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1. Assay Kit Ver 2 2 ycLex 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 Ver 2 Cat CY 1150V2 CycLex SIRT1 Sir2 Deacetylase Fluorometric Assay Kit Ver 2 Cat CY 1151V2 CycLex SIRT2 Deacetylase Fluorometric Assay Kit Ver 2 Cat CY 1152V2 CycLex SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 Cat CY 1153V2 CycLex SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 Cat CY 1156V2 CycLex HDACS8 Deacetylase Fluorometric Assay Kit Ver 2 Cat CY 1158V2 Anti Acetylated Histone p53 K382 Mouse Monoclonal Antibody Cat CY M1029 Anti Histone Deacetylase 1 HDAC1 Rabbit Polyclonal Antibody Cat CY P1011 Anti Histone Deacetylase 2 HDAC2 Rabbit Polyclonal Antibody Cat CY P1012 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 E1251 NMNATI1 Nicotinamide Mononucleotide Adenylyltransferase 1 s at Y 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 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka In2. activity Effect of several compounds on SIRT6 activity 100 E 80 5 S 60 o r 40 5 amp Sirtinol gt 20 Resveratrol 5 x Quercetin gt 0 Z 0 200 400 600 800 1 000 Test compound uM C CY 1156V2 12 Version 141107 Pae SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 e yCLex User s Manual For Research Use Only Not for use in diagnostic procedures References North B J and Verdin E Sirtuins SIRT6 related NAD dependent protein deacetylases Ge Biol 5 224 2004 N Michishita E et al Evolutionarily conserved and nonconserved cellular localizations a n human SIRT proteins Mol Biol Cell 16 4623 2005 W Mostoslavsky R et al Genomic instability and aging like phenotype in the absence o alian SIRT6 Cell 124 315 2006 4 Michishita E et al SIRT6 is a histone H3 lysine 9 deacetylase that modulates dun chromatin Nature 452 492 2008 nn Yang B et al The sirtuin SIRT6 deacetylates H3 K56Ac in vivo to promo ic stability Cell Cycle 8 2662 2009 6 Kawahara TL et al SIRT6 links histone H3 lysine 9 deacetylati appaB dependent gene expression and organismal life span Cell 136 62 2009 N Van Gool F et al Intracellular NAD levels regulate tumom nec factor protein synthesis in a sirtuin dependent manner Nat Med 15 206 2009 C CY 1156V2 13 Version 141107 oy SIRT6 Deacetylase Fluorometric
3. kit is for research us only and not for use in diagnostic or therapeutic procedures Storage e Upon receipt stored 5 Developer and 6 Recombinant SIRT6 at 70 C and all other components below 20 C4 e Do not expose eagentsfto excessive light Cat CY 1156V2 1 Version 141107 SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 jde Uers Mi GA y gt X ser s Manu 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 buddimg yeast and nematode In 2000 it was reported that the yeast Sir2 protein is a NAD dependent histone deacetylase that plays a critical role in transcriptional silencing genome stability and longevity In mammals the homologs of Sir2 have been named Sirtuins SIRTs with seven members in a family termed SIRT 1 through SIRT7 They share a conserved central deacetylase domain but have different N and C termini and display distinct subcellular localization suggesting different biological functions 1 The distant mammalian Sir2 homolog SIRT6 is a broadly expressed predominantly nuclear protein 2 like SIRT1 Inactivation of the Sirt6 gene in mice leads to dramatically shortened life span and a premature aging like phenotype 3 It was shown that SIRT6 is a histone H3 lysine 9 deacetylase and identified a role for SIRT6 activity at telomeric chromatin where it prevents telomete dysfunction in huma
4. quality e Do not mix reagents from different kits e Do not mouth pipet or ingest any of the reagents e Do not smoke eat or drink when performing the assay or in argas where samples or reagents are handled e Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly NOTE THE FOLLOWING PROCEDURES ARE INTENDED ONLY AS A GUIDELINE THE OPTIMAL EXPERIMENTAL CONDITIONS WILL VARY DEPENDING ON THE PARAMETERS BEING INVESTIGATED AND MUST BE DETERMINED BY THE INDIVIDUAL USER For research use only not for use in diagnostic or therapeutic procedures Cat CY 1156V2 5 Version 141107 oy SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol CycLex SIRT6 Deacetylase Fluorometric Assay Kit can measure the enzyme activity of SIRT6 witha homogeneous method In this method the reaction is initiated and the fluorescence intensity is measured by mixing simultaneously fluorescence labeled acetylated peptide which is a substrate SIRT6 NAD and the developer Since the reaction is not stopped it is necessary to measure fluorescenge intensity at regular intervals after the reaction is initiated and to determine reaction velocity Alternatively within a time in which the reaction velocity is kept constant it is also poss
5. strongly inhibit the peptidase activity in the development reaction pl as avoid using any protease peptidase inhibitors during the process of protein purification Note 5 If enzyme samples have an inhibitory effect on the peptidase in the development reaction the final fluorescence intensity will not increase Please use 3 Flu re D acetylated Peptide instead of 2 Fluoro Substrate Peptide and conduct a control experiment 2 Assay Procedures for Inhibitor Activator Screening 1 Following Table 2 below first add Distilled water s 1 SIRT6 Assay Buffer 2 Fluoro Substrate Peptide or 3 Fluoro Deacetylated Peptide add 4 NAD to microtiter plate wells Second add Test Compound or Solvent of Test Compound or Control Compound not provided and 5 Developer to each wellof the microtiter plate and mix well Table 2 Reaction mixture for inhibitor activator screening Assay reagentse Test Test Solvent Control No Enzyme Development Assay reagents Compound Control Compound Control Control Assay Assay Assay Assay Assay Distilled water 20 pL 20 uL 20 pL 25 pL 30 pL 1 SIRT6 Assay Buffer 5 phe 5 pL 5 pL 5 uL 5 uL 2 Fluoro Substrate Peptide 5 uL 5 uL 5 pL 5 pL 3 Fluoro Deacetylated Peptide 5 uL 4 NAD SuL 5 uL 5 pL 5 pL Test Compound 5ub 5 uL Solvent of Test Compound 5 uL 5 uL Control Compound not provided
6. 4 The recombinant SIRT6 should be run in duplicate using the protogol described in the Detailed Protocol Incubation times or temperatures significantly different from those specified may give erroneous results 5 The reaction curve is nearly a straight line if the kinetics of the assayis of the first order Variations in the protocol can lead to non linearity of the curve as can assay kinetics that are other than first order For a non linear curve point to point or quadratic curve fit methods should be used 6 Poor duplicates indicate inaccurate dispensing If all imstructions 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 Cyckex Research Product CycLex SIRT6 Deacetylase Fluorometric Assay Kit have been tested for stability Reagents should not be used beyond the stated expiration date Upon receipt store the 5 Developer and 6 Recombinant SIRT6 at 70 C all other kit reagents should be stored below 420 C Cat CY 1156V2 9 Version 141107 SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 GA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependent curve of recombinant SIRT6 activity Dose dependent curve 2 000 50 min D 25 min 2 1 500 T 10 min a A 5
7. 5 uL 5 Developer 5 uL 5 uL 5 uL 5 uL 5 uL 6 Recombinant SIRT6 or Enzyme Sample gt uL SuL a Total Volume of th mixture 50 uL 50 uL 50 uL 50 uL 50 uL 2 Initiate reactions by adding 5 uL of 6 Recombinant SIRT6 or your Enzyme Sample to each well and mixing thoroughly at room temperature 3 Read fluorescence intensity for 30 to 60 minutes at 1 to 2 minute intervals using microtiter plate fluorometer with excitation at 480 500 nm and emission at 520 540 nm Measure and calculate the rate of reaction while the reaction velocity remains constant Cat CY 1156V2 T Version 141107 SR SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 f yeLex User s Manual For Research Use Only Not for use in diagnostic procedures Alternate procedure 3 While the reaction rate is kept constant add 20 uL of 7 Stop Solution to each well at appropriate time to stop the reaction and measure fluorescence intensity in a microplate fluorescence reader capable of excitation at a wavelength in the range 480 500 nm and detection of emitted lightsin the range 520 540 nm Note 1 During the time in which SIRT6 reaction rate is maintained the difference in fluor scence intensity between Solvent Control Assay and No Enzyme Control Assay indicates the SIRT6 activity Note 2 In order to estimate the active or inhibitory effect on SIRT6 activity by thept st compounds correctly it is nec
8. a Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co jp URL http www cyelex 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 1156V2 14 Version 141107
9. ce will be emitt d 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 theameasur ment of SIRT6 activity in most laboratories is possible if they are equipped with a fluores entefeader for microtiter plates Considering that the use of fully automatic apparatus to measure fluorescence intensity has become widespread SIRT6 activity measurement which could not be mad byth Conventional method is now possible with the CycLex SIRT6 Deacetylase Fluorometric Assay Kit using the same equipment This new method of measurement should dramatically raise the efficiencyyof inhibitor screening and biochemical analysis of these enzymes Measuring Principle of The CycLex SIRT6 Deacetylase Fluorometric Assay Kit fluorophore X X X Lys Ac X X qu ncher fl cM Deacetylase fluorophore X X X Lys X X quencher lt _ Peptidase fluorophore X X X Lys X X quencher v Measurement of fluorescence intensity Note This measuring principle and kit are covered under CycLex s patents U S PatentsNow7 033 778 and No 7256013 European Patent No 1243658 Japanese Patent No 4267043 Canadian Patent No 2392711 Cat CY 1156V2 3 Version 141107 ry SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Provided Components of K
10. essary to conduct the control experiment of Solvent Control Assay at least once for every experiment and Control Compound Assay at least once for the first experiment in addition to Test Compound Assay as indicated im the Table 2 When test compounds cause an active or inhibitory effect on SIRT6 activity the level of increase of fluorescence intensity is strengthened or weakened as compared_withSolvent Control Assay Note 3 The efficacy of the test compounds on the SIRT6 actiyity is the difference in fluorescence intensity between Test Compound Assay minus No Enzyme Control Assay and Solvent Control Assay minus No Enzyme Control Assay Note 4 If test compounds have an inhibitory effect on proteas peptidase resulting that the increase in fluorescence intensity is not or a little observed in Development Control Assay the effect on SIRT6 activity cannot be evaluated correctly Note 5 Although the above tables indicate the volume of addition of Test Compound or Solvent of Test Compound or Control Compound not provided as 5 uL the concentration and the volume of the reagents to add can b Changed so that the concentration of test compounds becomes the setting concentration For examiple since the final volume of reaction is 50 uL here it is also possible to add 10 uL_of Test Compound or Solvent of Test Compound or Control Compound not provided In
11. ible to stop the reaction byjadding stop solution and to measure fluorescence intensity 1 Assay Method for Measurement of SIRT6 Activity 1 Following Table 1 below first add Distilled water 1 SIRT6 Assay Buffer 2 Fluoro Substrate Peptide and 4 NAD to microtiter plate wells Second 5 Developer to each well of the microtiter plate and mix well Table 1 Reaction mixture for measurement of SIRT6 activity Enzyme No Enzyme Positive No NAD Assay reagents Sample Control Control Control Assay Assay Assay Assay Distilled water 25 uL 25uL 25 uL 30 pL 1 SIRT6 Assay Buffer 5 uL 5 aD 5 uL 5 uL 2 Fluoro Substrate Peptide 5uL SuL 5 uL 5 uL 4 NAD 5SuL 5 uL 5 uL 5 Developer 5 uL 5 uL 5 uL 5 uL Enzyme Sample 5 uL 5 uL Buffer of Enzyme Sample 5 uL 6 Recombinant SIRT6 gt 5 uL Total Volume of the mixture 50 uh 50 uL 50 uL 50 uL 2 Initiate reactions by adding 5 uL of your Enzyme Sample or Buffer of Enzyme Sample or 6 Recombinant SIRT6 to each wellgand mixing thoroughly at room temperature 3 Read fluorescence intensity for 30 to 60 minutes at 1 to 2 minute intervals using microtiter plate fluorometer with excitation at 480 500 nm and emission at 520 540 nm Measure and calculate the rate of reaction while the reaction velocity remains constant Alternate procedure 3 While the reaction rat is kept constant add 20 uL
12. it 500 uL x1 Below 20 C 1 mL 2 Below 20 C Instruction manual 1 Room temp Materials Required but not Provided Microplate for fluorometer Microplate reading fluorometer capable of excitation at a wavelength in the range 480 500 nm and detection of emitted light in the range 520 540 nm Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Multi channel pipette Microplate shaker Deionized water of the highest quality e 500 or 1000 mL graduated cylinder e Reagent reservoirs e Control compound s Cat CY 1156V2 4 Version 141107 oy SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Precautions e Please thaw 2 Fluoro Substrate Peptide and 3 Fluoro Deacetylated Peptide at room 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 5 Developer and 6 Recombinant SIRT6 There is a possibility that the enzyme activity may be inactivated Aliquot to 10 20 uL and store at 70 C e Please avoid mixing of protease peptidase inhibitors such as PMSF or alkyl amine in samples that will be measured SIRT6 activity e Do not use kit components beyond the indicated kit expiration date e Rinse all detergent residue from glassware e Use deionized water of the highest
13. min o0 wt amp 1 000 Lv a la N gt 500 E aa 0 0 00 0 50 1 00 1 50 SIRT6 ug Fig 2 Time course of SIRT6 substrate deacetylation by recombinant SIRT6 Time course 2 000 gt 1 5 pg a 0 75 ug 2 1 500 A 0 38 ug ia 0 19 ug o0 lt 0 pg amp 1 000 wy lag N am 2 500 Z 0 0 10 20 30 40 50 Reaction time min Cat CY 1156V2 10 Version 141107 SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 Ca NycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 NAD dependency of recombinant SIRT6 activity NAD dependency XN 2 000 800uM cA E 400uM 1 500 A 200uM O 100uM 0uM RFU F535 F485 x10 n Onr oeoa e 0 10 20 30 40 50 reaction time min Fig 4 Effect of Trichostatin A and NAD on nome Ser W ivity 45 000 T 40 000 35 000 30 000 gt 25 000 3 20 000 V Nn 10 000 5 000 Reaction ve NAD NAD NAD NAD TSA TSA C CY 1156V2 11 Version 141107 SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 wy ANyCLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 5 Effect of several compounds on recombinant SITR6
14. n cells 4 In addition SIRT6 also deacetylates histone H3 acetylated lysiney56 imiWivo to promote genomic stability 5 In term of function SIRT6 binds to the NF KB subunit RELA and attenuates NF KB signaling by modifying chromatin at NF B target genes 6 Thus SIRT6 mediated control of NF B prevents aging associated hyperactive NF B dependent gene expression and inhibition of NF B can rescue the early lethality of Sirt6 knockout mice Overexpression of wild type Sirt6 but not the catalytically inactive form consistently resulted in increasedstumoOr necrosis factor protein production relative to its mRNA levels 7 The conventional method for measuring SIRT6 activity is very complicated and laborious In order to measure SIRT6 enzyme activity it is necessary to prepare radioactive acetylated histone H3 as a substrate First cells have to be labeled metabolically with radioactivity by adding radioactive acetic acid to the culture medium Second radioactive acetylated histone has to be purified from the cells Following the reaction it is necessary to extract and sepatate the radioactive acetyl group which has been released from acetylated histone using ethyl acetate to measure the activity of the enzyme based on the radioactivity Although a method for measuring the activity of dea etylase without the use of radioactive substances was reported in recent years owing to the use of fluores ent labeled acetylated lysine as a subs
15. of 7 Stop Solution to each well at appropriate time to stop th feaction and measure fluorescence intensity in a microplate fluorescence reader capable of excitation at a wavelength in the range 480 500 nm and detection of emitted light in the range 520 540im Note 1 During the time in which SIRT6 reaction rate is maintained the difference in fluorescence intensity between Enzyme Sample Assay and No Enzyme Control Assay indicates the SIRT6 activity of your Enzyme Sample Cat CY 1156V2 6 Version 141107 oy SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Note 2 Although the volume of addition of Enzyme Sample or Buffer of Enzyme Sample or 6 Recombinant SIRT6 is set to 5 uL in Table 1 it may be changed to a volume up to 20 uL at your discretion In that case please reduce the volume of Distilled water to set the final reaction volume of 50 uL Note 3 If enzyme samples contain some protease peptidase able to break down 2 Fluor6 Stibstrate Peptide resulting in an increase of fluorescence intensity in No NAD Control Assay the SIRT6 activity in the samples cannot be evaluated correctly Note 4 If enzyme samples contain inhibitors for protease peptidase precise SIRT6 enzyme activity cannot be measured Since protease peptidase inhibitors used in the usual pfOtein purification process
16. oy SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Quantitative test kit for NAD dependent histone deacetylase activity CycLex SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 b00 Assays Cat CY 1156V2 Intended USe cceeesccceeesseceeseseeeeeeteeeeees 1 SOOO SE A A 1 TintroductiOn ccceeeescceesesseeeseeeceesteeeessnees 2 Principle of the Assay 3 Materials Provided cccsccceesseceeeseeeeees 4 Materials Required but not Provided 4 Precattions esince ites 5 Detailed Protocol eececceessseeeeseteeeeeees 6 8 PrOUDIE SHO OMI cciiancraurseissdesreeaueriiacarentss 9 Reagent SIADU Yi igiacasahivpauniiveascusansnsnaaseuecive 9 Example of Test Results sciisaisscccnsasnssberavsens 10 12 Referen Ce sivcciseSisessacstieanseteaci eee beteccceseaea ts 13 Related Products ccccsccccessseeeeseseeeeeeees 14 Intended Use The CycLex Research Product CycLex SERT6 Deacetylase Fluorometric Assay Kit detects deacetylase activity of recombinant SIRT6 Primarily the CycLex Research Product CycLex SIRT6 Deacetylase Fluorometric Assay Kit is designed for the rapid and sensitive evaluation of SIRT6 inhibitors or activators using recombinanySIRT6 or purified SIRT6 Applications for this kit include 1 Screening inhibitors or activators of SIRT6 2 Detecting the effects of pharmacological agents on SIRT6 This assay
17. this case please reduce the volume of Distilled water to set the final reaction volume of 50 uL Note 6 Although the volume of addition of Recombinant SIRT6 or your Enzyme Sample is set to 5 uL in above tables it may be changed to a volume up to 20 uL at your discretion In that case please reduce the volume of Distilled water to set the final reaction volume of 50 uL Cat CY 1156V2 8 Version 141107 y A SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Troubleshooting 1 When chemicals that have an inhibitory effect on the peptidase are mixed in a crude SIRT6 fraction purified from various cells or the immunoprecipitate using a specific antibody against SIRT6 orother proteins precise SIRT6 enzyme activity cannot be measured Since the protease peptidase_inhibitors used in the usual protein purification process inhibit the peptidase activity strongly please avoid the use of any protease peptidase inhibitors during the protein purification process 2 Final fluorescence intensity will not increase both when test chemicals have an inhibitory effect on SIRT6 and also when there is an inhibitory effect on the peptidase 3 If the test reagents themselves emit fluorescence at excitation wavelength 4802500 nm and fluorescence wavelength 520 540 nm the inhibitory effect of the test assay cannot be evaluated correctly
18. trate the reaction product must be separated from the intact substrate and the fluorescent intensity measured by reverse phase HPLC As mentioned abovetheSe measurement systems are difficult to adapt for processing many samples under a variety of conditions because of their complicated operation Thus a simple system for biochemical analysis asewell asifor inhibitor screening without the use of radioactive substances is preferred Cat CY 1156V2 2 Version 141107 oe SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay CycLex SIRT6 Deacetylase Fluorometric Assay Kit measures the activity of SIRT6 by the basic principle of changing a SIRT6 reaction into the activity of the peptidase In order to measure the enzyme activity of SIRT6 which is the NAD dependent Histone deacetylase and its homolog this kit is designed so that the activity of NAD dependent Histone deacetylase can be measured undef existence of Trichostatin A which is the powerful inhibitor of HDACs In this kit fluorophore and quencher are coupled to amino terminal and carboxyl terminalof substrate peptide respectively and before reaction of deacetylase the fluorescence cannot be emitted However if SIRT6 performs deacetylation substrate peptide will become cut by the action of peptidase added simultaneously quencher will separate from fluorophore and fluorescen
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