CY-1158V2
Contents
1. CycLex SIRT3 Deacetylase Fluorometric Assay Kit Ver 2 Cat CY 1153V2 CycLex SIRT6 Deacetylase Fluorometric Assay Kit Ver 2 Cat CY 1156V2 CycLex HDACS8 Deacetylase Fluorometric Assay Kit Ver 2 Cat CY 1158V2 Anti Acetylated Histone p53 K382 Mouse Monoclonal Antibody Cat CY M1029 Anti Histone Deacetylase 1 HDAC1 Rabbit Polyclonal Antibody Cat CY P1011 Anti Histone Deacetylase 2 HDAC2 Rabbit Polyclonal Antibody Cat CY P1012 Anti Human SIRT1 Rabbit Polyclonal Antibody Cat CY P1016 NAD Dependent Deacetylase SIRT1 Cat CY E1151 NAD Dependent Deacetylase SIRT2 Cat CY E1152 NAD Dependent Deacetylase SIRT3 Cat CY E1153 NAMPT Nicotinamide Phosphoribosyltransferase Cat CY E1251 NMNATI1 Nicotinamide Mononucleotide Adenylyltransferase 1 s at Y E1252 Note This product is covered under CycLex s patents U S Patent No 7 033 778 and No 7256013 European Patent No 1243658 Japanese Patent No 4267043 Canadian Patent No 2392711 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co jp URL http www cyelex co jp CycLex CircuLex products are supplied for research use only CycLex CircuLex products and components thereof may not be resold modified for resale or used to manufacture commercial products without prior written approval from CycLex Co Ltd To inquire about licensing for such commercial use please2. acetyl group which has been released from acetylated histone using ethyl ac tate 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 fluoreseent 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 abovegtheSe measurement systems are difficult to adapt for processing many samples under a variety of Conditions because of their complicated operation Thus a simple system for biochemical analysis asewell asifor inhibitor screening without the use of radioactive substances is preferred Cat CY 1158V2 2 Version 141107 ry HDAC8 Deacetylase Fluorometric Assay Kit Ver 2 oS ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay CycLex HDAC8 Deacetylase Fluorometric Assay Kit measures the activity of HDAC by the basic principle of changing an HDAC reaction into the activity of protease 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 f
3. assayed for HDAC8 activity with the GycLex Research Product CycLex HDAC8 Deacetylase Fluorometric Assay Kit after immunoprecipitation with an appropriate HDAC8 specific antibody Applications for this kit include 1 Monitoring the purification of HDACs including HDAC1 2 3 and 8 2 Screening inhibitors or activators of HDAC8 3 Detecting the effects of pharmacological agents on HDAC8 This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage e Upon receiptistore 5 Developer and 6 Recombinant HDAC8 at 70 C and all other components below 20 C e Domot exposeweagents to excessive light Cat CY 1158V2 1 Version 141107 HDAC8 Deacetylase Fluorometric Assay Kit Ver 2 jde Urs Mi GA y gt X ser s Manu For Research Use Only Not for use in diagnostic procedures Introduction HDAC proteins are vital regulators of fandamental cellular events including cell cycle progression differentiation and tumorigenesis 1 2 A small molecule inhibitor of HDAC trichostatin A JSA arrests mammalian cells in both Gl and G2 3 4 while overexpression of HDAC1 in mouse Cells reduces their growth rate by lengthening the duration of G2 and M 5 TSA induces te minal differentiation of mouse erythroleukemia cells and apoptosis of lymphoid and colorectal cancer cells In addition TSA treatment of cells expressing the PML zinc finger protein derepresses transeription and allows cells
4. contact us via email Cat CY 1158V2 12 Version 141107
5. e multi channel pipette e Microplate shaker e Deionized water of the highest quality 500 or 1000 mL graduated cylinder e 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 after theytare completely thawed Please avoid repeated freezing and thawing of 5 Developer afd 6 Recombinant HDAC8 There is a possibility that the enzyme activity may be inactivated Aliquot to 10 20 uL and store at 70 C Please avoid mixing of protease peptidase inhibitors such as 2MSF or alkyl amine in samples that will be measured HDAC activity e If enzyme samples or test compounds themselves mit fluores ence at excitation wavelength 350 380 nm and fluorescence wavelength 440 460 nm the assays cannot be evaluated correctly Do not use kit components beyond the indicated kit expitation 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 drink whem 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 F
6. to differentiate normally 6 With this precedent HDAC inhibitors are being actively explored as potential agents for the treatment of certain forms of cancer 7 9 The human HDACs are organized into three different classes based on their similafity to yeast HDAC proteins 1 2 Class I enzymes are ubiquitously expressed and include HDAC1 2343 fand 8 which are homologous to the yeast RPD3 protein Class II includes HDAC4 5 6 7g 9 and 10 which are similar to yeast HDA1 and are expressed in a tissue specific manner The Sit2 lik yclass II HDACs including SIRT 1 to 7 require NAD for enzymatic activity It has been reported that HDAC8 is important for the growth of hum n tumor cell lines and has a distinct inhibition pattern that differs from that of HDACI and 3 which both share 43 sequence identity with HDAC8 These findings lead to open the way to the development of selective inhibitors of this subtype as potential novel anticancer therapeutics However the conventional method for measuring HDAC activity s very complicated and laborious In order to measure HDAC enzyme activity it is necessary to 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 sepatate th radioactive
7. LEX User s Manual A For Research Use Only Not for use in diagnostic procedures Fig 3 Effect of Trichostatin A on HDAC8 activity One step method 1 000 000 800 000 600 000 400 000 F355 F460 counts 200 000 C CY 1158V2 10 Version 141107 oe HDAC8 Deacetylase Fluorometric Assay Kit Ver 2 cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures References Thiagalingam S K H Cheng H J Lee N Mineva A Thiagalingam and J F Ponte 2003 Histone deacetylases unique players in shaping the epigenetic histone code Ann N Y Acad Sci 983 84 100 2 Yang X J and E Seto 2003 Collaborative spirit of histone deacetylases in regulating chromatin structure and gene expression Curr Opin Genet Dev 13 143 153 3 Ogryzko V V T H Hirai V R Russanova D A Barbie and B H Howard 1996 Human fibroblast commitment to a senescence like state in response to histone deacetylase inhibitors is cell cycle dependent Mol Cell Biol 16 5210 5218 4 Wharton W J Savell W D Cress E Seto and W J Pledger 2000 Inhibition offmitogenesis in Balb c 3T3 cells by trichostatin A Multiple alterations in the induction and activation of cyclin cyclin dependent kinase complexes J Biol Chem 275 33981 33987 5 Bartl S J Taplick G Lagger H Khier K Kuchler and C Seiser 1997 Identification of mouse histone deacetyla
8. OLDOWING 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 us only not for use in diagnostic or therapeutic procedures Cat CY 1158V2 4 Version 141107 oy HDAC8 Deacetylase Fluorometric Assay Kit Ver 2 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol CycLex HDAC8 Deacetylase Fluorometric Assay Kit can measure the enzyme activity of HDAG8 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 HDAC8 and the developer Since the reaction is not stopped it is necessary to measureffluorescence 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 possibl to stop the reaction by adding the Stop solution and to measure fluorescence intensity 1 Assay Procedures for Measurement of HDAC Activity 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
9. Table 1 Reaction mixture of One Step Method for measurement of HDAC activity Enzyme No Enzyme Positive Inhibitor Assay reagents Sample Control Control Control Assay Assay Assay Assay Distilled water 30 uL 30 ub 30 uL 25 uL 1 HDAC Assay buffer 5 uL SuL 5 uL 5 uL 2 Fluoro Substrate Peptide 5 uL 5 pL 5uL 5 uL 4 Trichostatin A 2 5 uL 5 Developer 5 uh 5 uL 5 uL 5 uL Enzyme Sample 5 uL 5 uL Buffer of Enzyme Sample 5 ub 6 Recombinant HDAC8 5 pL Total Volume of the mixture 50 uL 50 pL 50 pL 50 uL 2 Initiate reactions by adding 5 uL ofsyour Enzyme Sample or Buffer of Enzyme Sample or 6 Recombinant HDAC8 to each welland mixing thoroughly at room temperature 3 Read fluorescence intensitygfor30 to 60 minutes at 1 to 2 minute intervals using microtiter plate fluorometer with excitation at 3502380 nm and emission at 440 460 nm Measure and calculate the rate of reaction while the feactionfvelocity remains constant Alternate procedure 3 While the reaction rates 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 ex itation Jat a wavelength in the range 350 380 nm and detection of emitted light in the range 440 460 am 2 Assay Procedures for Inhibitor Screening Cat CY 1158V2 5 Version 141107 oy HDAC8 Deac
10. etylase Fluorometric Assay Kit Ver 2 YCLEX User s Manual For Research Use Only Not for use in diagnostic procedures 1 Following Table 1 below first add Distilled water 1 HDAC Assay Buffer and 2 Fluoro Substrate Peptide or 3 Fluoro Deacetylated Peptide to microtiter plate wells Second add Test Compound or Solvent of Test Compound or 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 inhibitor screening Test Solvent Inhibitor No Enzyme Development Assay reagents Compound Control Control Control Control Assay Assay Assay Assay Assay Distilled water 25 uL 25 pL 25 uL 30 pl 30 pL 1 HDAC Assay buffer 5 uL 5 uL 5 uL 5 aL 5 uL 2 Fluoro Substrate Peptide 5 uL 5uL 5 uL Sul 3 Fluoro Deacetylated Peptide 5 uL Test Compound 5uL 5 uL Solvent of Test Compound 5uL Sq 4 Trichostatin A 5 uL 5 Developer 5 uL 5 uL 5 uL 5 pL 5 pL 6 Recombinant HDAC8 or Enzyme Sample SaL SuL NL 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 HDA 8 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 excitatio
11. h Use Only Not for use in diagnostic procedures Troubleshooting j When compounds that have an inhibitory effect on the peptidase in the development reactiongare 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 Simce 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 excitationgWayelength 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 lineari
12. luorescence intensity has become widespread HDAC activity measurement which could not be made by the Conventional method is now possible with the CycLex HDAC8 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 HDAC8 Deacetylase Fluorometric Assay Kit X X X Lys Ac MCA peo X X X Lys MCA Ja X X X Lys AMC Measurement of fluoresceng jintensity 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 500uLx1 70 C 1 Room temp Cat CY 1158V2 3 Version 141107 oy HDAC8 Deacetylase Fluorometric Assay Kit Ver 2 P ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided e 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
13. n 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 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 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 HDAC activitys Note 2 In order t stimate 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 effect Om IDAC activity the level of increase of fluorescence intensity is weakened as compared with Solvent Control Assay The increase in fluorescence intensity is not observed in Inhibitor Control Assay Cat CY 1158V2 Version 141107 HDAC8 Deacetylase Fluorometric Assay Kit Ver 2 9 Ce ycLex User s Manual N For Research Use O
14. nly Not for use in diagnostic procedures Note 3 The efficacy of the test compounds on the HDAC activity is the difference in fluorescence intensity between Test Compound Assay minus No Enzyme Control Assay a Solvent Control Assay minus No Enzyme Control Assay Note 4 If test compounds have an inhibitory effect on protease peptidase resulting that the incr in fluorescence intensity is not or a little observed in Development Control Assay HDAC activity cannot be evaluated correctly Note 5 Although the above tables indicate the volume of addition of Test Compound o t of Test Compound or 4 Trichostatin A as 5 uL the concentration and the volume of the reagents to add can be changed so that the concentration of test compounds es the setting concentration For example since the final volume of reaction is 50 uL he lso possible to add 10 uL of Test Compound or Solvent of Test Compound is case please reduce the volume of Distilled water to set the final reaction ims Note 6 Although the volume of addition of 6 Recombinant HDA C8 yme Sample is set to 5 uL in above tables it may be changed to a volume up to your discretion In that case please reduce the volume of Distilled water to set the fin on volume of 50 uL amp C CY 1158V2 7 Version 141107 oe HDAC8 Deacetylase Fluorometric Assay Kit Ver 2 aa ycLex User s Manual For Researc
15. se 1 as a growth factor inducible gene Mol Cell Biol 1795033 5043 6 He L Z F Guidez C Tribioli D Peruzzi M Ruthardt A Zelentyand P P Pandolfi 1998 Distinct interactions of PML RAR alpha and PLZF RAR alpha wath co repressors determine differential responses to RA in APL Nat Genet 18 126 135 7 Johnstone R W 2002 Histone deacetylase inhibitors M ovel drugs for the treatment of cancer Nat Rev Drug Discov 1 287 299 CrossRef Medline 8 Kelly W K O A O Connor and P A Marks 2002 Histone deacetylase inhibitors from target to clinical trials Expert Opin Investig Drugs 11 1695 1713 9 Melnick A and J D Licht 2002 Histone deacetylases as therapeutic targets in hematologic malignancies Curr Opin Hematol 9 322 332 10 Gao L M A Cueto F Asselbergs and P Atadja 2002 Cloning and functional characterization of HDACI1 a novel member of the human histone deacetylase family J Biol Chem 277 25748 25755 Cat CY 1158V2 11 Version 141107 oy HDAC8 Deacetylase Fluorometric 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
16. ty of the curve as can assay 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 Cyelex Research Product CycLex HDAC8 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 Recombinant HDAC8 at 70 C all other kit reagents should be stored below 208 Cat CY 1158V2 8 Version 141107 AA NCLex HDAC8 Deacetylase Fluorometric Assay Kit Ver 2 User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency of recombinant HDAC8 30min F355 F460 counts 600 000 500 000 400 000 300 000 200 000 100 000 15 20 25 30 GST HDACS8 conc ug ml 40 45 Fig 2 Time course of HDAC8 reaction F355 F460 counts 1 000 000 800 000 600 000 400 000 200 000 e 40 ug ml 20 ug ml H 5 ug ml 2 5 ug ml VO ug l ac 125 ug ml TaT AA OTOT TTT s 30 40 Reaction Time min 60 70 Cat CY 1158V2 Version 141107 HDAC8 Deacetylase Fluorometric Assay Kit Ver 2 YC
17. y A HDAC8 Deacetylase Fluorometric Assay Kit Ver 2 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Quantitative test kit for histone deacetylase activity CycLex HDAC8 Deacetylase Fluorometric Assay Kit Ver 2 100 Assays Cat CY 1158V2 Intended Use cccccccccceccceceeeeeceeeeseees 1 Be oat nsatsinanigayonmnysanegocsiciasiadsoaepriaaen sine 1 Tntroduction ce ceececcceeeeeessssseeesneesneeees 2 Principle Of the Assay 3 Materials Provided cceccceeeseeeeeeeeeees 3 Materials Required but not Provided 4 PLeCAUTIONS 00ccceeeeeeeeeseceesseeeeeeeeeeeeeeees 4 Detailed Protocol eccceeccecessseeeeeeeeees 5 7 CAUtlONS 0cccccceescceccccecessceseceesceeseeeeseees 8 TrOUbleSHO OUI ssssissessnrcennecsnsoineasteaynoatnaes 8 Reagent SCADILY pcscenonsasaeadsanenatancsamnannaentneien 8 Example of Test RESults issccsssasesersseiccanns 9 10 RefCLENCES veiessccissisissaassesesesiseissdeaesveassneventvans 14 Related Products eeseeeeeeeeseeesseseesseseseee 12 Intended Use The CycLex Research Product CycLex MDAC8 Deacetylase Fluorometric Assay Kit detects HDAC activity in lysates Primarily the CyeLex Research Product CycLex HDAC8 Deacetylase Fluorometric Assay Kit is designed forgh rapid and sensitive evaluation of HDAC inhibitors using recombinant HDAC8 Additionally any cultuted primary cell cell line or tissue homogenate can be
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