Epigenase™ 5-mC Hydroxylase TET Activity/Inhibition
Contents
1. EpiQuik DNA Methyltransferase Activity Inhibition Assay Kit Base Catalog P 3001 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiQuik DNA Methyltransferase Activity Inhibition Assay Kit is suitable for non radioactively measuring DNMT activity or inhibition from a broad range of species including mammals plants and bacteria 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 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed nee Epigentek Group Inc All rights reserved Products are for research use only P 3001 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 3001 1 Cat P 3001 2 Upon Receipt fewelassaySrns winFamey fe e fe For maximum recovery of the products centrifuge the original vial after thawing prior to opening the cap SHIPPING amp STORAGE The kit is shipped in two parts one part at ambient room temperature and the second part on frozen ice packs at 4 C Upon receipt store M3 M4 and M6 at 20 C away from light store all other components at 4 C away from light Note Check if wash buffer M1 co2. g advantages and features e Extremely fast procedure which can be completed within 3 hours e Innovative colorimetric assay without radioactivity extraction or chromatography e Strip microplate format makes the assay flexible allowing manual or high throughput analysis e Simple reliable and consistent modification 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 DNA Methyltransferase Activity Inhibition Assay Kit is designed for measuring total DNMT activity de novo maintenance via a non radioactive 96 well plate format In an assay with this kit the unique cytosine rich DNA substrate is stably coated on the strip wells These wells are specifically treated to have a high DNA absorption ability DNMT enzymes transfer a methyl group to cytosine from Adomet to methylate the DNA substrate The methylated DNA can be recognized with a 5 methylcytosine antibody The ratio or amount of methylated DNA which is proportional to enzyme activity can then be colorimetrically quantified through an ELISA like reaction Page 3 Printed 2014 10 06 P 3001 Prepare nuclear extract 100 80 T Incubate with substrate E and assay buffer for 1h 5 40 z o Ee Add capture antibody 0 after washing wells untrea
3. gn Usage Limitation The EpiQuik DNA Methyltransferase Activity Inhibition Assay Kit is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The EpiQuik DNA Methyltransferase Activity Inhibition Assay Kit and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW Epigenetic inactivation of genes plays a critical role in many important human diseases especially in cancer A core mechanism for epigenetic inactivation of the genes is methylation of CpG islands in genome DNA Methylation of CpG islands involves the course in which DNA methyltransferases Dnmts transfer a methyl group from S adenosyl L methionine to the fifth carbon position of the cytosines Four active DNMTs have been identified in mammals DNMT1 DNMT2 DNMTS3A and DNMT3B The inhibition of DNMTs may lead to demethylation and expression of the silenced genes DNMT inhibitors are currently being developed as potential anticancer agents Currently there are very narrow selections of methods to use for measuring DNMT activity or inhibition These methods are time consuming labor intensive have low throughput or produce radioactive waste Epigentek addresses these problems with the EpiQuik DNA Methyltransferase Activity Inhibition Assay Kit by using a unique procedure to measure DNMT activity or inhibition The EpiQuik DNA Methyltransferase Activity Inhibition Assay Kit has the followin
4. le NY 11735 Page 4 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 06 Epigentek Group Inc All rights reserved Products are for research use only P 3001 10 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Dilute M3 with M2 at a 1 5 ratio to 1 6 mM Add 21 ul of M2 to each of your DNMT inhibition wells Add 24 ul of M2 to each of the other wells Add 3 ul of the Diluted M3 to each well Sample add 3 ul of your nuclear extracts 4 to 20 ug or purified DNMT enzymes to the sample wells Positive Control add 0 5 to 1 ul of M4 and 2 to 2 5 ul of M2 to each of the positive control wells DNMT Inhibition add 3 ul of tested inhibitors to each of the DNMT inhibition wells at different concentrations Blank add 3 ul of M2 to each of the blank wells Mix and cover all wells with Parafilm M and incubate at 37 C for 1 5 hours Aspirate and wash each well with 150 ul of 1X M1 3 times Dilute the M5 at a 1 1000 ratio to 1 ug ml with 1X M1 Add 50 ul of Diluted M5 to each well and incubate at room temperature for 60 min on an orbital shaker 50 to 100 rpm Aspirate and wash each well 4 times with 150 ul of 1X M1 each time This can be done by simply pipetting 1X M1 in and out of the wells Dilute M6 at a 1 1000 ratio to 0 2 ug ml with 1X M1 Add 50 ul of Diluted M6 to each well and incubate at room temperature for 30 min Aspirate and wash each well wi
5. ntains salt precipitates before using If so warm at room temperature or 37 C and shake the buffer until the salts are re dissolved The kit is stable for up to 6 months from the shipment date when stored properly MATERIALS REQUIRED BUT NOT SUPPLIED Microplate reader with the ability to read at 450 nm O Orbital shaker O Pipette and pipette tips O 1 5 ml microcentrifuge tubes GENERAL PRODUCT INFORMATION Quality Control Each lot of EpiQuik DNA Methyltransferase Activity Inhibition Assay Kitis tested against predetermined specifications to ensure consistent product quality Epigentek guarantees the performance of all products in the manner described in our product instructions 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 2 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 10 06 Epigentek Group Inc All rights reserved Products are for research use only P 3001 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 desi
6. scribed in the protocol are followed correctly No signal or weak signal The M4 positive control enzyme Ensure sufficient amount of M4 in only the positive control is insufficiently added to the well positive control enzyme is added in wells in Step 3e Step 3e The positive control enzyme has Follow the Shipping amp Storage lost activity due to improper guidance in this User Guide for storage storage of positive control No signal in only the The protein sample is not Ensure the nuclear protein sample well properly extracted extraction protocol is suitable for DNMT protein extraction Sodium chloride concentration of the extraction buffer should not be more than 100 mM Alternatively use the EpiQuik Nuclear Extraction Kit Cat No OP 0002 1 Insufficient protein amount added Ensure extract contains a sufficient to well amount of protein The sample is not prepared from The nuclear extracts obtained fresh cells or tissues frozen cells or tissues significantly lose enzyme activity Fresh samples should be used 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 Web www epigentek com Printed 2014 10 06 Epigentek Group Inc All rights reserved Products are for research use only P 3001 Nuclear extracts are improperly Ensure the nuclear extracts are stored or have been stored for an stored at 80 C for no greater than e
7. ted Zebularine HSC 3 cells were incubated with without zebularine 220 uM for 48 h Nuclear proteins were extracted and total Dnmt Add detection antibody activity was measured after washing wells 0 5 5 Add color developing g solution for color develop 0 4 ment then measure 8 absorbance gt 5 0 35 Schematic procedure of the EpiQuik 8 DNA Methy Itransf erase E Activ ity Inhibition Assay Kit 65 0 2 0 1 T T T T i 1 2 4 10 20 Nuclear extracts ug Nuclear extracts were prepared from MCF 7 cells using EpiQuik Nuclear Extraction Kit and total Dnmt activ ity was measured PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment 1 Prepare nuclear extracts by using your own successful method For your convenience and the best results Epigentek offers the EpiQuik Nuclear Extraction Kit Cat No OP 0002 1 optimized for use with this product Nuclear extracts can be used immediately or stored at 80 C for future use 2 a Predetermine the number of strip wells required for your experiment 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 Predetermine your sample wells positive control wells DNMT inhibition wells and blank wells c Dilute M1 whichis 10X concentration with distilled water pH 7 2 7 5 into 1X concentration 1X M1 110 Bi County Blvd Ste 122 Farmingda
8. th 150 ul of 1X M1 5 times Add 100 ul of M7 into each of the wells and incubate at room temperature for 2 10 min away from light Begin monitoring color development in the sample and the positive control well for a medium blue color Add 50 ul of M8 to each well to stop enzyme reaction when the color in the standard wells containing the higher concentrations of standard control turns medium blue The color will change to yellow and the absorbance should be read on a microplate reader at 450 nm within 2 to 15 min Page 5 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 10 06 Epigentek Group Inc All rights reserved Products are for research use only P 3001 Calculate DNMT activity or inhibition using the following formulas No Inhibitor OD Blank OD DNMT Activity OD h mg _ x 1000 Protein Amount ug Xx hour Protein amount added into the reaction at Step 3d Incubation time used at Step 3h Inhibitor Sample OD Blank OD DNMT Inhibition x 100 No Inhibitor Sample OD Blank OD TROUBLESHOOTING Possible Causes Suggestions No signal in both the Reagents are added incorrectly Check if reagents are added in the positive control and the proper order and if some steps in sample wells the protocol may have been omitted by mistake Incubation time and temperature Ensure the incubation time and are incorrect temperature de
9. xceedingly long period 6 weeks Absence of DNMT activity inthe NA sample due to treatment High background in blank Insufficient washing of wells Check if washing recommendations at each step is performed according to the protocol Contaminated by the positive Ensure the well is not contaminated control from adding enzyme accidentally or from using enzyme contaminated tips Over development Decrease the color development time incubation time in Step 9 before adding M8 Stop Solution in Step 10 RELATED PRODUCTS Sample Preparation OP 0002 EpiQuik Nuclear Extraction Kit OP 0022 EpiQuik Nuclear Extraction Kit II Nucleic Acid Free OP 0003 EpiQuik Whole Cell Extraction Kit DNA Methyltransferase Assay P 3004 EpiQuik DNA Methyltransferase Activity Inhibition Assay Kit Fluorometric P 3006 EpiQuik DNA Methyltransferase 1 Activity Inhibitor Screening Assay Kit P 3007 EpiQuik DNA Methyltransferase 3B Activity Inhibitor Screening Assay Kit P 3011 EpiQuik DNMT1 Assay Kit P 3012 EpiQuik DNMTS3A Assay Kit P 3013 EpiQuik DNMTS3B Assay Kit P 3021 EpiQuik MBD2 Binding Activity Inhibition Assay Kit 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Pag e7 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 10 06 Epigentek Group Inc All rights reserved Products are for research use only P 3001
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