Data Sheet
Contents
1. Below 20 C 5 Developer 500 uL x1 70 C 6 Recombinant SIRT1 500 uL x1 20 7 Stop Solution 1 mL x2 Below 20 C Instruction manual 1 Room temp Microplate for fluorometer detection of emitted light in the range 440 460 nm Multi channel pipette Microplate shaker Deionized water of the highest quality 500 or 1000 mL graduated cylinder Reagent reservoirs Control compound s Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Materials Required but not Provided Microplate reading fluorometer capable of excitation at a wayelength in the range 340 360 nm and Cat CY 1151V2 4 Version 150520 H9 SIRTI Sir2 Deacetylase Fluorometric Assay Kit Ver 2 e User s Manual For Research Use Only Not for use in diagnostic procedures Precautions 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 SIRT1 There is a possibility that the enzyme activity may be inactivated Aliquot to 10 20 uL and store at 70 Please avoid mixing of protease peptidase inhibitors such as PMSF or alkyl amine in samples that will be measured SIRTI Sir2 activity Do not use kit components beyond the indicated kit expirati2. Discard the supernatant Wash the nuclei pellet oneeswithicold 10 mM Tris HCI pH7 5 10 mM NaCl OVP Extraction of Nuclei 1 Suspend the isolated nuclei in 50 100 uL of extraction buffer Sonicate for 30 seconds Stand on ice fof 30aminutes Microcentrifuge at 20 000 x g for 10 minutes Take supernatant the crude nuclear extract Determine protein conc by Bradford method or equivalent Store tlie crude nuclear extract at 70 C until use SOY GRE 93 PS Note Do not use any kind of protease peptidase inhibitor Cat CY 1151V2 10 Version 150520 oe SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit Ver 2 e User s Manual For Research Use Only Not for use in diagnostic procedures SIRT1 Activity in an Immunoprecipitate Immunoprecipitation Followed by Measuring SITR1 Activity Protocol 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 Solutions and Reagents Note Prepare solutions with Milli Q or equivalently purified water Cell Lysis Buffer 1X 20 mM Tris HCI pH 7 5 250 mM NaCl 1 mM T mM EGTA 1 Triton X 100 1 mM DTT 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 wee
3. o syunod 201 X 09rA sseA A S O 3 s gt bn IL Cat CY 1151V2 Pat For Research Use Only Not for use in diagnostic procedures SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit Ver 2 User s Manual Fig 5 Substrate Preference of HDAC and SIRTI F355 F460 x 10 counts Sir2 substrate CycLex Sir2 Assay kit 10 000 p 9 000 8 000 7 000 6 000 5 000 4 000 3 000 2 000 1 000 e crude HDAC Recombinant SIRT1 Time min F355 F460 x 10 counts lt HDAC substrate CycLex HDAC Assay kit gt crude HDAC e Recombinant SIRT1 20 30 40 50 60 Time min Fig 6 Stability of Fluorescence Intensity after Stop the Reaction F355 F460 x 10 counts SIRTI 250ng HF SIRTI 93ng amp SIRTI 46 5ng SIRTI 15 625ng Time min Cat CY 1151V2 14 Version 150520 SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit Ver 2 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 SIRTI antibody CY P1016 400 e 350 x B9 T gE 250 O o S gg 2 o gt 150 100 sm E 0 o SEA E 8
4. 5 XE T 223 112 Es FES ww C CY 1151V2 15 Version 150520 Fg SIRTI Sir2 Deacetylase Fluorometric Assay Kit Ver 2 ee 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 A 97 6658 6663 2000 4 Vaziri H et al Cell 107 149 159 2001 5 Luo J et al Cell 107 137 148 2001 Qy 6 Langley E et al EMBO J 21 2383 2396 2002 7 Smith J Trends Cell Biol 12 404 2002 8 Grozinger CM and Schreiber SL Chem Biol 9 3 16 2002 Q 9 Sereno A et al Antimicrob Agents Chemother 49 808 812 200 10 Nicola Ferrara et al Rejuvenation Research 11 139 150 20 11 Rameshwar U et al Chemical Biology amp Drug Design 71 501 506 2008 12 Fan Lan et al J Biol Chem 283 27628 2008 13 Joana TAVARES et al Biochemical Journal 41 86 2008 14 Yue Zhao et al Mol Cell Biol Dec 2008 10 1128 MCB 02123 07 C CY 1151V2 16 Version 150520 H9 SIRTI Sir2 Deacetylase Fluorometric Assay Kit Ver 2 e 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
5. important role for SIRTI in modulating the sensitivity of cells in p53 dependent apoptotic response and the possible effect in cancer therapy Since the function of p53 is made to strengthen powerfully by using together with DNA damaging reagent it is expected that inhibitor of SIRT 1 becomes an effective anticancer drug However the conventional method for measuring SIRTI Sir2 activity is vety complicated and laborious In order to measure SIRTI Sir2 enzyme activity it is necessaryzto prepare radioactive acetylated histone 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 separate 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 deacetylase without the use of radioactive substances was reported in recent years owing to the use of fluorescent labeled acetylated lysine as a substrate the reaction product must be separated from the intact substrate 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 variety of conditions becaus of their complicated operation Th
6. 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 NMNATI Nicotinamide Mononucleotide Adenylyltransferase 1 s at CY 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 Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co jp URL http www cy lex co j p 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
7. RT1 The following protogols have been shown to work with a number of different cells and enzyme sources and are provided as examples of suitable methods Crude samples can frequently be used without dilution while more concentrated or highly purified SIRT1 should be diluted It is strongly advised that the user always perform an initial experiment to determine the proper dilution to be used in subsequent experiments This need not be any more than a single time point assay using serial dilutions of the crude extract cell 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 activity 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 Buffers Lysis Buffer Sucrose cushion Extraction buffer 10 mM Tris HCl pH7 5 30 Sucrose 50 mM Hepes KOH pH 7 5 10 mM NaCl 10 mM Tris HCl pH7 5 420 mM NaCl 15 mM MgCl 10 mM NaCl 0 5 mM EDTA Na 250 mM Sucrose 3 mM MgCl 0 1 mM EGTA 0 5 NP 40 10 glycerol 0 1 mM EGTA Procedure Isolation of Nuclei 1 Suspend 1x 10 cells 100 mm dishg iib confluent into 1ml of Lysis buffer Vortex for 10 seconds Keep on ice for 15 minutes Spin the cells through 4 ml of suerose cushion at 1 300 g for 10 minutes at 4
8. a ff orescent reader for microtiter plates Considering that the use of fully automatic apparatus to measure fluorescence intensity has become widespread SIRTI Sir2 activity measurement which gould ret be made by the conventional method is now possible with the CycLex SIRTI Sir2 Deacetylase Fluorometric Assay Kit using the same equipment This new method of measurement should dramatically raise the efficiency of inhibitor screening and biochemical analysis of these enzymes Measuring Principle of The CycLex SIRT1 Sir2 Deacetylase Fluorometric Assay Kit fluorophore X X X Lys Ac X X qu ncher Deacetylase fluorophore X X X Lys X X quencher lt _ Peptidase fluorophore X X X Lys X X quencher Measurement of fluorescence intensity Note This measuring principle and kit are covered under CycLex s patents U S Patent N0 7 033 778 and No 7256013 European Patent No 1243658 Japanese Patent No 4267043 Canadian Patent No 2392711 Cat CY 1151V2 3 Version 150520 A SIRT1 Sir2 Deacetylase Fluorometric Assay Kit Ver 2 User s Manual For Research Use Only Not for use in diagnostic procedures Materials Provided Components of Kit Quantit Storage 1 SIRT1 Assay Buffer 1mLx2 2 Fluoro Substrate Peptide 0 2 mM 500 uL x1 Below 20 C Below 20 C Below 20 C 3 Fluoro Deacetylated Peptide 0 2 mM 100 pLx1 4 NAD 2 mM 500 uL x1 5 Developrr
9. etermine reaction velocity Alternatively within a time in which the reaction velocity is kept constant it is also possible to stop the reaction by adding stop solution and to measure fluorescence intensity 1 Assay Method for Measurement of SIRT1 Sir2 Activity 1 Following Table 1 below first add Distilled water 1 SIRT1 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 SIRT1 Sir2 activity Enzyme No Enzyme Positive No NAD Assay reagents Sample Control Control Control Assay Assay Assay Assay Distilled water 25 uL 2541 25 uL 30 uL 1 SIRT1 Assay Buffer 5uL 5 5 5 2 Fluoro Substrate Peptide 5uL SuL 5 uL 5 uL 4 NAD 5uL 5uL 5uL 5 Developer 5uL 5uL 5uL 5 Enzyme Sample 5uL 5 Buffer of Enzyme Sample 5 6 Recombinant SIRT1 gt 5 uL Total Volume of the mixture 50 ub 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 SIRTT 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 a 340 360 nm and emission at 440 460 nm Measure and calculate the rate of reaction while the reaction ve
10. f protease peptidase inhibitor Cat CY 1151V2 11 Version 150520 ry SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit Ver 2 c User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency curve of recombinant SIRTI activity F355 F460 x10 counts SIRT1 ng Fig 2 Time course of SIRT 1 substrate deacetylation by recombinant SIRT1 10 000 8 000 E 2 6 000 a SIRTIfull 7 8125ng x 4 000 SIRTIfull 15 625ng m NSIRTI full 31 25ng wi 2 000 EF SIRTIfull 62 5ng E 9 SIRT full 125ng 0 0 10 20 30 Time min Cat CY 1151V2 12 Version 150520 150520 Version User s Manual SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit Ver 2 For Research Use Only Not for use in diagnostic procedures 12 000 40 60 80 S 20 13 0 0005 20 Fig 3 Effect of Trichostatin A and NAD on recombinant SIRTI activity a lt lt S z eo of oN RSE fon a s KS Q I m Il gt e i 2 2 y x 5 3 es S E i x n J w lt w w lt w z z s s e en N e lt T e gt S e lt 8 S lt d F d lt lt lt amp lt 2 Qe E c e e e lt c
11. idase precise SIRT 1 Sir2 enzyme activity cannot be measured Since protease peptidase inhibitors used in the usual pfotein purification process strongly inhibit the peptidase activity in the development reaction pl as avoid using any protease peptidase inhibitors during the process of protein purification 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 1 STRT1 Assay Buffer 2 Fluoro Substrate Peptide 3 Fluoro Deacetylated Peptide and 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 well of the microtiter plate and mix well Table 2 Reaction mixture for inhibitor activator screening Test Solvent Control No Enzyme Development Assay reagents Compound Control Compound Control Control Assay Assay Assay Assay Assay Distilled water 20 uL 20 pL 20 uL 25 uL 30 uL 1 SIRT1 Assay Buffer 5 uk 5 uL 5 uL 5 uL 5 uL 2 Fluoro Substrate Peptide 5 aL 5uL 5uL 5 3 Fluoro Deacetylated Peptide 5 4 SpE 5 5 5 Test Comp
12. ks at 4 C Preparing Cell Lysates 1 Aspirate media Treat cells by adding fresh media containing test compound for desired time 2 To harvest cells under nondenaturing conditions remov media and rinse 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 microcentrifuge 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 besstored at 70 C Immunoprecipitation 1 Take 200 uL cell lysate and add aftt SIRTT antibody CY P1016 1 2 ug incubate with gentle rocking for 2 hours or overnight at 4 C 2 Add protein A agarose beads 2050 68509 bead slurry Incubate with gentle rocking for 1 3 hours at 4 C 3 Microcentrifuge for 30 seconds at 4 C Wash pellet 3 times with 500 uL of 1X Cell Lysis Buffer and once with 500 uL of c ld 50 mM Tris HCl pH 8 8 0 5 mM DTT Keep on ice during washes 4 After immunoprecipitation according to the procedure of 1 Assay Method for Measurement of SIRTI Sir2 Activity in the Detailed Protocol add reaction mixture containing Fluoro Substrate peptide solution to protein A agarose beads as an Enzyme Sample and measure NAD dependent deacetylase acti vity Note Do not use any kind o
13. locity remains constant Alternate procedure 3 While the reaction rat is kept constant add 20 uL of 7 Stop Solution to each well at appropriate time to stop the reactior and measure fluorescence intensity in a microplate fluorescence reader capable of excitation at a wavelength in the range 340 360 nm and detection of emitted light in the range 440 460 nim Note 1z During theftime in which SIRT 1 Sir2 reaction rate is maintained the difference in fluorescence intensity between Enzyme Sample Assay and No Enzyme Control Assay indicates the SIRTT Sir2 activity of your Enzyme Sample Cat CY 1151V2 6 Version 150520 Pat Note 2 Note 3 Note 4 Note 5 SIRTI Sir2 Deacetylase Fluorometric Assay Kit Ver 2 User s Manual For Research Use Only Not for use in diagnostic procedures Although the volume of addition of Enzyme Sample or Buffer of Enzyme Sample 6 Recombinant SIRTT 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 If enzyme samples contain some protease peptidase able to break down 2 FIuor Substrate Peptide resulting in an increase of fluorescence intensity in No NAD Control Assay the SIRT 1 Sir2 activity in the samples cannot be evaluated correctly If enzyme samples contain inhibitors for protease pept
14. nd fluorescence wavelength 440 460 nm the inhibitory effect of the test assay annot be evaluated correctly 4 The recombinant SIRT1 should be run in duplicate using the protocol described in the Detailed Protocol Incubation times or temperatures significantly different frombstbose specified may give erroneous results 5 The reaction curve is nearly a straight line if the kinetics of the assay Is 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 Af all instructions in the Detailed Protocol were followed accurately such results indicate a needfor multi channel pipettor maintenance Reagent Stability All of the reagents included in the CycLex Research Product CycLex SIRTI Sir2 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 SIRT1 at 70 C all other kit reagents should be stored below 20 Cat CY 1151V2 9 Version 150520 oe SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit Ver 2 e yclex 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 SI
15. oe SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit Ver 2 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 Ver 2 100 Assays Cat CY 1151V2 Intended USE c 1 SOE BE M PR PN 1 2 Principle of the Assay 3 Materials Provided 4 Materials Required but not Provided 4 lies rej Me DUET 5 Detailed Protocol 6 8 Troubleshooting 9 Reagent Stability 9 Sample Preparation 10 SIRTI Activity in an Immunoprecipitate 1 1 Example of Test Results 1245 ion e 16 Related Products a 17 Intended Use The CycLex Research Product CycLex SIRTI Sir2 Deacetylase Fluorometric Assay Kit detects SIRTI Sir2 activity in lysates Primarily the CycLex Research Product CycLex SIRT1 Sir2 Deacetylase Fluorometric Assay Kit is designed for the rapid and sensitive evaluation of SIRT1 Sir2 inhibitors or activators using crude SIRT1 Sir2 fraction or purified SIRTI Sir2 Additionally any cultured primary cell cell line or tissue homogenate can be a
16. on date Rinse all detergent residue from glassware Use deionized water of the highest quality 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 or in areas where samples or reagents are handled 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 1151V2 5 Versionft 150520 H9 SIRTI Sir2 Deacetylase Fluorometric Assay Kit Ver 2 v User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol CycLex SIRT1 Sir2 Deacetylase Fluorometric Assay Kit can measure the enzyme activitygof SIRT1 Sir2 with a 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 SIRTI Sir2 NAD and the developer Since the reaction is not stopped it is pecessary to measure fluorescence intensity at regular intervals after the reaction is initiated and to d
17. ound Sub 5 Solvent of Test Compound 5uL 5 Control Compound not provided 5 5 Developer 5uL 5uL 5uL 5uL 5uL 6 Recombinant SIRT1 or Enzyme Sample mph us ian T i Total Volume of the mixture 50 uL 50 uL 50 uL 50 uL 50 uL 2 Initiate reactions by adding 5 uL of 6 Recombinant SIRT1 or your Enzyme Sample to each well and mixing thoroughly at room temperature 3 Read fluorescence intensity for 30 to 60 minutes at to 2 minute intervals using microtiter plate fluorometer With excitation at 340 360 nm and emission at 440 460 nm Measure and calculate the rate reaction while the reaction velocity remains constant Version 150520 Cat 1151 2 7 7 SIRTI Sir2 Deacetylase Fluorometric Assay Kit Ver 2 Vclex 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 Teader capable of excitation at a wavelength in the range 340 360 nm and detection of emitted light in the range 440 460 nm Note 1 During the time in which SIRT1 Sir2 reaction rate is maintained the difference in fluor scence intensity between Solvent Control Assay and No Enzyme Control Assay indicates the SIRT1 Sir2 activity Note 2 In order to es
18. prior written approval from CycLex Co Ltd To inquire about licensing for such commercial use please contact us via email Cat CY 1151V2 17 Versionft 150520
19. ssayed for SIRTI Sir2 activity with the CycLex Research Product CycLex SIRTI Sir2 Deacetylase Fluorometric Assay Kit if the appropriate antibody direct against SIRT1 or Sir2 is used for immunoprecipitation Applications for this kit include 1 Monitoring the purification o SIRTI Sir2 2 Screening inhibitors r activators of SIRTI Sir2 3 Detecting the effeetsyof pharmacological agents on SIRT1 Sir2 This assay kit is fomresearch use only and not for use in diagnostic or therapeutic procedures Storage Upon receipt store 5 Developer and 6 Recombinant SIRTI at 70 C and all other components below 20 C Do not expose reagents to excessive light Cat CY 1151V2 1 Version 150520 SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit Ver 2 bers i y gt 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 A human homologue of Sir2 SIRT1 also functions as a NAD dependent p53 deacetylase as well as a NAD dependent histone deacetylase SIRT1 was shown to regulate the activity of the p53 tumor suppressor and inhibits apoptosis These results have significant implications regarding an
20. t Compound or Solvent of Test Compound or Control Compound not provided In 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 SIRT1 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 th volume of Distilled water to set the final reaction volume of 50 uL Cat CY 1151V2 8 Versionft 150520 oe SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit Ver 2 e 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 SIRT 1 Si2 fraction purified from various cells or the immunoprecipitate using a specific antibody against SIRTI1 Sir2 or other proteins precise SIRTI Sir2 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 SIRTI Sir2 and also when there is an inhibitory effect on the peptidase 3 If the test reagents themselves emit fluorescence at excitation wavelength 340 360 nm a
21. timate the active or inhibitory effect on SIRT 1 Sir2 activitysby thestest compounds correctly it is necessary 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 in the Table 2 When test compounds cause an active or inhibitory effect on SIRTI Sir2 activity the level of increase of fluorescence intensity is strengthened or weakened as compared with Solvent Control Assay Note 3 The efficacy of the test compounds on the SIRT1 Sir2 activity is the difference in fluorescence intensity between Test Compound Assay minus No Enzyme Control Assay and 4Solvent Control Assay minus No Enzyme ControlAssay Note 4 If test compounds have an inhibitory effect on protease peptidase resulting that the increase in fluorescence intensity is not or a little observed in Development Control Assay the effect on SIRT1 Sir2 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 example since the final volume of reaction is 50 uL here it is also possible to add 10 uL of Tes
22. us a simple system for biochemical analysis as well as for inhibitor Screening without the use of radioactive substances is preferred Cat CY 1151V2 2 Version 150520 oe SIRT 1 Sir2 Deacetylase Fluorometric Assay Kit Ver 2 yclex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay CycLex SIRT1 Sir2 Deacetylase Fluorometric Assay Kit measures the activity of SIRTI Sir2 byahe basic principle of changing a SIRT 1 Sir2 reaction into the activity of the peptidase In order to measure the enzyme activity of SIRTI Sir2 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 measuredjunder existence of Trichostatin A which is the powerful inhibitor of HDACs In this kit fluorophore and quencher are coupled to amino terminal and carboxyl terminal of substrate peptide respectively and before reaction of deacetylase the fluorescence cannot be emitted However if SIRTI Sir2 performs deacetylation substrate peptide will become cut by the action of peptidase added simultaneously quencher will separate from fluorophore and fluorescence 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 pricey the Measurement of SIRTI Sir2 activity in most laboratories is possible if they are equipped with
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