CY-1150V2
Contents
1. S ul Buffer of Enzyme Sample 5uL 6 HDACs 5uL Total Volume of the mixture 50 uL 50 uL 50 uL 50 uL 2 Initiate reactions bya dding SuL of your Enzyme Sample or Buffer of Enzyme Sample or 6 HDACs to each well andamixing thoroughly at room temperature 3 Read fluorescence intensity for 30 to 60 minutes at 1 to 2 minute intervals using microtiter plate fluorometer gwithpexeitation at 350 380 nm and emission at 440 460 nm Measure and calculate the rate of reactionowhile the reaction velocity remains constant Altetnate procedure 3 While the reaction rate is kept constant add 20 uL of 7 Stop Solution to each well at appropriate Cat CY 1150V2 5 Version 141107 oe HDACs Deacetylase Fluorometric Assay Kit Ver 2 e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures time to stop the reaction and measure fluorescence intensity in a microplate fluorescence reader capable of excitation at a wavelength in the range 350 380 nm and detection of emitted light in the range 440 460 nm 2 Two Step Method 1 Following Table 2 below first add Distilled water 4 1 HDAC Assay Buffer and 4 2 Fluoro Substrate Peptide to microtiter plate wells Second add 4 Trichostatin A to each well of the microtiter plate and mix well Table 2 Reaction mixture of Two Step Method for measurement of HDACS actiyity Enzyme No Enzyme2. 141107 HDACs Deacetylase Fluorometric Assay Kit Ver 2 a Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 5 Substrate preference of HDAC and SIRTI using CycLex SIRTI Sir2 Deacetylase Fluorometric Assay Kit Cat CY 1151 lt Sir2 substrate CycLex Sir2 Assay kit 10 000 525552eIefSPERSREPLPOPOBRCPM se 9 000 5 8 000 7 000 2 6 000 s crude HDAC LC 5 000 S 4 000 e Recombinant E 3 000 SIRTI i 2 000 E 1 000 0 0 10 20 30 40 50 60 Time min Fig 6 Time course of 2 reaction in Two Step Meth d development reaction 400 350 300 250 200 150 e No enzyme control _ 9 No inhibitor control 100 amp Inhibitor control F355 F460 x10 counts 50 0 10 20 30 40 Reaction Time min Cat CY 1150V2 14 Version 141107 fc HDACs Deacetylase Fluorometric Assay Kit Ver 2 H Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 7 Measurement of HeLa cell endogenous HDACI in an immunoprecipitate using anti HDAC I antibody Cat CY P1011 by means of this CycLex HDACs Deacetylase Fluorometric Assay e Anti HDAC 1 pAb 6 000 000 e Normal rabbit IgG 5 000 000 4 000 000 3 000 000 Dd 2 000 000 Fluorescence Intensity F355 F460 1 000 000 0
3. Screening inhibitors or ctivators of HDACs 3 Detecting the effects Of pharmacological agents on HDACs This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage Upon receiptistore 5 Developer and 6 HDACs at 70 C and all other components below 20 C Do not expos reagents to excessive light Cat CY 1150V2 1 Version 141107 HDACs Deacetylase Fluorometric Assay Kit Ver 2 Ale Ee e y gt X ser s Manu For Research Use Only Not for use in diagnostic procedures Introduction Histone deacetylase HDAC is considered to play a crucial role in regulating gene expressiongby changing nucleosome structure HDAC is also thought to participate in regulation of cell cycle and differentiation and it has been reported that the failure of this regulation leads to some types_of cancer Inhibition of HDAC activity by HDAC inhibitors such as Trichostatin A TSA and suberoylanilide hydroxamic acid SAHA induce differentiation and or apoptosis of transformed cells in vitro and inhibit tumor growth in a mouse model It has been reported that HDAC inhibitors are effective for the medical treatment of acute promyelocytic leukemia APL and various cancers Thus HDAC inhibitors are expected to function as new anti tumor drugs and antibacterial reagents It is thought that screening of histone deacetylase inhibitors is likely to be further carried out as one way to discover additional subs
4. Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info 2 cyclex co jp URL http www cyelex co j p CycLex CircuLex produets 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 witliout prior written approval from CycLex Co Ltd To inquire about licensing for such commercial use please contact us via email Cat CY 1150V2 17 Version 141107
5. nm th assays cannot be evaluated correctly Do not use kit components beyond the indicated kit expiration date Rinse all detergent residue from glassware Use deionized water of the highest quality e Do not mix reagents from different kits Do not mouth pipette or ingest any of the reagents e Do not smoke eat or drinkgWhen 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 FOLLLOWING 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 r search use only not for use in diagnostic or therapeutic procedures Cat CY 1150V2 4 Version 141107 Ee HDACs Deacetylase Fluorometric Assay Kit Ver 2 e ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol CycLex HDACs Deacetylase Fluorometric Assay Kit can measure the enzyme activity of HDAC with two kinds of measuring methods one step method and two step method In the one step method the reaction is initiated and the fluorescence intensity is measured by mixing simultaneously fluorescence labeled acetylated peptide which is a substrate HDAC and the developer Since the react
6. 0 5 10 15 20 25 30 Reaction Time min x Fig 8 Measurement of HeLa cell endogenous H mmunoprecipitate using anti HDAC2 antibody Cat CY P1012 by means of this HDACs Deacetylase Fluorometric Assay Kit e Anti HDAC 2 pAb 6 000 000 e Normal rabbit IgG S 5 000 000 Kai t E uw uw amp 4 000 000 E E 3 000 000 o 2 o o Z 2 000 000 S E ra 1 000 000 0 0 5 10 15 20 25 30 Reaction Time min C CY 1150V2 15 Version 141107 Fg HDACs Deacetylase Fluorometric Assay Kit Ver 2 yclex User s Manual For Research Use Only Not for use in diagnostic procedures References Davie J R amp Chadee D N J Cell Biochem Suppl 30 31 203 213 1998 2 Kouzarides T Curr Opin Genet Dev 9 40 84 1999 3 Fenrick R amp Hiebert S W J Cell Biochem Suppl 30 31 194 202 1998 4 Yoshida M Horinouchi S amp Beppu T Bioassays 17 423 430 1995 5 Richon V M et al Proc Natl Acad Sci USA 93 5705 5708 1996 Ki 6 Richon V M et al Proc Natl Acad Sci USA 95 3003 3007 1998 amp 7 Cohen L et al Proc AACR 39 108 abstr 736 1998 Q 8 Desai D El Bayoumy K amp Amin S Proc AACR 40 239 1999 9 Laherty C D Yang W M et al Cell 89 349 356 1997 10 Hassig C Fleischer T C et al Cell 89 341 34 11 Hoffmann K Grosch G amp Jung M Nucleic Acid 27 2057 2058 1999 C CY 1150V2 16 Version 141107 Ee HDACs Deacetylase Fluorometr
7. 1 70 C 16 HDACS crude nuclear extract from HeLa 500 uL x1 70 C 7 Stop Solution 1mLx2 Below 20 C Instruction manual 1 Room temp Cat CY 1150V2 3 Version 141107 Ee HDACs Deacetylase Fluorometric Assay Kit Ver 2 e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided Microplate for fluorometer e Microplate reading fluorometer capable of excitation at a wavelength in the range 350 380 nmi and detection of emitted light in the range 440 460 nm e 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 500 or 1000 mL graduated cylinder Reagent reservoirs 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 aft rthey are completely thawed Please avoid repeated freezing and thawing of 5 Developer and 6 HDACs There is a possibility that the enzyme activity may be inactivated Aliquot to 10 20 pL and store at 70 C Please avoid mixing of protease peptidase inhibitors such as PMSF or alkyl amine in samples that will be measured HDAC activity If enzyme samples or test compounds themselves emitfluorescence at excitation wavelength 350 380 nm and fluorescence wavelength 440 460
8. Positiye Inhibitor Assay reagents Sample Control Control Control Assay Assay ASSay Assay Distilled water 35 uL 35 uL 35 uL 30 uL 1 HDAC Assay buffer 5 uL 5 uL SuL 5 uL 2 Fluoro Substrate Peptide 5 uL 5 uL SL 5uL 4 Trichostatin A 5uL Enzyme Sample 5uL 5 ul Buffer of Enzyme Sample SuL 6 HDACs 5 uL Total Volume of the mixture 50 uL 5SOuL 50 uL 50 uL 2 Initiate reactions by adding 5 uL of your Enzyme Sample or Buffer of Enzyme Sample or 6 HDACs to each well and mixing thoroughly at room temperature 3 Incubate for 20 min or desired length of time at room temperature 4 Add 20 uL of 7 Stop Solution to each well of the microtiter plate and mix thoroughly 5 Add 5uL of 5 Developer to each well of the microtiter plate and mix thoroughly 6 Incubate for at least 10 min or desiredlength of time at room temperature Measurement should be made between 10 minutes and 40 minutes 7 Read fluorescence intensity using microtiter plate fluorometer with excitation at 350 380 nm and emission at 440 460 nm Note 1 During the timein which HDAC reaction rate is maintained the difference in fluorescence intensity between Enzyme Sample Assay and No Enzyme Control Assay indicates the HDAC activity of your Enzyme Sample Note 2 Althoughjthe volume of addition of Enzyme Sample or Buffer of Enzyme Sample or 6 HDACs isset to 5 uL in the tables
9. ase Fluorometric Assay Kit Ver 2 e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures time to stop the reaction and measure fluorescence intensity in a microplate fluorescence reader capable of excitation at a wavelength in the range 350 380 nm and detection of emitted light in the range 440 460 nm 2 Two Step Method 1 Following Table 2 below first add Distilled water 4 1 HDAC Assay Buffer and 4 2 Fluoro Substrate Peptide or 3 Fluoro Deacetylated Peptide to microtiter plate wells Second add your Test Compound or Solvent of Test Compound or 4 Trichostatin A to each well of a microtiter plate and mix well Table 2 Reaction mixture of Two Step Method for inhibitor screening Test Solvent Inhibitor No Enzyme Development Assay reagents Compound Control Control Control Control Assay Assay Assay Assay Assay Distilled water 30 pL 30 pL 30 pL 35 uL 35 uL 1 HDAC Assay Buffer 5 uL 5 uL 5 uL 5 uL 5 uL 2 Fluoro Substrate Peptide 5 uL 5 uL 5 uL 5 ul 3 Fluoro Deacetylated Peptide 5 uL Test Compound 5 uL 5 ul Solvent of Test Compound 5 uL 5 ul 4 Trichostatin A 5 pL 6 HDACs or Enzyme Sample 5uL 5yuL 5 uL Total Volume of the mixture 50 uL 50 uL 50 uL 50 uL 50 uL 2 Initiate reactions by adding 5 uL of 6 HDACs or your Enzyme Sample to each well and mixing thoroughly at room temperatu
10. d Peptide tomnierotiter plate wells Second add Test Compound or Solvent of Test Compound of 4 Trichostatin A and 5 Developer to each well of the microtiter plate and mix well Table 1 Reaction mixture of One Step Method for inhibitohscreening Test Solvent Inhibitor No Enzyme Development Assay reagents Compound Control Control Control Control Assay Assay Assay Assay Assay Distilled water 25 uL 25 uL 25 uL 30 pL 30 pL 1 HDAC Assay buffer 5uL 5uL 5uL 5uL 5uL 2 Fluoro Substrate Peptide 5uL 5uL 5uL 5 ul 3 Fluoro Deacetylated Peptide 5 ul Test Compound 5uL 5 ul Solvent of Test Compound 5uL 5 ul 4 Trichostatin A 5uL 5 Developer SuL 5 uL 5 uL 5 uL 5 uL 6 HDACS or Enzyme Sample 5 uE 5uL 5uL Total Volume of the mixture 50 uL 50 uL 50 uL 50 uL 50 uL 2 Initiate reactions by adding 5 uL of 6 HDACs or your Enzyme Sample to each well and mixing thoroughly aroom temperature 3 Read fluorescence intensity for 30 to 60 minutes at 1 to 2 minute intervals using microtiter plate fluorometer with excitation at 350 380 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 rate is kept constant add 20 uL of 7 Stop Solution to each well at appropriate Cat CY 1150V2 7 Version 141107 oe HDACs Deacetyl
11. e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency of HDACs Two Step Method 500 450 400 350 300 250 200 150 100 50 F355 F460 x10 5 counts 6 HDACs uL Fig 2 Time course of HDAC reaction Two Step M 600 500 S EE E G No enzyme control SE EEN No inhibitor control E amp Inhibitor control ER A 200 p cecus en fu 0 Q 0 20 40 60 80 HDAC reaction time min C CY 1150V2 12 Version 141107 D e Mey HDACs Deacetylase Fluorometric Assay Kit Ver 2 User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Effect of Trichostatin A on HDAC activity One Step Method F355 F460 x10 counts 300 Trichostatin A 100nM Trichostatin A 10nM Trichostatin A 1nM e DMSO 250 200 150 100 50 20 40 60 80 100 120 HDAC reaction time min Fig 4 Substrate preference of HDAC and SIRTI using this GyeLex HDACSs Deacetylase Fluorometric Assay Kit 70 000 60 000 50 000 40 000 30 000 20 000 10 000 F355 F460 x 10 counts HDAC substrate CycLex HDAC Assay kit crude HDAC e Recombinant SIRTI 30 40 50 60 Time min Cap CY 1150V2 13 Version
12. hould 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 10 mM NaCl 10 mM Tris HCl pH7 5 1 5 15 mM MgCl 10 mM NaCl 420 mM NaCl 250 mM Sucrose 3 mM MgCl 0 5 mM EDTA Na 0 5 96 NP 40 0 1 mM EGTA 0 1 mM EGTA 10 glycerol Procedure Isolation of Nuclei 1 Suspend 1x 10 cells 100 mm dish tib confluent into 1ml of lysis buffer Vortex for 10 second Keep on ice for 15 min Spin the cells through 4 ml of sterose cushion at 1 300 x g for 10 min at 4 C Discard the supernatant Wash the nuclei pellet oneeswithicold 10 mM Tris HCI pH7 5 10 mM NaCl Oy Oe bo Extraction of Nuclei 1 Suspend the isolated nuclei in 50 100 uL of extraction buffer 2 Sonic ate for 30 seconds 3 Stand on ice fof 30min 4 c f g at 20 000x for 10 min 5 Take supernatant the crude nuclear extract 6 Determineprotein conc by Bradford method or equivalent 7 Store tlie crude nuclear extract at 70 C until use Not Do motiise any kind of protease peptidase inhibitor Cat CY 1150V2 11 Version 141107 Pe HDACs Deacetylase Fluorometric Assay Kit Ver 2 f H
13. ic 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 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 HDACS 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 Catit 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
14. ion 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 the stop solution and to measure fluorescence intensity Conversely the two step method begins by initiating a reaction of fluorescenc labeled acetylated peptide and HDAC within a set time period to remove an acetyl group from substr te peptide and then in the second step adds the stop solution to stop the HDAC reaction while simultaneously cleaving the resultant deacetylated fluorescence labeled peptide by the developer I Assay Procedures for Measurement of HDAC Activity 1 One Step Method 1 Following Table 1 below first add Distilled water 1 HDAC Assay Buffer and 2 Fluoro Substrate Peptide to microtiter plate wells Second add 4 Trichostatin A and 5 Developer to each well of the microtiter plate and mix well Table 1 Reaction mixture of One Step Method forgn asurement of HDACS activity Enzyme No Enzyme Positive Inhibitor Assay reagents Sample Control Control Control Assay Assay Assay Assay Distilled water 30uL 30 uL 30 uL 25 uL 11 HDAC Assay buffer SuL 5uL 5uL 5uL 2 Fluoro Substrate Peptide 5uL Su 5uL Su 4 Trichostatin A 5uL 5 Developer 5uL 5uL S ul 5 ul Enzyme Sample 5uL
15. it may be changed to a volume up to 20 uL at your diseretion dn 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 Fluoro Substrate Cat CY 1150V2 6 Versionft 141107 v oy HDACs Deacetylase Fluorometric Assay Kit Ver 2 Vclex User s Manual For Research Use Only Not for use in diagnostic procedures Peptide resulting in an increase of fluorescence intensity in Inhibitor Control Assay the HDAC activity in the samples cannot be evaluated correctly Note 4 If enzyme samples contain inhibitors for protease peptidase precise HDAC enzyme activity cannot be measured Since protease peptidase inhibitors used in the usual protein purification process strongly inhibit the peptidase activity in the development reaction please void 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 Fluoro Deacetylated Peptide instead of 2 Fluoro Substrate Peptide and conduct a control experiment II Assay Procedures for Inhibitor Screening 1 One Step Method 1 Following Table 1 below first add Distilled water 1 HDAC Assay Buffer and 2 Fluoro Substrate Peptide or 3 Fluoro Deacetylate
16. oe HDACs 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 histone deacetylase activity CycLex HDACs Deacetylase Fluorometric Assay Kit Ver 2 100 Assays Cat CY 1150V2 Intended US 1 Be M 1 TOA CUO E 2 Principle of the Assay isse tnos 3 Materials Provided eerte 3 Materials Required but not Provided 4 DEO QUOI Suus eese A ERI gege EXIM qu S 4 Detailed Protocol eset oer meters 5 9 Troubleshooting isses eset bran tina Se ort 10 Reagent DEEN agedroe beant intu 10 Sample Preparation ees errato 11 Example of Test RESHMS iscccscassesenstanicains 12 15 licis 0 M 16 Related PrOGHOUS iecur ie trea tg EUER nui I Intended Use The CycLex Research Product CycLex HDACs Deacetylase Fluorometric Assay Kit detects HDAC activity in lysates Primarily the CycLex Res ar h Product CycLex HDACs Deacetylase Fluorometric Assay Kit is designed for the rapid and ensitive evaluation of HDAC inhibitors using crude HDAC fraction Additionally any cultured primary ell cell line or tissue homogenate can be assayed for HDAC activity with the CycLex Research Product CycLex HDACs Deacetylase Fluorometric Assay Kit if the appropriate dose of HDAC specific inhibitor e g Trichostatin A is used Applications for this kit include 1 Monitoring the purification of HDACs including HDACI 2 3 and 8 2
17. ol Assay The increase in fluorescence intensity is not observ in Inhibitor Control Assay The efficacy of the test compounds on the HDAC activity is the difference in fluor ce intensity between Test Compound Assay minus No Enzyme Control 4 Solvent Control Assay minus No Enzyme Control Assay If test compounds have an inhibitory effect on protease peptidase resulting that t se in fluorescence intensity is not or a little observed in Development Control Assay the effect on HDAC activity cannot be evaluated correctly Although the above tables indicate the volume of addition of Test Co r Solvent of Test Compound or 4 Trichostatin A as 5 uL the concentrati he volume of the reagents to add can be changed so that the concentration of test comp comes the setting concentration For example since the final volume of reaction is to add 10 uL of Test Compound or Solvent of Test Co reduce the volume of Distilled water to set the final reaction vo It is also possible In this case please of 50 uL Although the volume of addition of 6 HDACs or you e Sample is set to 5 uL in above tables it may be changed to a volume up to 20 pL at iscretion In that case please reduce the volume of Distilled water to set the final ion me of 50 uL C CY 1150V2 9 Version 141107 oe HDACs Deacetylase Fluorometric Assay Kit Ver 2 e Vclex User s Manual For Research Use Only Not for u
18. re 3 Incubate for 20 min or desired length of time t room temperature 4 Add 20 uL of 7 Stop Solution to each well of the microtiter plate and mix thoroughly 5 Add 5uL of 5 Developer to each well of the microtiter plate and mix thoroughly 6 Incubate for at least 10 ming fdesired length of time at room temperature Measurement should be made between 10 minutes and 40 minutes 7 Read fluorescence int nsity using microtiter plate fluorometer with excitation at 350 380 nm and emission at 440 460 nm Note 1 During the time in which HDAC reaction rate is maintained the difference in fluorescence intensity between Solvent Control Assay and No Enzyme Control Assay indicates the HDA Gativity Note 2 dn order to estimate the inhibitory effect on HDAC activity by the test compounds correctly it is necessary to conduct the control experiment of Solvent Control Assay at least once for every experiment and Inhibitor Control Assay at least once for the first experiment in addition to Test Compound Assay as indicated in the tables When test compounds cause an inhibitory Cat CY 1150V2 8 Version 141107 HDACs Deacetylase Fluorometric Assay Kit Ver 2 For Research Use Only Not for use in diagnostic procedures v Mey User s Manual AN Note 3 Note 4 Note 5 Note 6 effect on HDAC activity the level of increase of fluorescence intensity is weakened as compared with Solvent Contr
19. se in diagnostic procedures Troubleshooting j When compounds that have an inhibitory effect on the peptidase in the development reactionzare mixed in a crude HDACs fraction purified from various cells or the immunoprecipitate using a specific antibody against HDACs or other proteins precise HDAC activity cannot be measured Stace protease peptidase inhibitors used in the usual protein purification process strongly inhibit the peptidase in the development reaction please avoid the use of any protease peptidase inhibitors during the protein purification process 2 Final fluorescence intensity will not increase both when test compounds have an inhibitory effect on HDAC activity and also when there is an inhibitory effect on the peptidase ithe development reaction 3 If enzyme samples or test compounds themselves emit fluorescence at excitation s vayelength 360 380 nm and fluorescence wavelength 440 460 nm the inhibitory effect of the test assay cannot be evaluated correctly 4 The assays should be run in duplicate using the protocol described in the Detailed Protocol Poor duplicates indicate inaccurate dispensing If all instructions in the Detailed Protocol were followed accurately such results may indicate a need for multi channel pipettor maintenance 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 a
20. ssay Kinetics that are other than first order For a non linear curve point to point or quadratic curvedit methods should be used 6 Incubation times or temperatures significantly different from those specified may give erroneous results Reagent Stability All of the reagents included in the Cyel ex Research Product CycLex HDACs Deacetylase Fluorometric Assay Kit have been tested for stability Reagents should not be used beyond the stated expiration date Upon receipt store 5 Developer and 6 HDACs at 70 C all other kit reagents should be stored below 20 C Cat CY 1150V2 10 Version 141107 oe HDACs Deacetylase Fluorometric Assay Kit Ver 2 e Vclex 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 HDACs The following protocols 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 HDACs 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 erude extract cell lysate or sample fraction taken prior to a purification step All sample preparation s
21. tances with similar properties However the conventional method for measuring HDAC activity is very complicated and laborious In order to measure HDAC enzyme activity it is necessary to prepare radioactive a tylated 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 40 bep rified 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 b cause of their complicated operation Thus a simple system for biochemical analysis as well as fof mhibitomscreening without the use of radioactive substances is preferred Cat CY 1150V2 2 Version 141107 ry HDACs Deacetylase Fluorometric Assay Kit Ver 2 c Vclex User s Manual For Research Use Only Not for use in diagnostic proced
22. ures Principle of the Assay CycLex HDACs Deacetylase Fluorometric Assay Kit measures the activity of HDAC by the basic principle of changing an HDAC reaction into the activity of the peptidase Since it is very simple to measure common protease peptidase activity and it can be performed at a low price the measurement of HDAC activity in most laboratories is possible if they are equipped with a fluorescent reader for microtiter plates Considering that the use of fully automatic apparatus to measure fluorescence intensity has become widespread HDAC activity measurement which could not be made by the Conventional method is now possible with the CycLex HDACs 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 HDACs Deacetylase Fluorometfic Assay Kit X X X Lys Ac MCA peo X X X Lys MCA Ja X X X Lys AMC Measurement of fluorescene intensity Deacetylase Peptidase Note This measuring principle and kit are 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 Materials Provided All assays should be run in duplicate The following components are supplied and are sufficient for one hundred assays Components of Kit 5 Developer 500 uL x
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