CY-1152V2
Contents
1. C Cat CY 1152V2 9 Version 141107 ry SIRT2 Deacetylase Fluorometric Assay Kit Ver 2 oS 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 4 000 Ca N 2 3 000 i N S 2 000 1 000 0 0 0 0 5 1 0 1 5 2 0 2 5 SIRT2 ug Fig 2 Time course of SIRT2 substrate deacetylation bywfecombinant SIRT2 14 000 p e 2 0 ug e 1 0 ug 12 000 e 0 5 ug 10 000 0 25 ug 2 x 8 000 N iA amp 6 000 N a0 wt 4 000 2 000 0 0 20 40 60 80 100 120 Reaction time min Cat CY 1152V2 10 Version 141107 Fu SIRT2 Deacetylase Fluorometric Assay Kit Ver 2 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Effect of Trichostatin A and NAD on recombinant SIRT2 activity 2 500 2 000 1 500 1 000 F485 F535 x10 500 TSA TSA NAD gt Fig 4 Effect of Sirtinol on recombinant SITR2 ov 6 000 r 5 000 4 000 w N F485 F535 x10 1 000 o MM i W m 0 15 6 62 5 250 1000 No No enzyme NAD Sirtinol conc uM C CY 1152V2 11 Version 141107 SIRT2 Deacetylase Fluorome2. Joseph Landry Ann Sutton Stefan T Tafrov Ryan C Heller John Stebbins Lorraine Pillus and Rolf Sternglanz The silencing protein SIR2 and its homologs are NAD dependent protein deacetylases Proc Natl Acad Sci U S A 97 5807 5811 2000 7 Jeffrey S Smith Carrie Baker Brachmann Ivana Celic Margaret A Kenna Shabazz Muhammad Vincent J Starai Jose L Avalos Jorge C Escala te Semerena Charles Grubmeyer Cynthia Wolberger and Jef D Boeke A phylogenetically conserved NAD dependent protein deacetylase activity in the Sir2 protein family Proc Natl Acad Sci US A 97 6658 6663 2000 oo H Vaziri SK Dessain E Ng Eaton SI Imai RA Frye TK Pandita L Guarente and RA Weinberg hSIR2 SIRT1 functions as an NAD dependent p53 deacetylase Cell 107 149 159 2001 9 J Luo AY Nikolaev S Imai D Chen F Su AShiloh L Guarente and W Gu Negative control of p53 by Sir2alpha promotes cell survival understressyCell 107 137 148 2001 10 E Langley M Pearson M Faretta UM Bauer RA Frye S Minucci PG Pelicci and T Kouzarides Human SIR2 deacetylates p53 ahd antagonizes PML p53 induced cellular senescence EMBO J 21 2383 2396 2002 11 J Smith Human Sir2 and the silencing of p53 activity Trends Cell Biol 12 404 2002 12 CM Grozinger and SL Schreiber Deacetylase enzymes biological functions and the use of small molecule inhibitors Chem Biol 9 3 16 2002 Cat CY 1152V2 13 Version 141107 oy SIRT2 Deacetylase F
3. nm and detection of emitted lightsin the range 520 540 nm Note 1 During the time in which SIRT2 reaction rate is maintained the difference in fluor scence intensity between Solvent Control Assay and No Enzyme Control Assay indicates the SIRT2 activity Note 2 In order to estimate the active or inhibitory effect on SIRT2 activity by thept 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 SIRT2 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 SIRT2 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 SIRT2 activity cannot be evaluated correctly Note 5 Although the above tables indicate the volume of addition of Test Compound or Solvent of Test Compoun
4. asawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex cop URL http www cyclex 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 1152V2 14 Version 141107
5. ase peptidase able to break down 2 Fluor6 Stibstrate Peptide resulting in an increase of fluorescence intensity in No NAD Control Assay the SIRT2 activity in the samples cannot be evaluated correctly Note 4 If enzyme samples contain inhibitors for protease peptidase precise SIRT2 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 s 1 SIRT2 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 Test Solvent Control No Enzyme Development Assay reagents Com
6. binant SIRT2 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 SIRT2 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 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 1152V2 5 Version 141107 oy SIRT2 Deacetylase Fluorometric Assay Kit Ver 2 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol CycLex SIRT2 Deacetylase Fluorometric Assay Kit can measure the enzyme activity of SIRT2 witha homogeneous method In this method the reaction is initiated and the
7. ction 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 price theameasur ment of SIRT2 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 SIRT2 activity measurement which could not be mad byth Conventional method is now possible with the CycLex SIRT2 Deacetylase Fluorometric Assay Kit using the same equipment This new method of measurement should dramatically raise the efficiencyjof inhibitor screening and biochemical analysis of these enzymes Measuring Principle of The CycLex SIRT2 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 1152V2 3 Version 141107 A SIRT2 Deacetylase Fluorometric Assay Kit Ver 2 ycLex User s Manual Fo
8. d 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 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 SIRT2 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 1152V2 8 Version 141107 y A SIRT2 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 SIRT2 fraction purified from various cells or the immunoprecipitate using a specific antibody against SIRT2 orother proteins precise SIRT2 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 p
9. fluorescence intensity is measured by mixing simultaneously fluorescence labeled acetylated peptide which is a substrate SIRT2 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 possible to stop the reaction byjadding stop solution and to measure fluorescence intensity 1 Assay Method for Measurement of SIRT2 Activity 1 Following Table 1 below first add Distilled water 1 SIRT2 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 SIRT2 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 SIRT2 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 SIRT2 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 Recomb
10. g a proteomic approach 4 The SIRT2 gene which is located at chromosome 19q13 2 lies within a region that is ff quently deleted in human gliomas and levels of SIRT2 mRNA and protein expression are severely reduced in a large fraction of human glioma cell lines 4 Ectopic expression of SIRT2 if th se cell lines suppressed colony formation and modified the microtubule network These results indicate that SIRT2 may act as a tumor suppressor and may function to control the cell cycle by acetylationfof alpha tubulin It was reported that SIRT2 inhibitor rescued alpha synuclein toxicity and modified inclusion morphology in a cellular model of Parkinson s disease however the exact mechanism remains uncertain However the conventional method for measuring SIRT2 activity is very complicated and laborious In order to measure SIRT2 enzyme activity it is necessary to prepare radioactive acetylated histone H4 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 repo
11. gents on SIRT2 This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage e Upon receipt store 5 _Developer and 6 Recombinant SIRT2 at 70 C and all other components below 20 C e Do not exposewreagents 0 excessive light Cat CY 1152V2 1 Version 141107 SIRT2 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 SIRT 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 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 I HDAC and deacetylation by HDAC6 deadspto changes in cellular motility 3 A role for SIRT2 in cancer pathogenesis was demonstrated usin
12. inant SIRT2 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 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 SIRT2 reaction rate is maintained the difference in fluorescence intensity between Enzyme Sample Assay and No Enzyme Control Assay indicates the SIRT2 activity of your Enzyme Sample Cat CY 1152V2 6 Version 141107 oy SIRT2 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 SIRT2 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 prote
13. luorometric 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 Ter
14. oy SIRT2 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 SIRT2 Deacetylase Fluorometric Assay Kit Ver 2 100 Assays Cat CY 1152V2 Intended USe ccccccccesseccceeesessseceeeeeseees 1 UBT ES css E E ET 1 TntroductiOn ccccccsscccccessesseeceeeeseesteeees 2 Principle of the Assay 3 Materials Provided ccccccccccessssssseeeceeees 4 Materials Required but not Provided 4 PLECAULIONS cccsesseccceecesssssseeeeceesessseeeeees 5 Detailed Protocol ccccccccccsssssseeeceeeeeeees 6 8 Troubleshoot sisssccassccdssscscasnsosnsesancnareansens 9 Reagent SADLY ccvccsarcovsncasasesasesassernsasaareany 9 Example of Test Results eee 10 12 ReferencCes ccccesesccecescessseceeceeceesssaseeeeees 13 Related Products ccccccccccssssccceeeessseeeeees 14 Intended Use The CycLex Research Product CycLex SIRT2 Deacetylase Fluorometric Assay Kit detects deacetylase activity of recombinant SIRT2 Primarily the CycLex Research Product CycLex SIRT2 Deacetylase Fluorometric Assay Kit is designed for the rapid and sensitive evaluation of SIRT2 inhibitors or activators using recombinant SER T2 of purified SIRT2 Applications for this kit include 1 Screening inhibitors or activators ofSIRT2 2 Detecting the effects of pharmacological a
15. pound Control Compound Control Control Assay Assay Assay Assay Assay Distilled water 20 pL 20 uL 20 pL 25 pL 30 pL 1 SIRT2 Assay Buffer 5 nb 5 wL 5 uL 5 uL 5 uL 2 Fluoro Substrate Peptide 5 aL 5 pL 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 5 uL 5 Developer 5 uL 5 uL 5 uL 5 uL 5 uL 6 Recombinant SIRT2 or Enzyme Sample gt uL Syl SL i i 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 SIRT2 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 1152V2 T Version 141107 SR SIRT2 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
16. r Research Use Only Not for use in diagnostic procedures Materials Provided Components of Kit Quantit Storage 1 SIRT2 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 8 mM 500 uLx1 5 Developrr S O Below 20 C 5 Developer 500 uLx1 70 C 6 Recombinant SIRT2 500 uLx1 70 C 7 Stop Solution 1 mL x2 Below 20 C Instruction manual 1 Room temp Materials Required but not Provided Microplate for fluorometer detection of emitted light in the range 520 540 nm 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 1152V2 4 Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Version Microplate reading fluorometer capable of excitation at a wavelengthin the range 480 500 nm and 141107 oy SIRT2 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 Recom
17. rted in recent years owing to the use of fluorescent labeled acetylated lysine as a substrate the reaction product must be separated from the ghtact gt substrate and the fluorescent intensity measured by reverse phase HPLC As mentioned above thes 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 1152V2 2 Version 141107 oe SIRT2 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 SIRT2 Deacetylase Fluorometric Assay Kit measures the activity of SIRT2 by the basic principle of changing a SIRT2 reaction into the activity of the peptidase In order to measure the enzyme activity of SIRT2 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 terminaljof substrate peptide respectively and before reaction of deacetylase the fluorescence cannot be emitted However if SIRT2 performs deacetylation substrate peptide will become cut by the a
18. tric Assay Kit Ver 2 1 ANyCLex 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 x ra w 3 000 x wn 4 2 000 1 000 0 Vs 0 15 6 62 5 250 1000 No No enzyme NAD Reveratrol conc uM C CY 1152V2 12 Version 141107 SIRT2 Deacetylase Fluorometric Assay Kit Ver 2 jde Uers i GA y gt X ser s Manu For Research Use Only Not for use in diagnostic procedures References j North B J and Verdin E Sirtuins SIRT2 related NAD dependent protein deacetylases Genome Biol 5 224 2004 2 North BJ Marshall BL Borra MT Denu JM Verdin E The human SIRT2 ortholog ASIRT2 isan 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 T Ohama E Tahimic CG Kurimasa A Oshimura M Proteomics based identification of differentially expressed genes in human gliomas down regulation of SIRT2 gene BBRC 309 558 566 2003 A S Imai CM Armstrong M Kaeberlein and L Guarente Transcripti nal silencing and longevity protein Sir2 is an NAD dependent histone deacetylase Nature 403 795 800 2000 6
19. urification process 2 Final fluorescence intensity will not increase both when test chemicals have an inhibitory effect on SIRT2 and also when there is an inhibitory effect on the peptidase 3 If the test reagents themselves emit fluorescence at excitation wavelength 480 500 nm and fluorescence wavelength 520 540 nm the inhibitory effect of the test assay cannot be evaluated correctly 4 The recombinant SIRT2 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 SIRT2 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 SIRT2 at 70 C all other kit reagents should be stored below 420
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