Epigenase™ 5-mC Hydroxylase TET Activity/Inhibition Assay Kit
Contents
1. Epigenase JARID Demethylase Activity Inhibition Assay Kit Fluorometric Base Catalog P 3083 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The Epigenase JARID Demethylase Activity Inhibition Assay Kit Fluorometric is suitable for measuring activity or inhibition of total JARID using nuclear extracts or subtype JARID JARID1A through JARID1D purified enzymes from a broad range of species such as mammalians plant fungal and bacterial types in a variety of forms including cultured cells and fresh tissues Nuclear extracts can be prepared by using your own successful method For your convenience and the best results Epigentek offers a nuclear extraction kit Cat OP 0002 optimized for use with this kit Nuclear extracts can be used immediately or stored at 80 C for future use Purified enzymes can be active JARIDs from recombinant proteins or isolated from cell tissues Input Material Inout materials can be nuclear extracts or purified JARID enzymes The amount of nuclear extracts for each assay can be 0 5 ug to 20 yg with an optimal range of 2 ug to 10 ug The amount of purified enzymes can be 10 ng to 500 ng depending on the purity and subtypes of the enzymes Internal Control A JARID assay standard demethylated histone H3 K4 is provided in this kit for the quantification of JARID enzyme activity Because JARID activity can vary from tissue to tissue and from normal and diseased states it is advised to run replica2. 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 Page 9 Printed 2014 10 30 P 3083 the cap is tightly capped after each opening or use No signal or weak signal in only the standard curve wells The standard amount is insufficiently added to the well in Step 2c Ensure a sufficient amount of standard is added The standard is degraded due to improper storage conditions Follow the Shipping amp Storage guidance in this User Guide for storage of JD4 JARID Assay Standard 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 standard Ensure the well is not contaminated from adding sample or standard accidentally or from using contaminated tips Incubation time with Diluted JD6 is too long The incubation time at Step 3d should not exceed 45 min Over development of fluorescence Decrease the development time in Step 4a No signal or weak signal only in sample wells Protein sample is not properly extracted or purified Ensure your protocol is suitable for JARID protein extraction For the best results it is advised to use Epigentek s Nuclear Extraction Kit Cat No OP 000
3. Storage Nuclear extract or purified JARID enzyme should be stored in aliquots at 80 C until use 1 Working Buffer and Solution Preparation a Prepare Diluted JD1 1X Wash Buffer 48 Assay Kit Add 13 ml of JD1 10X Wash Buffer to 117 ml of distilled water and adjust pH to 7 2 7 5 110 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 Page 5 Printed 2014 10 30 P 3083 96 Assay Kit Add 26 ml of JD1 10X Wash Buffer to 234 ml of distilled water and adjust pH to 7 2 7 5 This Diluted JD1 1X Wash Buffer can now be stored at 4 C for up to six months Prepare CJD2 Completed JARID Assay Buffer Add Co Factor 1 Co Factor 2 and Co Factor 3 to JD2 JARID Assay Buffer at a ratio of 1 100 ex add 1 ul of each Co Factor to every 100 ul of JC2 Prepare Diluted JD5 Capture Antibody Solution Dilute JD5 Capture Antibody with Diluted JD1 1X Wash Buffer at a ratio of 1 1000 ex add 1 ul of JD5 to 1000 ul of Diluted JD1 1X Wash Buffer 50 ul of Diluted JC5 will be required for each assay well Prepare Diluted JD6 Detection Antibody Solution Dilute JD6 Detection Antibody with Diluted JD1 1X Wash Buffer at a ratio of 1 2000 ex add 1 ul of JD6 to 2000 ul of 1 X wash buffer 50 ul of Diluted JD6 will be required for each assay well Prepare Diluted JD4 Standar
4. amount of JARID converted demethylated product using the following formulas Sample RFU Blank RFU Demethylated product ng Slope Demethylated Product ng JARID Activity nghnin ng _ X 1000 Protein Amount ug X min Incubation time minutes at Step 2f For inhibition calculation Inhibitor Sample RFU Blank RFU Inhibition 1 1 X 100 No Inhibitor Sample RFU Blank RFU 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 Web www epigentek com Printed 2014 10 30 Epigentek Group Inc All rights reserved Products are for research use only P 3083 SUGGESTED WORKING BUFFER AND SOLUTION SETUP Table 2 Approximate amount of required buffers and solutions for defined assay wells Reagents 1 well 1 strip 2 strips 6 strips 12 strips 8 wells 16 wells 48 wells 96 wells Diluted JD1 2 5 ml 20 ml 40 ml 120 ml 240 ml CJD2 50 ul 400 ul 800 ul 2400 ul 4800 ul JD3 1 ul 8 ul 16 ul 50 ul 120 ul JD4 NA NA 1 pl optional 2 ul 2 ul Diluted JD5 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted JD6 50 ul 400 ul 800 ul 2400 ul 4800 ul Fluorescence 0 05 ml 0 4ml 0 8 ml 2 4 ml 4 8 ml Development Solution SUGGESTED STRIP WELL SETUP Table 3 The suggested strip well plate setup for JARID activity assay in a 48 assay format in a 96 assay format Strip
5. amp STORAGE The kit is shipped in two parts the first part at ambient room temperature and the second and third parts on frozen ice packs at 4 C Upon receipt 1 Store JD3 JD4 JD6 and JD7 at 20 C away from light 2 Store JD1 JD5 JD8 Co factor 1 Co factor 2 Co factor 3 and 8 Well Assay Strips at 4 C away from light 3 Store remaining components JD2 JD9 and Adhesive Covering Film at room temperature Note 1 Check if JD1 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 All components of the kit are stable for 6 months from the date of shipment when stored properly MATERIALS REQUIRED BUT NOT SUPPLIED m OHoadada Adjustable pipette or multiple channel pipette Multiple channel pipette reservoirs Aerosol resistant pipette tips Fluorescence microplate reader capable of reading fluorescence at 530ex 590em nm 1 5 ml microcentrifuge tubes Incubator for 37 C incubation 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 Page 2 Printed 2014 10 30 P 3083 Plate seal Distilled water Nuclear extract or purified enzymes oO Oo o O GENERAL PRODUCT INFORMATION Parafilm M or aluminium foil Quality Control Each
6. lot of the Epigenase JARID Demethylase Activity Inhibition Assay Kit Fluorometric 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 Epigenase JARID Demethylase Activity Inhibition Assay Kit Fluorometric is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The Epigenase JARID Demethylase Activity Inhibition Assay Kit Fluorometric and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW Lysine histone methylation is one of the most robust epigenetic marks and is essential for the regulation of multiple cellular processes The methylatio
7. 2 Also use fresh cells or tissues for protein extraction as frozen cells or tissues could lose enzyme activity Sample amount added into the wells is insufficient Ensure a sufficient amount of purified enzymes or nuclear extracts is used as indicated in Step 2 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 with no more than 6 weeks for nuclear extracts and 6 months for purified enzymes Avoid repeated freezing thawing Little or no activity of JARID contained in the sample This problem may be a result of many factors If the affecting factors cannot be determined use new or re prepared nuclear extracts or purified enzymes Uneven fluorescent development Insufficient washing of the wells Ensure the wells are washed according to the guidance of washing and residue washing buffer is removed as much as possible Delayed fluorescence development in the wells Ensure fluorescence development 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 1 110 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 u
8. RID1A expression is observed in gastric cancer and its inhibition triggers senescence of malignant cells Detection of activity and inhibition of JARID would be important in elucidating mechanisms of epigenetic regulation of gene activation and silencing and benefiting cancer diagnostics and therapeutics Prior to this kit there was only one method used for detecting JARID activity inhibition This method is based on the measurement of formaldehyde release a by product of JARID enzymatic reaction and has significant weaknesses 1 a large amount at ug level of substrate and enzyme are required 2 nuclear extracts from cell tissues cannot be used 3 redox sensitive JARID inhibitiors are not suitable for testing with this method 4 high interferance by SDS DMSO thiol containing chemicals and ions which are often contained in enzyme solution tested compound solvents and assay buffers and 5 Less accuracy than direct measurement of JARID converted demethylated products The Epigenase JARID Demethylase Activity Inhibition Assay Kit Fluorometric addresses all of these issues Compared to a formaldehyde release based method this kit has the following advantages e 3 hour fluorometric procedure in a 96 stripwell microplate format allows for either manual or high throughput analysis e Directly measures JARID activity via a straightforward detection of JARID converted demethylated products rather than by products thus eliminating assay in
9. d Solution Suggested Standard Curve Preparation First dilute JD4 with JD2 to 5 ng ul by adding 1 ul of JD4 to 9 ul of JD2 Then further prepare five concentrations by combining the 5 ng ul Diluted JD4 with JD2 into final concentrations of 0 2 0 5 1 2 and 5 ng ul according to the following dilution chart sos sng voe Resulting D8 1 1 0 ul 24 0ul 0 2 ng ul 2 1 0 ul 9 0 ul 0 5 ng ul 3 1 0 ul 4 0 ul 1 0 ng ul 4 2 0 ul 3 0 ul 2 0 ng ul 5 4 0 ul 0 0 ul 5 0 ng ul Prepare Fluorescence Development Solution Add 1 ul of JD7 Fluoro Developer and 1 ul of JD8 Fluoro Enhancer to each 500 ul of JD9 Fluoro Dilutor Note Keep each of the diluted solutions except Diluted JD1 1X Wash Buffer on ice until use Any remaining diluted solutions other than Diluted D1 should be discarded if not used within the same day 2 Enzymatic Reaction a Predetermine the number of strip wells required for your experiment It is advised to run replicate samples include blank and positive control 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 b Blank Wells Add 49 ul of CUD2 and 1 ul of JD3 to each blank well 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 Epigentek Group Inc All r
10. ights reserved Products are for research use only Printed 2014 10 30 P 3083 d e Standard Wells For a standard curve add 49 ul of CJD2 and 1 ul of JD4 to each standard well with a minimum of five wells each at different concentrations between 0 2 to 5 ng ul based on the dilution chart in Step 1e see Table 2 as an example Sample Wells Without Inhibitor Add 45 to 48 ul of CUD2 1 ul of JD3 and 1 to 4 ul of your nuclear extract or 1 to 4 ul of purified JARID enzyme to each sample well without inhibitors Total volume should be 50 ul per well Sample Wells With Inhibitor Add 40 to 43 ul of CJD2 1 ul of JD3 1 to 4 ul of nuclear extract or 1 to 4 ul of purified JARID enzyme and 5 ul of inhibitor solution Total volume should be 50 ul per well Note 1 Follow the suggested well setup diagrams 2 It is recommended to use 2 ug to 10 ug of nuclear extract per well or 10 ng to 100 ng of purified enzyme per well 3 The concentration of inhibitor to be added into the sample wells can be varied e g 1 uM to 1000 uM However the final concentration of the inhibitors before adding to the wells should be prepared with JD2 at a 1 10 ratio e g add 0 5 ul of inhibitor to 4 5 ul of JD2 so that the original solvent of the inhibitor can be reduced to 1 of the reaction solution or less The Jumonji demethylase general inhibitor N Oxalylglycine can be used as the control inhibitor Tightly cover the strip well microplate with Adhe
11. n of H3 K4 seems to be of particular significance as it is associated with active regions of the genome H3 K4 methylation was considered irreversible until the identification of a large number of histone demethylases indicated that demethylation events play an important role in histone modification dynamics So far at least 2 classes of H3 K4 specific histone demethylase LSD1 BHC110 KDM1 and JARIDs have been identified The JARID family except JARID2 JARID1A JARID1B JARID1C and JARID1D can remove tri methylation from H3 K4 JARID demethylases are Jumonji domain proteins and catalyze the removal of methylation by using a hydroxylation reaction with a requirement of iron and a ketoglutarate as cofactors Fig 1 Histone H3 K4 demethylation reaction catalyzed by JARID demethylase 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 Web www epigentek com Printed 2014 10 30 Epigentek Group Inc All rights reserved Products are for research use only P 3083 JARIDs function as transcription repressors and might participate in different biological processes through recruitment to different chromosomal regions and differing enzymatic activities JARID demethylases are also found to have potential oncogenic functions For example all 5 members of JARID are overexpressed in bladder cancer and may promote cancer progression by regulating E2F expression Increased JA
12. om m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 10 30 P 3083 Start with nuclear extract or purified JARID enzyme Incubate with substrate and assay buffer for 90 minutes Wash wells then add capture antibody Wash wells then add detection antibody l Add fluoro developing solution for fluorescence D development then measure RFU Schematic procedure of Epigenase JARID Demethylase Activity Inhibition Assay Kit Fluorometric PROTOCOL Total JARID activity RFU 8000 7000 6000 5000 4000 3000 2000 1000 T T T 1 0 5 10 15 20 Nuclear extracts ug Demonstration of high sensitivity of JARID activity assay achieved by using A549 nuclear extracts with Epigenase JARID Demethylase Activity Inhibition Assay Kit Fluorometric For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Input Amount The amount of nuclear extracts for each assay can be 0 5 ug to 20 ug with an optimal range of 2 10 ug The amount of purified enzymes can be 10 ng to 500 ng depending on the purity and catalytic activity of the enzymes Nuclear Extraction You can use your method of choice for preparing nuclear extracts Epigentek offers a nuclear extraction kit Cat OP 0002 optimized for use with this kit Nuclear Extract or Purified JARID
13. ounty 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 10 30 Epigentek Group Inc All rights reserved Products are for research use only P 3083 b Read the fluorescence on a fluorescence microplate reader within 2 to 10 min at 530ex 590em nm Note If the strip well plate frame does not fit in the fluorescence microplate reader transfer the solution to a standard 96 well microplate 5 JARID Activity Calculation a Calculate the average duplicate readings for the sample wells and blank wells b Calculate JARID activity or inhibition using the following formulas For simple calculation Sample RFU Blank RFU JARID Activity OD min mg _ x 1000 Protein Amount ug x min Protein amount ug added into the reaction at Step 2d Incubation time minutes at Step 2f Example calculation Average RFU of sample is 6800 Average RFU of blank is 800 Protein amount is 5 pg Incubation time is 2 hours 120 min 6800 800 JARID activity X 1000 10000 REFU min mg 5 X 120 For accurate or specific activity calculation es Generate a standard curve and plot RFU value versus amount of JD4 at each concentration point 2 Determine the slope as RFU ng you can use Microsoft Excel statistical functions for slope calculation then calculate the
14. s 7 to 12 can be configured as Sample The controls and samples can be measured in duplicate Well Strip 1 Strip 2 Strip 3 Strip 4 Strip 5 Strip 6 A Blank Blank Sample Sample Sample Sample B JD4 0 2 ng JD4 0 2 ng Sample Sample Sample Sample c JD4 0 5 ng JD4 0 5 ng Sample Sample Sample Sample D JD4 1 0 ng JD4 1 0 ng Sample Sample Sample Sample E JD4 2 0 ng JD4 2 0 ng Sample Sample Sample Sample F JD4 5 0 ng JD45 0 ng Sample Sample Sample Sample G Sample Sample Sample Sample Sample Sample H Sample Sample Sample Sample Sample Sample TROUBLESHOOTING Problem Possible Cause Suggestion No signal or weak signal in both the positive 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 The well is incorrectly washed before enzyme reaction Ensure the well is not washed prior to adding the positive control and sample Incubation time and temperature are incorrect Ensure the incubation time and temperature described in the protocol are followed correctly Incorrect fluorescence reading Check if appropriate fluorescence wavelength 530ex 590em is used Kit was not stored or handled properly Ensure all components of the kit were stored at the appropriate temperature and 110 Bi County Blvd Ste
15. se only Page 10 Printed 2014 10 30 P 3083 to well 12 RELATED PRODUCTS Nuclear Extract Preparation OP 0002 1 EpiQuik Nuclear Extraction Kit Histone Demethylase Activity inhibition Assay P 3077 P 3078 P 3079 P 3080 P 3081 P 3082 EpiQuik Histone Demthylase H3 K9 Specific Activity Inhibition Fast Assay Kit Epigenase LSD1 Demethylase Activity Inhibition Assay Kit Colorimetric Epigenase LSD1 Demethylase Activity Inhibition Assay Kit Fluorometric Epigenase JMJD2 Demethylase Activity Inhibition Assay Kit Colorimetric Epigenase JMJD2 Demethylase Activity Inhibition Assay Kit Fluorometric Epigenase JARID Demethylase Activity Inhibition Assay Kit Colorimetric 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 11 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 10 30 P 3083
16. sive Covering Film to avoid evaporation and incubate at 37 C for 60 to 120 min Note 1 The incubation time may depend on intrinsic JARID activity However in general 60 90 min incubation is suitable for active purified JARID enzyme and 90 120 min incubation is required for nuclear extract 2 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 JD1 1X Wash Buffer each time 3 Antibody Binding amp Signal Enhancing a Add 50 ul of the Diluted JD5 to each well then cover with Parafilm M or aluminium foil and incubate at room temperature for 60 min Remove the Diluted JD5 solution from each well Wash each well three times with 150 ul of the Diluted JD1 each time Add 50 ul of the Diluted JD6 to each well then cover with Parafilm M or aluminium foil and incubate at room temperature for 30 min Remove the Diluted JD6 solution from each well Wash each well four times with 150 ul of the Diluted JD1 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 50 ul of Fluorescence Development solution to each well and incubate at room temperature for 2 to 4 min away from light Fluorescence Development solution will turn pink in the presence of sufficient demethylated products 110 Bi C
17. te samples 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 Web www epigentek com Printed 2014 10 30 Epigentek Group Inc All rights reserved Products are for research use only P 3083 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 3083 48 Cat P 3083 96 Upon Receipt JD1 10X Wash Buffer 14 ml 28 ml 4 C JD2 JARID Assay Buffer 4ml 8 ml RT JD3 JARID Substrate 50 ug ml 60 ul 120 ul 20 C JD4 JARID Assay Standard 50 ug ml 10 ul 20 ul 20 C JD5 Capture Antibody 1000 yg ml 5 ul 10 pl 4 JD6 Detection Antibody 400 ug ml 6 ul 12 ul 20 C JD7 Fluoro Developer 10 ul 20 ul 20 C JD8 Fluoro Enhancer 10 ul 20 ul 47 JD9 Fluoro Dilution 4 ml 8 ml RT Co factor 1 30 ul 60 ul 4 C Co factor 2 30 ul 60 ul 4 C Co factor 3 30 ul 60 ul 4 C 8 Well Assay Strips with frame 6 12 4 C Adhesive Covering Film 1 1 RT User Guide 1 1 RT Spin the solution down to the bottom prior to use SHIPPING
18. terference caused by thiol containing chemicals such as DTT GSH and 2 mercaptoethanol or caused by detergents ions such as tween 20 SDS triton X 100 Fe and Na e Both cell tissue extracts and purified JARID proteins including JARID1A JARID1B JARID1C and JARID1D can be used which allows for the detection of inhibitory effects of JARID inhibitors in vivo and in vitro e Sensitivity is up to 2 000 times higher than formaldehyde release based JARID assays allowing activity to be fluorometrically detected from as low as 5 ng of purified JARID enzyme e Demethylated H3 K4 standard is included allowing specific activity of JARID to be quantified e Accurate reliable and consistent with extremely low background signals PRINCIPLE amp PROCEDURE In this assay a tri methylated histone H3 K4 substrate is stably coated onto microplate wells Active JARIDs bind to the substrate and remove methyl groups from the substrate The JARID demethylated products can be recognized with a specific antibody The ratio or amount of demethylated products which is proportional to enzyme activity can then be fluorometrically measured by reading the fluorescence in a fluorescent microplate reader at 530 excitation and 590 emission The activity of the JARID enzyme is in turn proportional to the relative fluorescent units measured 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 4 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek c
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