL 5 uL 50X Fluoro Deacetylated Peptide 1 1 uL 1 pL 3 10X NAD SuL 5 uL 4 10X TSA 5 uL 5 uL 5 10X Test sample 5 pL 6 X5 diluted recombinant SIRT2 5uL or Your enzyme sample dH 0 pL 26 5 uL 31 5 uL 2 X20 diluted Lysylendopeptidase 5 uL 2 5 uL 2 5 uL 1 Following the table above add 2 5 uL of 2 X20 diluted Lysyl ndop 2 Incubate for 60 min or desi 3 Add 50 uL of 7 2X 4 Read fluorescence emission at 53 C CY 1152 3 4 5 or 6 and dH O to each well Finally add dase to each well and mix thoroughly to initiate reaction solution to each well h of time at room temperature Ca 25 C Qj using microtiter plate fluorometer with excitation at 490 10 nm and
14. ing the effects of p ical agents on SIRT2 This assay kit is for resear y and not for use in diagnostic or therapeutic procedures inant SIRT2 at 70 C and all other components below 20 C xcessive light CyY 1152 1 Version 130130 p SIRT2 Deacetylase Fluorometric Assay Kit f cA yel ex 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 deacetylase that plays a critical role in transcriptional silencing genome stability and i mammals the homologs of Sir2 have been named sirtuins SIRT with seven membe termed SIRT1 through SIRT7 They share a conserved central deacetylase domain but ha and C termini and display distinct subcellular localization suggesting different biological f In contrast to SIRT1 mammalian SIRT2 is localized mainly in the cytoplasm SIRT2 colocalizes with the microtubule network and deacetylates Lys40 of alpha tubulin 2 The same residue of alpha tubulin is also deacetylated by HDAC6 a class II HDAC and deacetylation by o changes in cellular motility 3 A role for SIRT2 in cancer pathogenesis was demonstrated using a prot c_ approach 4 The SIRT2 gene which is located at chromosome 19q13 2 lies within a region th uently deleted in human gliomas and levels of SIRT2 m
15. inhibitory effect on lysylendo fraction purified from various cells or the immunoprecipitate or other proteins precise SIRT2 enzyme activity cannot used in the usual protein purification process inhibit lys the use of any protease inhibitors during the protei 2 Final fluorescence intensity will not increase bi en test chemicals have an inhibitory effect on SIRT2 and also when there is an inhibitory effect sylendopeptidase 3 If the test reagents themselves emit fl ence at excitation wavelength 480 500 nm and fluorescence wavelength 520 540 nm t itory effect of the test assay cannot be evaluated correctly 4 The recombinant SIRT2 should be Protocol Incubation times or t erroneous results in duplicate using the protocol described in the Detailed significantly different from those specified may give 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 n y of the curve as can assay kinetics that are other than first order For a non linear curve t nt or quadratic curve fit methods should be used 6 Poor duplicates in curate dispensing If all instructions in the Detailed Protocol were esults indicate a need for multi channel pipettor maintenance included in the CycLex Research Product CycLex SIRT2 Assay kit have been Reagents should not be used beyond the stated expiration date Upon receipt store inant SIRT2 at 70 C all other kit reage
16. metric Assay Kit Ce yCLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 5 Effect of Resveratrol on recombinant SITR2 activity 5 000 4 000 3 000 2 000 6 62 5 250 1000 No No enzyme NAD F485 F535 x10 Reveratrol conc uM C CY 1152 13 Version 130130 4 SIRT2 Deacetylase Fluorometric Assay Kit g yCLex User s Manual For Research Use Only Not for use in diagnostic procedures References North B J and Verdin E Sirtuins SIRT2 related NAD dependent protein deacetylases Ge Biol 5 224 2004 2 North BJ Marshall BL Borra MT Denu JM Verdin E The human SIRT2 ortholog 1S NAD dependent tubulin deacetylase Mol Cell 11 437 444 2003 w Hubbert C Guardiola A Shao R Kawaguchi Y Ito A Nixon A Yoshida M Wang XF Yao TP HDAC6 is a microtubule associated deacetylase Nature 417 455 458 2002 4 Hiratsuka M Inoue T Toda T Kimura N Shirayoshi Y Kamitani H Watanabe E Tahimic CG Kurimasa A Oshimura M Proteomics based identification of differenti ssed genes in human gliomas down regulation of SIRT2 gene BBRC 309 558 566 2003 n S Imai CM Armstrong M Kaeberlein and L Guarente Transcrip protein Sir2 is an NAD dependent histone deacetylase Nature 403 79 ilencing and longevity 000 6 Joseph Landry Ann Sutton Stefan T Tafrov Ryan C Heller Jo Sternglanz The silencing protein SIR2
17. nts should be stored below 20 C CyY 1152 Version 130130 SIRT2 Deacetylase Fluorometric Assay Kit Pa ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency curve of recombinant SIRT2 activity 6 000 r 5 000 D 4 000 f gt N 7 3 000 amp N 2 000 1 000 0 0 0 0 5 1 0 1 5 2 0 2 5 SIRT2 ug wy Fig 2 Time course of SIRT2 substrate deacetylation combinant SIRT2 14 000 r e 2 0 ug e 1 0 ug 12 000 e 0 5 ug 10 000 0 25 ug E x 8 000 wn ia amp 6 000 N 2 vt 4 000 2 000 0 0 20 40 60 80 100 120 Reaction time min C CY 1152 11 Version 130130 g SIRT2 Deacetylase Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Efect of Trichostatin A and NAD on recombinant SIRT2 activity 2 500 2 000 A Z 1 500 en amp 1 000 wv cs 500 a TSA TSA NAD Fig 4 Effect of Sirtinol on recombinant SITR2 activa 6 000 r 5 000 a 4 000 Y F485 F535 x10 N 1 000 0 15 6 62 5 250 1000 No No enzyme NAD Sirtinol conc uM C CY 1152 12 Version 130130 wn SIRT2 Deacetylase Fluoro
18. s where samples or reagents are handled Biological samples may be contaminated wit io wounds or breathe aerosols Wear protective ents Do not ingest expose to open d dispose of biological samples properly C CY 1152 5 Version 130130 4 SIRT2 Deacetylase Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol Description of assay system CycLex SIRT2 Deacetylase Fluorometric Assay Kit can measure the enzyme activity of homogeneous method In this method the reaction is initiated and the fluorescence intensi by mixing simultaneously fluorescence labeled acetylated peptide which is subs trichostatin A NAD and lysylendopeptidase Since the reaction is not stopped it is necessa fluorescence intensity at regular intervals after the reaction is initiated and to determine reactio Alternatively within a time in which the reaction velocity is kept constant it is also ible to stop the reaction by adding 2X stop solution and to measure fluorescence intensity Q Preparation Method for Assay Reagents Thaw 2 50X Fluoro Substrate Peptide and 50X Fluoro Deacetylated i room temperature Stand other reagents in ice to thaw Use them after they thaw completely 1 1X SIRT2 assay buffer 50 mM Tris HCl pH 8 8 0 5 mM D Quantity Required 50 uL assay Dilute the 10X SIRT2 assay buffer 1 10 with distilled wate Since this is the base buffer for the assay
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