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CY-1140

1. 140318 LS d Cellular Histone Acetylation Assay Kit 4 v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures amp n C Q Introduction 9 Acetylation and deacetylation of nucleosomal histones play an important role in the moifiton of chromatin structure chromatin function and in the regulation of gene expressio Histone acetyltransferases HATs and histone deacetylases HDACs are two opposing RR ui which tightly control the equilibrium of histone acetylation An imbalance in the equilibjym of histone acetylation has been associated with carcinogenesis and cancer progression So f amp amp a number of structurally distinct classes of compounds have been identified as HDAC inhibstors including the short chain fatty acids hydroxamates cyclic tetrapeptides and benzamides These pounds lead to an accumulation of acetylated histone proteins both in tumor cells and in normal tysues HDAC inhibitors are able to activate differentiation to arrest the cell cycle in G1 and or G2 afto induce apoptosis in transformed or cancer cells Attention is currently being drawn to molegQY mechanisms involving histone deacetylases An induction of p21W F and a suppression of angi ge fc stimulating factors have been observed in tumor cells following exposure to HDAC inhibitor xenograft models several HDAC inhibitors have demonstrated antitumor activity with only few alte effects Several clinical trials showed that H
2. Care should be taken to avoid direct contact with these reag s Dispose of tetra methylbenziding RIB containing solutions in compliance with local regulations Avoid contact with the sciqgStop Solution and Substrate Solution which contains hydrogen peroxide Wear gloves and eye pr amp t ction when handling immunodiagnostic materials and samples of human origin and these rea In case of contact with the Stop Solution and the Substrate Solution wash skin thoroughly wi ter and seek medical attention when necessary Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or br afhe aerosols Wear protective gloves and dispose of biological samples properly CAUTIONJSulfuric Acid is a strong acid Wear disposable gloves and eye protection when handling Stop Solution Q S Q Roan CY 1140 5 Version 140318 LS d A S Cellular Histone Acetylation Assay Kit e 4 v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures amp Detailed Protocol 9 The CycLex Research Product Cellular Histone Acetylation Assay Kit includes all r ts for detection of Histone acetylation in cultured cells except cell fixative Since experimental condg ns may vary treatment cells with Trichostatin A within the kit should be included in each REL Mm as a positive control for induction of histone acetylation Disposable pipette tips and reagent tpughs should be us
3. In cases where the cells are easy to d from 96 well plates even aftter 1st fixation I Remove media from wells with a wrist flick void touching the bottom of the well and removing cells amp 2 Immediately add 150 pL well of 95 vakon as a fixative Add the fixing solution slowly to insure cells are not detached from the plastig Qi stand for 10 minutes at room temperature ca 25 C 3 Remove fixing agent from wel ith a wrist flick While still inverted tap the plate gently onto absorbent paper to remove any ss fixing agent still within the wells 4 Add 150 uL well of 1 iepratormatdensa in PBS Add solution slowly to ensure cells are not dislodged from the wells stand for 5 minutes at room temperature ca 25 C 5 Remove paraformalQWe solution from wells with a wrist flick While still inverted gently tap the plate onto absorbe er to remove any excess liquid still in the wells 6 Add 200 uL vp of Wash Buffer Let stand for 1 minute at room temperature ca 25 C T Remove watter with a wrist flick While still inverted gently tap the plate onto absorbent paper to removefaby excess liquid g agg uL well of Blocking Reagent and incubate for 1 hour at room temperature ca 25 C o e Q Roan CY 1140 7 Version 140318 LS d 4 v ycLex User s Manual C 1 2 9 10 11 12 13 14 15 16 17 18 For Research Use Only Not for use in diagnostic proced
4. off scale samples The readings at 405 nm should not replace the on scale re s at 450 nm Troubleshooting OQ wV 1 The signals are influenced a great deal by cell line and cell number Mat you plated please ensure the appropriate cell number for your experiment See Ex e of Test Results Fig 2 and 3 MN 2 Unavoidable background at Treatment time 0 hr or vehicle Onirol is observed even if an appropriate cells number is used It is usually around 0 2 0 8 See Example of Test Results Fig 1 3 With some cell lines higher cell concentrations more than S cells well in case of adherent cells may lead to increasing absorbance values in vehicle trol background Please ensure the appropriate cell number for your experiment o 4 All treatments including treatment of Trichostatin amp should be run in duplicate using the protocol described in the Detailed Protocol Incubatioreffmes or temperatures significantly different from those specified may give erroneous results b gt 5 Poor duplicates accompanied by elevate Mwilues for wells containing non treated cells vehicle control indicate insufficient washing or rous washing Wash the plate thoroughly and gently 6 Overall low signal may indicate that tci of the plate has occurred between the final wash and addition of Substrate Reagent Do d low the plate to dry out Add Substrate Reagent immediately after wash o Reagent Stability P All of the reagents included
5. Primary Antibody Solution Anti Acetylated Histone p53 K382 Mafi lonal Antibody TM 5C5 One vial containing 12 mL of Anti Acetylated Histone p53 K382 Monegjonal Antibody Ready to use Secondary Antibody Solution HRP conjugated Anti Mouse One vial containing 12 mL of HRP horseradish peroxidase conjugated anti mouse IgG polyc antibody Ready to use Substrate Reagent One bottle containing 20 mL of the chffynogenic substrate tetra methylbenzidine TMB Ready to use I Stop Solution One bottle containing 20 mL of 1 N H2 4 Ready to use Materials Required but not Provid d Cell culture flasks for growing and splittinggcells Cell culture media Q Ice cold 95 Methanol for 1 fixatio ells 1 paraformaldehyde in PBS for 2 amp fiXation of cells IX PBS pH 72 Pipettors 2 20 uL 20 200 uL and o 1000 uL precision pipettors with disposable tips Precision repeating pipettor Orbital microplate shaker Microcentrifuge and tuber sample preparation Vortex mixer Microplate washer Software package fa 500 or 1000 mL g Reagent reservoirs Deionized wat the highest quality Absorbent p disposable paper towels Plate read pable of measuring absorbance in 96 well plates at dual wavelengths of 450 nm 540 nm Du velengths of 450 550 or 450 595 nm can also be used The plate can also be read at a le of 450 nm which will give a somewhat higher reading SA Manual washing is possible but
6. in the CycLex Research Product Cellular Histone Acetylation Assay Kit have been tested fo Jllity Reagents should not be used beyond the stated expiration date Upon receipt kit reagents III stored at 4 C For research use Q not for use in diagnostic or therapeutic procedures A S gt o o A S Cellular Histone Acetylation Assay Kit e 4 v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures amp Example of Test Results 9 Fig 1 Typical result of time course experiment using MCF7 a breast cancer cell line treated qm 0 25 uM Trichostatin A k C Time Course of Trichostatin A Treatment Q S A450 0 1 2 3 4 5 TSA Treatment Time hr Fig 2 Typical result of time course experiment NS ur HeLa and BALB c 3T3 cell lines treated with 0 25 uM Trichostatin A xO A Time Course of Trichostatin A Treatment 2 0 4 MCF7 9 HeLa 1 5 BALB c 373 1 0 A450 Q 0 5 Q o ge 0 1 2 3 4 5 TSA Treatment Time hr S Q Roan CY 1140 10 Version 140318 g amp C Cellular Histone Acetylation Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures amp Fig 3 Effect of cell number on ELISA value in MCF7 cells treated with Trichostatin A for 5hr 3 FI Effect of cell number on ELISA value 2 9 esa nares itur diede S 2 0 p
7. DAC inhibitors in well tolerated doses have RE antitumoral activities A combination of HDAC inhibitors with differentiation inducin ents and cytotoxic drugs is an innovative therapeutic strategy that carries the potential for signe improvements in the treatment of cancer 4 G Principle of the Assay FC The CycLex s Histone Acetylation Kit is a Qil based ELISA formatted for conventional chromometric detection of relative levels of acet histones in microplate cells cultures Adherent cells are cultured in conventional 96 well microplates treated with agents that induce histone acetylation such as Trichostatin A a specific HDAC inhibi and are then fixed and permeabilized Several types of histones including histone H3 and H4 whith are acetylated at several lysine residues are detected by anti acetylated histone p53 K382 monoc antibody clone TM 5C5 and an anti mouse IgG HRP horseradish peroxidase conjugate The mogenic HRP substrate TMB is then added and signal is measured in a microplate reader o Rox CY 1140 2 Version 140318 LS d A 4 Vclex User s Manual For Research Use Only Not for use in diagnostic procedures S Cellular Histone Acetylation Assay Kit e amp The CycLex Research Product Cellular Histone Acetylation Assay Kit is designed to measurgithe relative levels of histone acetylation in situ The summary of the assay is shown in below eM YV Summary of Procedure K o Culture adherent cells
8. S Cellular Histone Acetylation Assay Kit e 4 e Vclex User s Manual For Research Use Only Not for use in diagnostic procedures gt Cell Based ELISA Kit for Measuring Acetylation of Histones in situ 3 CycLex Cellular Histone Acetylati Assay Kit amp AS Assays x 2 Catit CY 1140 O ww y Intended Use cccccccccccccceceeeeeseeeeseees 1 C Sij c 1 w Introduction tecto dtr een 2 SS Principle of the Assay 2 3 e Materials Provided esses 4 n Materials Required but not Provided 4 amp Precautions and Recommendations 5 4 Detailed Protocol eeeeeeee 6 9 Q TroublesBoOUM 5255s aamsansbutaveonsad aad etis 9 KG Reagent 5EIDIBEV uis ern sesto ono n ntcgcis 9 Example or Test Results svo taroen ice References o PERDU ain Me Related Products ssss Oe amp Intended Use o The CycLex Research Product qe ar Histone Acetylation Assay Kit is used for the semi quantitative measurement of histon acetylation level in situ by means of cell based ELISA Applications for this kit include o 1 Monitoring the effects of acological agents on histone acetylation in cells 2 Screening inhibitors of in cells This assay kit is for researth use only and not for use in diagnostic or therapeutic procedures Storage Upon receipt s all components at 4 C Don t expos eagents to excessive light S E KS Q Roan CY 1140 1 Version
9. e cccccccIccccfcliiI L aks Peer nnn T E uv lt Se beh fette 40 000 cells well o 20 000 cells well 0 0 0 0 1 0 2 0 3 TSA conc uM o S S o References o 1 Davie J R amp Chadee D N J GS Biochem Suppl 30 31 203 213 1998 2 Kouzarides T Curr Opin Ge Dev 9 40 84 1999 3 Fenrick R amp Hiebert S WaJ amp f ell Biochem Suppl 30 31 194 202 1998 4 Yoshida M Horinouchi Beppu T Bioassays 17 423 430 1995 5 Richon V M et al P eo ie Sci USA 93 5705 5708 1996 6 Richon V M et al Natl Acad Sci USA 95 3003 3007 1998 7 Cohen L et al P G AACR 39 108 abstr 736 1998 8 Desai D El Bqygumy K amp Amin S Proc AACR 40 2396 abstr 362 1999 9 Laherty C D amp fang W M et al Cell 89 349 356 1997 10 Hassig eischer T C et al Cell 89 341 347 1997 11 Hoffmgfig K Grosch G amp Jung M Nucleic Acids Res 27 2057 2058 1999 e Q Roa CY 1140 11 Version 140318 Q amp C Cellular Histone Acetylation Assay Kit d Vclex User s Manual Q For Research Use Only Not for use in diagnostic procedures amp Related Products 9 CycLex Cellular Histone Acetylation Assay Kit Cat CY 1140 CycLex HDACs Deacetylase Fluorometric Assay Kit Cat CY 1150 CycLex HDACS Deacetylase Fluorometric Assay Kit Cat CY 1158 S CycLex SIRT1 Sir2 Deacetylase Fluorometric Assay Kit Cat CY 1151 Q CycLex SIRT2 Deacetylase Fluoromet
10. ed for all liquid transfers to avoid cross contamination of reagents or samples amp Preparation of Working Solutions Y All reagents need to be brought to room temperature prior to the assay Ass amp geagents in the kit are supplied ready to use with the exception of 10X Wash Buffer Preparet 5 MetOH and 1 paraformaldehyde separately RS 1 Wash Buffer Add 100 mL of the 10X Wash Buffer provided to MP mL of deionized distilled water Mix well Store at 4 C for two weeks or 20 C for long term age 2 95 MetOH For each 96 well plate add 1 mL H20 to 19 CF methanol Cool in 20 C freezer This solution must be prepared fresh Discard unused portion 4QNowing assay completion 3 Alternatively See blelow B Fixing cells to 96 well icroplate and blocking Double Fixation protocol 1 paraformaldehyde in PBS For each 96 plate dissolve 0 2 g of paraformaldehyde in 20 mL of PBS pH7 2 This solution must be prepafed fresh Discard unused portion following assay completion Q Se Assay Procedure re Ww A Culture adherent cells in 96 well mic ate and treatment with compounds N 1 Plate adherent cells in 96 well miron at 40 60 confluency 2 Incubate the microplate at 37 C eR high in CO incubator 3 Add appropriate amount EM ipon to each well Trichostatin A treatment should be run in duplicate as a positive cool for induction of histone acetylation Please include vehicle control e g DMSO in case o
11. f Trichestatin A 4 Incubate the microplgseht 37 C for appropriate time Trichostatin A trggjment Treat cells with 0 5 uM Trichostatin A for 0 0 25 0 5 1 2 3 4 and 5 hours A S gt o o Roan CY 1140 6 Version 140318 g amp C Cellular Histone Acetylation Assay Kit 4 v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures B Fixing cells to 96 well microplate and blocking Single Fixation protocol 3 Fixing of the cells in the 96 well plates should be done as soon as the desired treatment has CINE 1 Remove media from wells with a wrist flick Avoid touching the bottom of the well aft removing cells Q 2 Immediately add 150 uL well of 95 MetOH as a fixative Add the fixing soluti cells are not detached from the plastic Let stand for 10 minutes at room tempe lowly to insure 3 Remove fixing agent from wells with a wrist flick While still inverted Mhe plate gently onto absorbent paper to remove any excess fixing agent still within the wells ww 4 Add 200 uL well of Wash Buffer Let stand for 1 minute at room tegiperature ca 25 C 5 Remove wash buffer with a wrist flick While still inverted seal the plate onto absorbent paper to remove any excess liquid m 6 Add 200 uL well of Blocking Reagent and incubate for 1 MSS room temperature ca 25 C o B Fixing cells to 96 well microplate and blocking Qpuble Fixation protocol Alternatively
12. in microplate at 40 60 confluency amp 4 Incubate O N at 37 C in CO2 incubator c Add appropriate amount of test compound for inductio histone acetylation i Incubate appropriate time at 37 C 1 2 incubator b Discard the culture medium and wash the microfsMate Add 150 uL of ice cold 95 methanol fopftttion 4 Stand for 10 min at roo p Discard the methanol e Add 200 uL of Blocking Reage 4 Incubate rac at room temp ca 25 C Add 50 uL of Anti acet A histone p53 K382 Monoclonal Antibody MC 1 hr at room temp ca 25 C the wells Add 50 uL of conjugated Anti Mouse IgG i Q Incubate for O N at 4 C o Wash the wells Add af of Substrate Reagent t E 50 uL of Stop Solution S od Measure absorbance at 450 nm o e S o A Roan CY 1140 3 Version 140318 g amp C n S Cellular Histone Acetylation Assay Kit e 4 cA ycLex User s Manual For Research Use Only Not for use in diagnostic procedures amp Materials Provided 9 All compounds treatment and positive control Trichostatin A treatment should be ed in duplicate The following components are supplied and are sufficient for the two 96 well micreplates Microplate Two 96 well cell culture plates Q 100X Trichostatin A One vial containing 50 uL of 50 uM Trichostatin A in ous 10X Wash Buffer One 100 mL bottle of 10X buffer containing 2 Tween 20 Blocking Reagent Two bottles containing 20 mL of 1X Blocking Reagent Rey to use
13. not preferable ing data generation and analysis optional ated cylinder S Q Roan CY 1140 4 Version 140318 LS d A S Cellular Histone Acetylation Assay Kit e 4 Vclex User s Manual For Research Use Only Not for use in diagnostic procedures amp Precautions and Recommendations 9 Technical Notes gt V 1 When performing washes manually avoid introducing bubbles when dispensing liquids sak the wells and ensure each well is filled with buffer but not overflowing to avoid cross contamifigtion between wells Empty wells with a wrist flick motion over an appropriate receptacle and w still inverted blot any remaining moisture onto clean absorbent paper qx 2 A brief 1X PBS rinse is recommended prior to the addition of the HRP substrate to remove any traces of the Tween 20 with can interfere with the HRP activity Yon S 3 Do not allow the wells to dry out during the protocol Y 4 Incubation temperatures for Primary Antibody and Detection Antib jy can be varied and should be empirically determined amp 4 General Notes amp Allow all the components to come to room temperature betse Do not use kit components beyond the indicated kit expiidtion date Rinse all detergent residue from glassware lt Ww Use deionized water of the highest quality xO Do not mix reagents from different kits e The buffers and reagents in this kit m ntain preservatives or other chemicals
14. ric Assay Kit Cat CY 1152 amp CycLex SIRT3 Deacetylase Fluorometric Assay Kit Cat CY 1153 Y CycLex SIRT6 Deacetylase Fluorometric Assay Kit Cat CY 1156 Anti Acetylated Histone p53 K382 Mouse Monoclonal Antibody Cat C 029 Anti Histone Deacetylase 1 HDAC1 Rabbit Polyclonal Antibody Cat QY P1011 Anti Histone Deacetylase 2 HDAC2 Rabbit Polyclonal Antibody C Y P1012 Anti Human SIRT1 Rabbit Polyclonal Antibody Cat CY P1016 NAD Dependent Deacetylase SIRT1 Cat CY E1151 Y NAD Dependent Deacetylase SIRT2 Cat CY E1152 wy NAD Dependent Deacetylase SIRT3 Cat CY E1153 Note This product is covered under CycLex s pats U S Patent No 6 884 597 amp PRODUCED BY CycLex Co Ltd RS 1063 103 Terasawapig Ina Nagano 396 0 Japan Fax 181 265 7918 e mail info Gyzlex co j URL http w cyclex co j Creag euL ex 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 pro s without prior written approval from CycLex Co Ltd To inquire about licensing for S ommercial use please contact us via email Q m CY 1140 12 Version 140318 o c
15. t Avoid exposing the microplate to direct sunlight Covering the plate with e g jnum foil is recommended Return Substrate Reagent to 4 C immediately after the necessar Qhume is removed Incubate the plate for 10 15 minutes at room temperature ca 25 C The incubation time may be extended up minutes if the reaction temperature is below than 20 C Add 50 ga of Stop Solution in the same order as the previously added Substrate Reagent M MY absorbance in each well using a spectrophotometric microplate reader at dual wavelengths of 480 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the microplate a 2450 nm if only a single wavelength can be used Wells must be read within 30 minutes of adding Stop Solution CNote 1 Complete removal of liquid at each step is essential to good performance After the last wash S att CY 1140 8 Version 140318 gs CY 1140 9 Version 140318 d 4 v Vclex User s Manual For Research Use Only Not for use in diagnostic procedures n S Cellular Histone Acetylation Assay Kit e amp remove any remaining Wash Buffer by aspirating or decanting Invert the plate and Qi against clean paper towels Note 2 If the microplate reader is not capable of reading absorbance greater than the absor of the highest standard perform a second reading at 405 nm A new standard curv nstructed using the values measured at 405 nm is used to determine of histone acetylation level of
16. ures n S Cellular Histone Acetylation Assay Kit e amp Detection of Signals Addition of Primary and Secondary Antibodies and Substrate ree Remove Blocking Reagent with a wrist flick QN Rinse the wells once with 200 uL well of Wash Buffer This can be achieved eithefby using a multichannel pipette or a manifold Q Remove Wash Buffer with a wrist flick While the plate is still inverted tap ong toten paper to remove any excess buffer within the wells Add 50 uL well of Primary Antibody Solution and incubate for 1 houn room temperature ca 25 C n c W Remove Primary Antibody with a wrist flick gt Rinse the wells once with 200ul well of Wash Buffer Ad Remove Wash Buffer with a wrist flick While still inverted md plate onto absorbent paper Wash wells 4 times with 200 uL well of Wash Buffer ve Wash Buffer in between each wash with a wrist flick o Add 50 uL well of Secondary Antibody Solution aud incubate for O N at 4 C Remove Secondary Antibody with a wrist nia S Rinse wells once with 200 pL well of waste Remove Wash Buffer with wrist flick Ap plate onto absorbent paper Wash wells 4 times with 200 uL w hkor Wash Buffer Remove Wash Buffer in between each wash with a wrist flick o After last wash with Wash er rinse wells once with 300 ul well of 1X PBS Remove with a wrist flick and tap onto a ent paper Ensure that that no liquid remains in the well Add 50 uL well of S rate Reagen

CY-1140

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