EpiQuik Histone H3 Modification Multiplex Assay Kit
Contents
1. EpiQuik Histone H3 Modification Multiplex Assay Kit Colorimetric Base Catalog P 3100 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiQuik Histone H3 Modification Multiplex Assay Kit Colorimetric is suitable for specifically measuring up to 21 histone H3 modifications simultaneously from human mouse rat and other species including most plants fungi and bacteria based on high sequence homology of histone H3 Starting materials can be in a variety of forms including cultured cells and fresh frozen tissues Histone extracts can be prepared by using your own successful method The prepared histone extracts should not contain detergents Each kit can be used for two different samples or a pair of samples control and treated normal and diseased and other paired comparisons Input Material Input materials can be histone extracts or purified histone H3 proteins The amount of histone extracts for each assay can be 20 ng to 500 ng with an optimal range of 50 to 100 ng depending on the purity of histone extracts The amount of purified histone H3 proteins for each assay can be 1 ng to 25 ng with an optimal range of 4 to 5 ng Internal Control An assay control is provided in this kit It can be used for signal intensity comparison between the assay control and the samples to indicate the amount of each histone H3 modification captured from the samples Because the content of each histone H3 modification can vary from tissue2. 20 C away from light 2 Store WB AB DS 96 Well Strip Plate and Extra 8 Well Strips at 4 C away from light and 3 Store remaining components SS and Adhesive Covering Film at room temperature away from light All components of the kit are stable for 6 months from the date of shipment when stored properly Note 1 Check if WB 10X Wash Buffer contains salt precipitates before use If so warm at room temperature or 37 C and shake the buffer until the salts are re dissolved and 2 Check if a blue color is present in DS Developer Solution which would indicate contamination of the solution and should not be used To avoid contamination transfer the amount of DS required into a secondary container tube or vial before adding DS into the assay wells MATERIALS REQUIRED BUT NOT SUPPLIED O Adjustable pipette or multiple channel pipette Aerosol resistant pipette tips 1 5 ml microcentrifuge tubes Incubator for 37 C incubation Oo OF OF QO 2 OQ Distilled water 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Multiple channel pipette reservoirs Microplate reader capable of reading absorbance at 450 nm Page 2 Printed 2014 09 22 P 3100 O Histone extracts or purified histone proteins O Parafilm M or aluminum foil GENERAL PRODUC
3. NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 11 Printed 2014 09 22 P 3100 especially those with small volumes e g 1 ul are completely added into the wells Solutions or antibodies were not Do not allow the pipette tip to touch the actually added into the wells outer edges or inner sides of the wells to prevent solutions from sticking to the surface SS Stop Solution was not evenly Gently and evenly shake the plate frame distributed in the wells in Step across a flat surface so that the solutions in 4b the wells are better distributed Do not stir Did not use the same pipette Use the same multi channel pipette device device throughout the throughout the entire experiment as experiment different pipette devices may have slight variations in performance RELATED PRODUCTS Histone Extract Preparation OP 0006 EpiQuik Total Histone Extraction Kit OP 0007 EpiQuik Total Histone Extraction HT Kit Singleplex Histone Modification Quantification P 3022 EpiQuik Global Di Methyl Histone H3K4 Quantification Kit Colorimetric P 3023 EpiQuik Global Di Methyl Histone H3K4 Quantification Kit Fluorometric P 3024 EpiQuik Global Mono Methyl Histone H3K4 Quantification Kit Colorimetric P 3025 EpiQuik Global Mono Methyl Histone H3K4 Quantification Kit F
4. Global Tri Methyl Histone H3K79 Quantification Kit Fluorometric P 3060 EpiQuik Global Pan Methyl Histone H3K79 Quantification Kit Colorimetric P 3061 EpiQuik Global Pan Methyl Histone H3K79 Quantification Kit Fluorometric P 3062 EpiQuik Total Histone H3 Quantification Kit Colorimetric P 3063 EpiQuik Total Histone H3 Quantification Kit Fluorometric P 4010 EpiQuik Global Acetyl Histone H3K9 Quantification Kit Colorimetric P 4011 EpiQuik Global Acetyl Histone H3K9 Quantification Kit Fluorometric P 4012 EpiQuik Global Acetyl Histone H3K14 Quantification Kit Colorimetric P 4013 EpiQuik Global Acetyl Histone H3K14 Quantification Kit Fluorometric P 4014 EpiQuik Global Acetyl Histone H3K18 Quantification Kit Colorimetric P 4015 EpiQuik Global Acetyl Histone H3K18 Quantification Kit Fluorometric P 4016 EpiQuik Global Acetyl Histone H3K23 Quantification Kit Colorimetric P 4017 EpiQuik Global Acetyl Histone H3K23 Quantification Kit Fluorometric P 4018 EpiQuik Global Acetyl Histone H3K36 Quantification Kit Colorimetric P 4019 EpiQuik Global Acetyl Histone H3K36 Quantification Kit Fluorometric P 4020 EpiQuik Global Acetyl Histone H3K56 Quantification Kit Colorimetric P 4021 EpiQuik Global Acetyl Histone H3K56 Quantification Kit Fluorometric P 4030 EpiQuik Total Histone H3 Acetylation Detection Fast Kit Colorimetric P 4031 EpiQuik Total Histone H3 Acetylation Detection Fast Kit Fluorometric P 4032 EpiQuik Total Histone H4 Acetylatio
5. H3K36me3 H3K79me2 H3K9ac H3K18ac H3ser10P control 5 ng C Assay Blank H3K4me2 H3K9me1 H3K9me3 H3K27me2 H3K36me1 H3K36me3 H3K79me2 H3K9ac H3K18ac H3ser10P control 25 ng D Assay Blank H3K4me2 H3K9me1 H3K9me3 H3K27me2 H3K36me1 H3K36me3 H3K79me2 H3K9ac H3K18ac H3ser10P control 25 ng E Total H3 H3K4me1 H3K4me3 H3K9me2 H3K27me1 H3K27me3 H3K36me2 H3K79me1 H3K79me3 H3K14ac H3K56ac H3ser28P F Total H3 H3K4me1 H3K4me3 H3K9me2 H3K27me1 H3K27me3 H3K36me2 H3K79me1 H3K79me3 H3K14ac H3K56ac H3ser28P G Total H3 H3K4me1 H3K4me3 H3K9me2 H3K27me1 H3K27me3 H3K36me2 H3K79me1 H3K79me3 H3K14ac H3K56ac H3ser28P H Total H3 H3K4me1 H3K4me3 H3K9me2 H3K27me1 H3K27me3 H3K36me2 H3K79me1 H3K79me3 H3K14ac H3K56ac H3ser28P Page 9 Printed 2014 09 22 P 3100 Table 4 Alternatively the two extra strip wells can be set up as controls for detection of select H3 Sample1 Sample 2 A Blank Blank B Assay control 25 ng Assay control 25 ng 0 50 ng 50 ng D 50 ng 50 ng E 100 ng 100 ng F 100 ng 100 ng G 200 ng 200 ng H 200 ng 200 ng modifications each strip can be used as an extra control for the assay TROUBLESHOOTING 1 2 Assay Control 5 ng Assay Control 5 ng Assay Control 5 ng Assay Control 5 ng Assay Control 25 ng Assay Control 25 ng Assay Control 25 ng Assay Control 25 ng Total H3 sample 1 Total H3 sample 1 Total H3 sample 1 Total H3 sa
6. can be between 20 ng to 500 ng with an optimal range of 50 ng to 100 ng Histone Extraction You can use your method of choice for preparing histone extracts from treated and untreated samples The prepared histone extracts should not contain detergents such as SDS Tween Triton X 100 or NP 40 Epigentek also offers a histone extraction kit Cat OP 0006 optimized for use with this kit Histone extracts should be stored in aliquots at 80 C until use Use of Extra Strips If necessary the extra strips included in the kit can be used for input amount pre optimization or used as controls if only a few histone H3 modifications are selected for detection The strips can be set up as indicated in Table 3 and Table 4 under the Extra Strip Well Setup section and carried out by using the same assay protocol described below 1 Working Buffer and Solution Preparation a Prepare Diluted WB 1X Wash Buffer 96 Assays Add 26 ml of WB 10X Wash Buffer to 234 ml of distilled water and adjust pH to 7 2 7 5 This Diluted WB can now be stored at 4 C for up to six months b Prepare Diluted DA Detection Antibody solution Dilute DA Detection Antibody with Diluted WB at a ratio of 1 1000 i e add 1 ul of DA to 1000 ul of Diluted WB 50 ul of Diluted DA will be required for each assay well c Prepare Diluted Assay Control Protein Suggested Assay Control Preparation Prepare 2 concentrations by combining the 100 ng l Assay Cont
7. enables analysis of a single modification or total 21 modification patterns within the same samples e Two extra 8 well strips are included in the kit which can be used if necessary for sample amount pre optimization to determine the input amount ex 50 100 200 ng well needed to fall within the detection limits of the assay Extra strips may also be used as assay controls and total histone level controls if selective detection of some histone H3 modifications from the total 21 modification pattern is desired e Simple reliable and consistent assay conditions PRINCIPLE amp PROCEDURE 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only The EpiQuik Histone H3 Modification Multiplex Assay Kit Colorimetric is designed for measuring multiple histone H3 modifications simultaneously In an assay with this kit each histone H3 modified at specific sites will be captured by an antibody that is coated on the strip wells and specifically targets the appropriate histone modification pattern The captured histone modified at specific sites will be detected with a detection antibody followed by a color development reagent The ratio of modified Page 4 Printed 2014 09 22 P 3100 histone is proportional to the intensity of absorbance measured by a microplate reader a
8. leads to transcriptional inactivation Other histone demethylases such as jumonji SHDM2A are responsible for H3K9 demethylation whereas JHDM1 has the ability to convert active chromatin marks such as H3 K36me2 to an unmodified state Lysine residues can be mono di or trimethylated each of which can differentially regulate chromatin structure and transcription Along with other histone modifications such as phosphorylation this enormous variation leads to a multiplicity of possible combinations of different modifications This may constitute a histone code which can be read and interpreted by different cellular factors 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 3 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3100 Abnormal histone modification patterns have been associated with many different diseases such as cancer autoimmune disorders and inflammatory and neurological diseases Therefore detection of histone H3 modifications would provide useful information for a better understanding of epigenetic regulation of gene activation and silencing histone modification associated pathological disease process as well as for developing histone modification targeted drugs As the leading provider of histone modification assay products Epigentek was the first company to offer
9. Pan Methyl Histone H3K27 Quantification Kit Colorimetric P 3045 EpiQuik Global Pan Methyl Histone H3K27 Quantification Kit Fluorometric P 3046 EpiQuik Global Mono Methyl Histone H3K36 Quantification Kit Colorimetric P 3047 EpiQuik Global Mono Methy Histone H3K36 Quantification Kit Fluorometric P 3048 EpiQuik Global Di Methy Histone H3K36 Quantification Kit Colorimetric P 3049 EpiQuik Global Di Methy Histone H3K36 Quantification Kit Fluorometric 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 12 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3100 P 3050 EpiQuik Global Tri Methyl Histone H3K36 Quantification Kit Colorimetric P 3051 EpiQuik Global Tri Methyl Histone H3K36 Quantification Kit Fluorometric P 3052 EpiQuik Global Pan Methyl Histone H3K36 Quantification Kit Colorimetric P 3053 EpiQuik Global Pan Methyl Histone H3K36 Quantification Kit Fluorometric P 3054 EpiQuik Global Mono Methyl Histone H3K79 Quantification Kit Colorimetric P 3055 EpiQuik Global Mono Methy Histone H3K79 Quantification Kit Fluorometric P 3056 EpiQuik Global Di Methyl Histone H3K79 Quantification Kit Colorimetric P 3057 EpiQuik Global Di Methy Histone H3K79 Quantification Kit Fluorometric P 3058 EpiQuik Global Tri Methyl Histone H3K79 Quantification Kit Colorimetric P 3059 EpiQuik
10. T INFORMATION Quality Control Each lot of the EpiQuik Histone H3 Modification Multiplex Assay Kit Colorimetric is tested against predetermined specifications to ensure consistent product quality Epigentek guarantees the performance of all products in the manner described in our product instructions Product Warranty If this product does not meet your expectations simply contact our technical support unit or your regional distributor We also encourage you to contact us if you have any suggestions about product performance or new applications and techniques Safety Suitable lab coat disposable gloves and proper eye protection are required when working with this product Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design The information in this User Guide is subject to change at any time without notice Thus only use the User Guide that was supplied with the kit when using that kit Usage Limitation The EpiQuik Histone H3 Modification Multiplex Assay Kit Colorimetric is for research use only and is not intended for diagnostic or therapeutic application A BRIEF OVERVIEW Histone modifications have been defined as epigenetic modifiers Post translational modifications of histones include the acetylation of specific lysine residues by histone acetyltransferases HATs deacetylation by histone deacetylase HDACs the methylation of lysine and arginine residues by h
11. a series of kits for singleplex quantification of each histone H3 modification We have further refined our assay expertise by developing the EpiQuik Histone H3 Modification Multiplex Assay Kit As the first multiplex assay for detecting up to 21 modified histone H3 patterns simultaneously this kit has the following advantages e Simultaneously measure 21 different histone H3 modifications which include all of the most important and the most well characterized patterns H3K4me1 H3K4me2 H3K4me3 H3K9me1 H3K9me2 H3K9me3 H3K27me1 H3K27me2 H3K27me3 H3K36me1 H3K386me2 H3K36me3 H3K79me1 H3K79me2 H3K79me3 H3K9ac H3K14ac H3K18ac H3K56ac H3ser10P H3ser28P Total H3 e Quick and efficient procedure which can be finished within 2 5 hours e Innovative colorimetric assay without the need for radioactivity electrophoresis chromatography or expensive equipment e High sensitivity with a detection limit as low as 0 5 ng well for each modification pattern and a detection range from 20 ng to 500 ng well of histone extracts e Anassay control is conveniently included for quantification of each histone H3 modification e Total histone H3 sets are included which can be used for normalizing total histone H3 levels for relative comparison of histone H3 content between different samples or different treatment conditions e Strip microplate format makes the assay flexible manual or high throughput which
12. ate at room temperature for 1 to 10 min away from light Begin monitoring color change in the sample wells and control wells within one minute The DS solution will turn blue in the presence of sufficient modified products 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 7 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3100 Note Average color development time is 2 5 minutes Use control wells and blank wells as a reference for color development Add 100 ul of SS to each well to stop enzyme reaction when color in the positive control wells turns medium blue The color will change to yellow after adding SS and the absorbance should be read ona microplate reader within 2 to 10 min at 450 nm with an optional reference wavelength of 655 nm Note 1 Most microplate readers have the capability to carry out dual wavelength analysis and will automatically subtract reference wavelength absorbance from the test wavelength absorbance If your plate reader does not have this capability the plate can be read twice once at 450 nm and once at 655 nm Then manually subtract the 655 nm ODs from 450 nm ODs 2 If the strip well microplate frame does not fit in the microplate reader transfer the solution to a standard 96 well microplate 5 Histone H3 Modification Calculation Calculate the ave
13. istone methytransferases HMTs the demethylation of lysine residues by histone demethylases HDMTs and the phosphorylation of specific serine groups by histone kinases HKs Additional histone modifications include the attachment of ubiquitin Ub small ubiquitin like modifiers SUMOs and poly ADP ribose PAR units Next to DNA methylation histone acetylation and histone methylation are the most well characterized epigenetic marks Generally tri methylation at H3 K4 H3 K36 or H3 K79 results in an open chromatin configuration and is therefore characteristic of euchromatin Euchromatin is also characterized by a high level of histone acetylation which is mediated by histone acetyltransferases Conversely histone deacetylases have the ability to remove this epigenetic mark which leads to transcriptional repression Condensed heterochromatin is enriched in tri methylation of H3 K9 and H3 K27 and silencing of euchromatin loci caused by histone deacetylation involves the recruitment of specific K9 histone methyltransferases Methylated H3 K9 provides a binding site for the chromodomain containing heterochromatin protein 1 HP1 which induces transcriptional repression and heterochromatinization At euchromatic loci this process is mediated by co repressors such as retinoblastoma protein pRb or KAP1 Histone demethylases have the opposite effect on transcription For example the histone demethylase LSD1 is responsible for H3K4 demethylation which
14. la 3 H3 modification in sample 1 or treated sample Relative Change x 100 H3 modification in sample 2 or control sample SUGGESTED BUFFER AND SOLUTION SETUP Table 1 Approximate amount of required buffers and solutions for defined assay wells based on the protocol Reagents 1 well 1 strip 2 strips 6 strips 12 strips 8 wells 16 wells 48 wells 96 wells Diluted WB 1 2 ml 10 ml 20 ml 60 ml 120 ml Diluted DA 50 ul 400 ul 800 ul 2400 ul 4800 ul Assay Control Protein N A N A 2 ul 2 ul 2 ul DA Developer Solution 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml SS Stop Solution 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml STRIP WELL SETUP Table 2 An antibody for each H3 modification is coated onto the indicated wells accordingly Table 3 Two extra strip wells can be set up for input amount pre optimization Different concentrations of samples can be added to wells C through H as shown below 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only 1 2 3 4 5 6 7 8 9 10 11 12 A Assay Blank H3K4me2 H3K9me1 H3K9me3 H3K27me2 H3K36me1 H3K36me3 H3K79me2 H3K9ac H3K18ac H3ser10P control 5 ng B Assay Blank H3K4me2 H3K9me1 H3K9me3 H3K27me2 H3K36me1
15. luorometric P 3026 EpiQuik Global Tri Methyl Histone H3K4 Quantification Kit Colorimetric P 3027 EpiQuik Global Tri Methyl Histone H3K4 Quantification Kit Fluorometric P 3028 EpiQuik Global Pan Methyl Histone H3K4 Quantification Kit Colorimetric P 3029 EpiQuik Global Pan Methyl Histone H3K4 Quantification Kit Fluorometric P 3030 EpiQuik Global Mono Methyl Histone H3K9 Quantification Kit Colorimetric P 3031 EpiQuik Global Mono Methyl Histone H3K9 Quantification Kit Fluorometric P 3032 EpiQuik Global Di Methyl Histone H3K9 Quantification Kit Colorimetric P 3033 EpiQuik Global Di Methyl Histone H3K9 Quantification Kit Fluorometric P 3034 EpiQuik Global Tri Methyl Histone H3K9 Quantification Kit Colorimetric P 3035 EpiQuik Global Tri Methyl Histone H3K9 Quantification Kit Fluorometric P 3036 EpiQuik Global Pan Methyl Histone H3K9 Quantification Kit Colorimetric P 3037 EpiQuik Global Pan Methyl Histone H3K9 Quantification Kit Fluorometric P 3038 EpiQuik Global Mono Methy Histone H3K27 Quantification Kit Colorimetric P 3039 EpiQuik Global Mono Methy Histone H3K27 Quantification Kit Fluorometric P 3040 EpiQuik Global Di Methy Histone H3K27 Quantification Kit Colorimetric P 3041 EpiQuik Global Di Methy Histone H3K27 Quantification Kit Fluorometric P 3042 EpiQuik Global Tri Methyl Histone H3K27 Quantification Kit Colorimetric P 3043 EpiQuik Global Tri Methyl Histone H3K27 Quantification Kit Fluorometric P 3044 EpiQuik Global
16. may be a result of many factors If the affecting factors cannot be determined use new or re prepared histone extracts Uneven color development Insufficient washing of the wells Ensure the wells are washed according to the guidance of washing and that the residual washing buffer is removed as much as possible Delayed color development or delayed stopping of color development in the wells Ensure DS Development Solution or SS Stop Solution is added sequentially and is consistent with the order you added the other reagents e g from well A to well G or from well 1 to well 12 Large variation between replicate wells Color reaction is not evenly stopped due to an inconsistency in pipetting time Ensure DS Developer Solution and SS Stop Solution are added at the same time between replicates or otherwise maintain a consistent timing in between each addition of solutions Color reaction is not evenly stopped due to an inconsistent order of adding solutions Ensure all solutions particularly DS Developer Solution and SS Stop Solution are added in the same order each time as all other solutions The solutions are not evenly added due to inconsistency in pipetting volume Ensure the solution in each pipette tip is equal in the multi channel pipette Equilibrate the pipette tip in any solutions before adding them Ensure the solutions 110 Bi County Blvd Ste 122 Farmingdale
17. mple 1 Total H3 Sample 2 Total H3 Sample 2 I o Tnm o oO w gt Total H3 Sample 2 Total H3 Sample 2 Problem Possible Cause Suggestion No signal or weak signal in both the Assay Control and sample wells Reagents are added incorrectly Check if reagents are added in the proper order with the right amount and if any steps in the protocol may have been omitted by mistake Incubation time and temperature are incorrect Ensure the incubation time and temperature described in the protocol are followed correctly Incorrect absorbance reading Check if appropriate absorbance wavelength 450 nm is used Kit was not stored or handled properly Ensure all components of the kit were stored at the appropriate temperature and the cap is tightly closed after each opening or use No signal or weak signal in only the Assay Control wells The Assay Control Protein amount is insufficiently added to the well in Step 2c Ensure a sufficient amount of Assay Control Protein is added The Assay Control Protein is degraded due to improper storage conditions Follow the Shipping amp Storage guidance in this User Guide for storage of Assay Control Protein 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Prod
18. n Detection Fast Kit Colorimetric P 4033 EpiQuik Total Histone H4 Acetylation Detection Fast Kit Fluorometric 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 13 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3100
19. rage duplicate readings for the sample wells assay control wells and blank wells Simple amount quantification of each H3 modification or total H3 in the samples can be carried out using Formula 1 shown below Formula 1 Sample OD Blank OD S H3 Modification or total H3 ng ug protein x 1000 Assay Control OD Blank OD P S is the amount of input sample protein in ng P is the amount of input assay control in ng use 25 ng Example calculation Average OD450 of blank is 0 115 Average OD450 of Assay control is 0 775 Average OD450 of Sample H3 modification or total H3 is 0 575 Sis 100 ng P is 25 ng 0 575 0 115 100 H3 Modification or total H3 ng ug protein x 1000 174 2 ng ug protein 0 775 0 115 25 Calculate the percentage of histone H3 modification in total H3 using Formula 2 shown below Formula 2 Amount of H3 modification ng rotein CEG Aet of HS modification ng ug protein sane Amount of total H3 ng ug protein The amount of H3 modification or total H3 is calculated from Formula 1 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 8 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 09 22 P 3100 d Calculate the relative change of each histone H3 modification between different samples using Formula 3 shown below Formu
20. represents a specific histone modification pattern according to Table 2 of the Strip Well Setup section Blank Wells Add 49 ul of AB to each blank well Control Wells Add 49 ul of AB and 1 ul of Diluted Assay Control Protein to each standard well using 2 wells for each concentration point 5 and 25 ng well based on the dilution chart in Step 1c see Table 2 as an example Sample Wells Add 46 to 49 ul of AB and 1 to 4 ul of your histone extracts Total volume should be 50 ul per well Note 1 Follow the strip well setup diagrams Table 2 2 It is recommended to use 50 100 ng or pre optimized amount of histone extract per well Tightly cover strip well microplate with Adhesive Covering Film to avoid evaporation and incubate at 37 C for 60 to 120 min Note The Adhesive Covering Film can be cut to the required size to cover the strips based on the number of strips to be used Remove the reaction solution from each well Wash each well three times with 150 ul of the Diluted WB each time 3 Antibody Binding a Add 50 ul of the Diluted DA to each well then cover with Parafilm M or aluminum foil and incubate at room temperature for 60 min Remove the Diluted DA solution from each well Wash each well four times with 150 ul of the Diluted WB each time Note Ensure any residual wash buffer in the wells is removed as much as possible at each wash step 4 Signal Detection a Add 100 ul of DS to each well and incub
21. rol Protein with AB into final concentrations of 5 and 25 ng ul according to the following dilution chart The high concentration 25 ng ul of the Assay Control Protein can be used for a simple amount quantification of histone H3 modification and total H3 The low concentration 5 ng ul along with high concentration is used to generate proportional concentration signal intensity for determining if the assay control works properly Assay Control Resulting Assay Control vue 100 ng pl Az Concentration 1 1 0 ul 19 0 ul 5 ng ul 2 1 0 ul 3 0 ul 25 ng ul 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 6 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3100 Note Keep each of the diluted solutions except Diluted WB on ice until use Any remaining diluted solutions other than Diluted WB should be discarded if not used within the same day 2 Histone Binding a Predetermine the number of strip wells required for your experiment It is advised to run replicate samples include blank and positive controls to ensure that the signal generated is validated Carefully remove un needed strip wells from the plate frame and place them back in the bag seal the bag tightly and store at 4 C Note If removing strip wells you must absolutely keep track of which wells have been removed as each well
22. t a wavelength of 450 nm Start with histone protein extracts Modified histone binds to the assay wells Wash wells then add detection antibody VW SM VW Add color developing solution for color development then measure absorbance Fig 1 Schematic procedure of the EpiQuik Histone H3 Fig 2 Working principle of EpiQuik Histone Modification Multiplex Assay Kit Colorimetric H3 Modification Multiplex Assay Colorimetric 1 2 1 15 4 m MCF 7 I MDA 231 0 9 4 0 8 5 E 4 0 7 D 0 6 a 0 5 0 4 0 3 5 0 2 5 0 1 4 of n the oS of gs RS s S i So me amp oe tf S 8 20 SPL FFF EES Ea Histone modifications Fig 3 Histone extracts were prepared from MCF 7 and MDA 231 cells using the EpiQuik Total Histone Extraction Kit Epigentek OP 0006 and 21 histone H3 modifications were measured using the EpiQuik Histone H3 Modification Multiolex Assav Kit Colorimetric 100 na of total histone proteins per well were used 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 5 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3100 PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Input Amount The amount of histone extracts for each assay
23. to tissue and from normal and diseased states or from different treatments it is advised to run duplicate for each sample to ensure that the signal generated is validated Precautions To avoid cross contamination carefully pipette the sample or solution into the strip wells Use aerosol barrier pipette tips and always change pipette tips between liquid transfers Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3100 KIT CONTENTS Component 96 Assays Storage Cat P 3100 96 Upon Receipt WB 10X Wash Buffer 28 ml 4 C AB Antibody Buffer 8 ml 4 C DA Detection Antibody 1000X 12 ul 20 C DS Developer Solution 12 ml 47 SS Stop Solution 12 ml RT Assay Control Protein 100 ug ml 20 ul 20 C 96 Well Strip Plate With Frame 1 4 Extra 8 Well Strips 2 4 C Adhesive Covering Film 1 RT User Guide 1 RT Spin the solution down to the bottom prior to use SHIPPING amp STORAGE The kit is shipped in two parts the first part at ambient room temperature and the second part on frozen ice packs at 4 C Upon receipt 1 Store DA and Assay Control Protein at
24. ucts are for research use only Page 10 Printed 2014 09 22 P 3100 High background present in the blank wells Insufficient washing of wells Check if washing recommendations at each step is performed according to the protocol Contaminated by sample or Assay Conirol Protein Ensure the well is not contaminated from adding sample or Assay Control Protein accidentally or from using contaminated tips Incubation time with Diluted DA is too long The incubation time at Step 3a should not exceed 90 min Over development of color Decrease the development time in Step 4a before adding SS Stop Solution in Step 4b No signal or weak signal only in sample wells or for some of H3 modification patterns Protein sample is not properly extracted or purified Ensure your protocol is suitable for histone protein extraction For the best results it is advised to use EpiQuik Total Histone Extraction Kit Epigentek OP 0006 Sample amount added into the wells is insufficient Ensure a sufficient amount of histone extracts is used as indicated in Step 2d The sample can be titrated to determine the optimal amount to use in the assay Sample was not stored properly or has been stored for too long Ensure sample is stored in aliquots at 80 C Histone extracts should be stored for up to 6 months at 80 C Little or no modified H3 at specific sites in the sample This problem
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