CY-1153V2
Contents
1. LIT LEE y OEE EEE P 62 5 250 1000 15 6 Resveratrol conc uM PPP EL GINI 7777777777777777 77777777777777 77777777777777 7777777777 pa 62 5 250 1000 15 6 Quercetin conc uM e gt e e gt e ei e oc N a e oc N peony AYIL ISELI peony APIAQIe aseAJaDvIG w 141107 Version 12 CY 1153V2 9 SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 t Vclex User s Manual For Research Use Only Not for use in diagnostic procedures References Frye R A Biochem Biophys Res Commun 260 273 279 1999 2 Luo J Nikolaev A Y Imai S Chen D Su F Shiloh A Guarente L and Gu W Cell 107 137 148 2001 3 Langley E Pearson M Faretta M Bauer U M Frye R A Minucci S Peli Q and Kouzarides T EMBO J 21 2383 2396 2002 4 Yeung F Hoberg J E Ramsey C S Keller M D Jones D R Frye R ayo M W EMBO J 23 2369 2380 2004 5 Motta M C Divecha N Lemieux M Kamel C Chen D Gu W B McBurney M and Guarente L Cell 116 551 563 2004 6 Brunet A Sweeney L B Sturgill J E Chua K E Greer P L Mostoslavsky R Cohen H Y Hu L S Cheng H L Jedryc D A Alt F W and Greenberg M E Science 303 2011 201 Tran H Ross S E P Gygi S P Sinclair 7 Vaquero A Scher2. Quantit Storage 1 SIRT3 Assay Buffer 1mLx2 2 Fluoro Substrate Peptide 0 2 mM 500 uLx1 Below 20 C Below 20 C Below 20 C 3 Fluoro Deacetylated Peptide 0 2 mM 100 pLx1 4 NAD 2 mM 500 uLx1 5 Developrr O Below 20 C 5 Developer 500 uL x1 70 C 6 Recombinant SIRT3 500 uL x1 70 C 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 Cas CY 1153V2 4 Version 141107 H9 SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 e Vclex 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 SIRT3 There is a possibility that the enzyme activity may be inactivated Aliquot to 10 2
3. 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 theameasurement of SIRT3 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 SIRT3 activity measurement which could not be made by th Conventional method is now possible with the CycLex SIRT3 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 SIRT3 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 1153V2 3 Version 141107 A SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Provided Components of Kit
4. 0 uL and store at 70 C Please avoid mixing of protease peptidase inhibitors such as PMSF or alkyl amine in samples that will be measured SIRT3 activity Do not use kit components beyond the indicated kit expiration 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 1153V2 5 Version 141107 H9 SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol CycLex SIRT3 Deacetylase Fluorometric Assay Kit can measure the enzyme activity of SIRT3 witha homogeneous method In this method the reaction is initiated and the fluorescence intensity is measured by mixing simultaneously fluorescence labeled acetylated peptide
5. 5 uL 5 uL 2 Fluoro Substrate Peptide 5 aL 5 uL 5 uL 5 uL 3 Fluoro Deacetylated Peptide 5 uL 4 NAD SuL 5 uL 5 uL 5 uL Test Compound Sub 5uL Solvent of Test Compound 5uL 5puL Control Compound not provided 5uL 5 Developer 5uL 5uL 5uL 5uL 5uL 6 Recombinant SIRT3 or Enzyme Sample mph us nb i 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 SIRT3 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 of reaction while the reaction velocity remains constant Version 141107 Cat CY 1153V2 7 7 SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 f 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 SIRT3 reaction rate is maintained the difference in fluor scence intensity
6. H9 SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Quantitative test kit for NAD dependent histone deacetylase activity CycLex SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 100 Assays Cat CY 1153V2 Intended Use eee 1 Mrd T 1 Introduction a ENAKAN KENEN a 2 Principle of the Assay eet onbres 3 Materials Provided ss 4 Materials Required but not Provided 4 PLECAULIONS ccccccccecccccccessesesssscsesseseeeseees 5 Detailed Protocol eee 6 8 Troubleshooting 0esenenenenenenanenenanene 9 Reagent Stability a0eeneneenene nenen rentas 9 Example of Test Results eee 10 12 Referentes oirrasan 13 Related Products eee 14 Intended Use The CycLex Research Product CycLex SIRT3 Deacetylase Fluorometric Assay Kit detects SIRT3 activity in lysates Primarily the CycLex Research Product CycLex SIRT3 Deacetylase Fluorometric Assay Kit is designed for the rapid and sen itive evaluation of SIRT3 inhibitors or activators using crude SIRT3 fraction or purified SIRT3 Applications for this kit include 1 Screening inhibitors or activators of SIRT3 2 Detecting the effects of pharmacological agents on SIRT3 This assay kit is for research use only and not for use in diagnostic or therapeutic proc
7. M Lee D Erdjument Bromage H Tempst 93 105 2004 d Reinberg D Mol Cell 16 8 Fulco M Schiltz R L Iezzi S King M T Zhao a aya Y Hoffman E Veech R L and Sartorelli V Mol Cell 12 51 62 2003 9 North B J Marshall B L Borra M T De M and Verdin E Mol Cell 11 437 444 2003 10 Dryden S C Nahhas F A Nowak J tin A S and Tainsky M A Mol Biol Cell 23 3173 3185 2003 11 Onyango P Celic I McCaffery J M Boeke J D and Feinberg A P Proc Natl Acad Sci U S A 99 13653 13658 2002 12 Schwer B North B J Frye Ott M and Verdin E J Cell Biol 158 647 657 2002 13 Yang Y H Chen Y C Y Nimmakayalu M A Ward D C and Weissman S Genomics 69 355 369 Q 2 C CY 1153V2 13 Version 141107 H9 SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 e Vclex 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 SIRTI 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 HDACS Deacetylase Fluorometr
8. between Solvent Control Assay and No Enzyme Control Assay indicates the SIRT3 activity Note 2 In order to estimate the active or inhibitory effect on SIRT3 activity by them st 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 im the Table 2 When test compounds cause an active or inhibitory effect on SIRT3 activity the level of increase of fluorescence intensity is strengthened or weakened as compared with sSolvent Control Assay Note 3 The efficacy of the test compounds on the SIRT3 activity is the difference in fluorescence intensity between Test Compound Assay minus No Enzyme Control Assay and 4Solvent 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 SIRT3 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 eXa
9. edures Storage e Upon receipt store 5 Developer and 6 Recombinant SIRT3 at 70 C and all other components below 20 C Do not exposejreagents to excessive light Cat CY 1153V2 1 Version 141107 SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 Nas bers 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 budding 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 There are seven mammalian Sir2 homologs 1 all of which maintain the catalytic core domain of Sir2 NAD dependent deacetylase activity has been demonstrated for mammalian SIRT1 SIRT2 SIRT3 SIRT5 and SIRT6 proteins The presence of NAD dependent ADP ribosylase and protein deacetylase activities of sirtuin proteins suggests that they may function as sensors of metabolic or oxidative states of cells and regulate cellular functions accordingly Mammalian SIRT1 which resides in the nucleus is the most elosely related to yeast Sir2 SIRT1 binds and deacetylates p53 2 3 NF kappa B 4 forkhead transcription factors 5 6 and histones 7 SIRT1 also suppresses muscle differentiation in response to thesredox state 8 SIRT2 is a cytoplasmic protein which colocalizes with mic
10. ic Assay Kit Ver 2 Cat CY 1158V2 Anti Acetylated Histone p53 K 382 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 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 Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co4p URL http www cyclex co jp CycLex CircuLex products are supplied for research use only CycLex CircuLex products and components th reof 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 1153V2 14 Version 141107
11. niple 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 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 SIRT3 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 1153V2 8 Version 141107 v Pat SIRT3 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 SIRT3 fraction purified from various cells or the immunoprecipitate using a specific antibody against SIRT3 or other proteins precise SIRT3 enzyme activity cannot be measured Since the protease peptidase_inhibitors used in the usual protein purification process inhibit the peptidase activity strongly ple se 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 SIRT3 and also when there is an inhibitory effect on the peptidase 3 If the test reagents
12. nutes 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 velocity 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 1 During the time in which SIRT3 reaction rate is maintained the difference in fluorescence intensity between Enzyme Sample Assay and No Enzyme Control Assay indicates the SIRT3 activity of your Enzyme Sample Cat CY 1153V2 6 Version 141107 SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 User s Manual For Research Use Only Not for use in diagnostic procedures Pat clex Note 2 Although the volume of addition of Enzyme Sample or Buffer of Enzyme Sample or 6 Recombinant SIRT3 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 Cont
13. pendency curve of recombinant SIRT3 activity SIMU Eee rere ettet tenerte 25 000 20 000 15 000 10 000 Deacetylase activity counts min 5 000 0 200 400 600 800 1 000 SIRT3 ng Fig 2 Time course of SIRT3 substrate deacetylation by fecombinant SIRT3 9 1000ng 500ng B 250ng 62 5ng UIN 31 25ng Cat CY 1153V2 10 Version 141107 User s Manual NG NANA NANA NAN ANNA NAN NAN NA NAN ESE EEE SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 For Research Use Only Not for use in diagnostic procedures 3 000 e a 4 amp wm N N sjuno2 OT X O9PA SSEA Fig 3 Effect of Trichostatin A and NAD on recombinant SIRT3 activity oy V 141107 Version 0 9939 30 1 uM TSA 3E 05x 4E 06 gp 200uM NAD 20 y Km 0 13 S 11 gt i 0 0008 0 0006 0 0004 0 0002 0 0000 0 0002 0 0004 0 0006 Fig 4 Km value of recombinant SIRT3 for Fluoro Substrate Peptide CY 1153V2 Cati E 3 a 9 e a X 6 2 3 5b A E o Q lt 5 o 34 o0 2 9 a on ES 22 5 Sua dir mg o ZZ awa E E ES 9 o v o N A gt en c E m E A e e E e E clex y y Fig 5 Effect of polyphenol on recombinant SIRT3 activity ge AT EEE
14. product must be separated from the iftact substrate and the fluorescent intensity measured by reverse phase HPLC As mentioned above tlies amp measurement systems are difficult to adapt for processing many samples under a variety ofycondifions because of their complicated operation Thus a simple system for biochemical analysis as welas for inhibitor screening without the use of radioactive substances is preferred Cat CY 1153V2 2 Version 141107 oe SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 e ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay CycLex SIRT3 Deacetylase Fluorometric Assay Kit measures the activity of SIRT3 by the basic principle of changing a SIRT3 reaction into the activity of the peptidase In order to measure the enzyme activity of SIRT3 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 terminal of substrate peptide respectively and before reaction of deacetylase the fluorescence cannot be emitted However if SIRT3 performs deacetylation substrate peptide will become cut by the action of peptidase added simultaneously quencher will separate from fluorophore and fluorescence will be emitt
15. rol Assay the SIRT3 activity in the samples cannot be evaluated correctly Note 4 If enzyme samples contain inhibitors for protease peptidase precise SIRT3 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 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 1 STIRT3 Assay Buffer 2 Fluoro Substrate Peptide or 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 uL 20 uL 25uL 30 uL 1 SIRT3 Assay Buffer 5 uL 5 uL 5 uL
16. rotubules and deacetylates alplfa tubulin 9 SIRT2 abundance increases during mitosis suggesting that the protein plays a role in cell cycle regulation 10 Human SIRT3 is a mitochondria protein with its N terminal 25 amino acid residues responsible for its mitochondrial localization 11 12 Synthesized as an enzymatically inactive protein human SIRT3 is activated by mitochondrial matrix processing peptidase to active 28 kD active enzyme 13 These Observations suggest that the existence of a latent class III deacetylase that becomes catalytically activated upon import into the human mitochondria However the conventional method for measuring SIRT3 activity is Very complicated and laborious In order to measure SIRT3 enzyme activity it is necessary to prepare radioactive acetylated histone or p53 as a substrate First cells have to be labeled metabolically with radioactivity by adding radioactive acetic acid to the culture medium Second radioactive acetylat d 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
17. themselves emit fluorescence at excitation wavelength 340 360 nm and fluorescence wavelength 440 460 nm the inhibitory effect of the test assay cannot be evaluated correctly 4 The recombinant SIRT1 should be run in duplicate using the protog l 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 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 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 CycLex Research Product CycLex SIRT3 Deacetylase Fluorometric Assay Kit have been tested fof 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 420 C Cat CY 1153V2 9 Version 141107 A SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose de
18. which is a substrate SIRI3 NAD and the developer Since the reaction is not stopped it 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 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 SIRT3 Activity 1 Following Table 1 below first add Distilled water 1 SIRT3 Z 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 SIRT3 activity Enzyme No Enzyme Positive No NAD Assay reagents Sample Control Control Control Assay Assay Assay Assay Distilled water 25 uL 254uL 25 uL 30 uL 1 SIRT3 Assay Buffer 5uL 5 aD 5uL 5 uL 2 Fluoro Substrate Peptide 5uL SuL 5 uL 5 uL 4 NAD 5uL 5uL 5uL 5 Developer 5uL 5uL 5uL 5 uL Enzyme Sample 5uL 5 uL Buffer of Enzyme Sample 5uL 6 Recombinant SIRT3 gt 5uL 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 SIRT3 to each wellgand mixing thoroughly at room temperature 3 Read fluorescence intensity for 30 to 60 mi
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