EpiQuik ™ DNMT Activity/Inhibition Assay Ultra Kit
Contents
1. Add 1X PBS buffer into the Capture Antibody vial until you restore the correct intended volume according to the Kit Contents described in this User Guide Mix and centrifuge prior to use 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 11 Printed 2014 09 22 P 3009 RELATED PRODUCTS Nuclear Extract Preparation OP 0002 1 EpiQuik Nuclear Extraction Kit DNMT Activity Inhibition Assay and Content Quantification P 3010 EpiQuik DNMT Activity Inhibition Assay Ultra Kit Fluorometric P 3011 EpiQuik DNMT1 Assay Kit P 3012 EpiQuik DNMTS3A Assay Kit P 3013 EpiQuik DNMTS3B Assay Kit DNMT Antibodies A 1001 DNMT1 Monoclonal Antibody A 1002 DNMT2 Polyclonal Antibody A 1101 DNMT2 Monoclonal Antibody A 1003 DNMTS3A Polyclonal Antibody A 1004 DNMTSB Polyclonal Antibody A 1005 DNMTS3L Polyclonal Antibody 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 12 Printed 2014 09 22 P 30092. 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 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 10 Printed 2014 09 22 P 3009 Little or no activity of DNMT 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 color development Insufficient washing of the wells Ensure the wells are washed according to the protocol Ensure any residues from the wash buffer are removed as much as possible Delayed color development or delayed stopping of color development in the wells Ensure color development solution is added sequentially and 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 MU8 Developer Solution and MUS Stop Solution are added at the same time between replicates or otherwise maintain a consistent t
3. EpiQuik DNMT Activity Inhibition Assay Ultra Kit Colorimetric Base Catalog P 3009 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiQuik DNMT Activity Inhibition Assay Ultra Kit Colorimetric is suitable for measuring total DNMT activity or inhibition using nuclear extracts or purified enzymes from a broad range of species such as mammalians plants fungi bacteria and viruses in a variety of forms including but not limited to cultured cells and fresh and frozen tissues Nuclear extracts can be prepared by using your own successful method For your convenience and the best results Epigentek also offers a nuclear extraction kit Cat No 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 DNMTs from recombinant proteins or isolated from cell tissues Input Material Inout materials can be nuclear extracts or purified DNMT enzymes The amount of nuclear extracts for each assay can be between 0 5 ug to 20 ug with an optimal range of 5 10 ug The amount of purified enzymes can be 0 5 ng to 200 ng depending on the purity and catalytic activity of the enzymes Internal Control A positive enzyme control is provided in this kit Because DNMT activity can vary from tissue to tissue and from normal and diseased states it is advised to run replicate samples to ensure that the signal generated is validated Precautions To avoid
4. d Add 50 ul of the Diluted MU6 to each well then carefully cover with Parafilm M or aluminium foil and incubate at room temperature for 30 min e Remove the Diluted MU6 solution from each well f Wash each well with 150 ul of the Diluted MU1 each time for four times 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 Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 09 22 P 3009 Add 50 ul of the Diluted MU7 to each well then carefully cover with Parafilm M or aluminium foil and incubate at room temperature for 30 min Remove the Diluted MU7 solution from each well Wash each well with 150 ul of the Diluted MU1 each time for five times Note Ensure any residual wash buffer in the wells is thoroughly removed as much as possible at each wash step 4 Signal Detection a Add 100 ul of MU8 to each well and incubate at room temperature for 1 to 10 min away from direct light Monitor color change in the sample wells and control wells The MU8 solution will turn blue in the presence of sufficient methylated DNA Add 100 ul of MU9 to each well to stop enzyme reaction when the color in the positive control wells turns medium blue Mix the solution by gently shaking the frame and wait 1 2 min to allow the color reaction to be completely stopped The color will change to yellow after addin
5. o o o o GENERAL PRODUCT INFORMATION Parafilm M or aluminium foil Quality Control Each lot of this product 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 This product is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property This product and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW 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 DNA methylation occur
6. 09 22 P 3009 c Calculate DNMT inhibition using the following formula Inhibitor Sample OD Blank OD DNMT Inhibition X 100 No Inhibitor Sample OD Blank OD SUGGESTED WORKING BUFFER AND SOLUTION SETUP Table 1 Approximate amount of required buffers and solutions for defined assay wells based on the protocol Reagenis 1 well 8 wells 16 wells 48 wells 96 wells 1 strip 2 strips 6 strips 12 strips Diluted MU1 2 5 ml 20 ml 40 ml 120 ml 240 ml Diluted MU3 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted MU5 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted MU6 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted MU7 50 ul 400 ul 800 ul 2400 ul 4800 ul Developer 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml Solution Stop Solution 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml DNMT Enzyme N A 0 25ul O 5ul 2ul 1yl 4yul j2ul 8uyl Conirol 1 ul SUGGESTED STRIPWELL SETUP Table 2 The suggested strip well plate setup for the DNMT activity assay in a 48 assay format in a 96 assay format Strips 7 to 12 can be configured as Sample The controls and samples can be measured in duplicates Well Strip 1 Strip 2 Strip 3 Strip 4 Strip 5 Strip 6 A Blank Blank Sample Sample Sample Sample B MU4 0 5 ul MU4 0 5 ul Sample Sample Sample Sample c MU4 1 ul MU4 1 0 ul Sample Sample Sample Sample D Sample Sample Sample Sample Sample Sampl
7. 7 5 96 Assay Kit Add 26 ml of MU1 10X Wash Buffer to 234 ml of distilled water and adjust pH to 7 2 7 5 This Diluted MU1 1X Wash Buffer can now be stored at 4 C for up to six months b Prepare Diluted MU3 Working Buffer Freshly prepare the Diluted MU3 Working Buffer required for the assay by adding 2 ul of MU3 into 98 ul of MU2 DNMT Assay Buffer About 50 ul of this Diluted MU3 will be required for each assay well c Prepare Diluted MU5 Capture Antibody Solution Dilute MU5 Capture Antibody with Diluted MU1 at a ratio of 1 1000 i e add 1 ul of MU5 to 1000 ul of Diluted MU1 About 50 ul of this Diluted MU5 will be required for each assay well d Prepare Diluted MU6 Detection Antibody Solution Dilute MU6 Detection Antibody with Diluted MU1 at a ratio of 1 2000 i e add 1 ul of MUG to 2000 ul of Diluted MU1 About 50 ul of this Diluted MU6 will be required for each assay well e Prepare Diluted MU7 Enhancer Solution Dilute MU7 Enhancer Solution with Diluted MU1 at a ratio of 1 5000 i e add 1 ul of MU7 to 5000 ul of Diluted MU1 About 50 ul of this Diluted MU7 will be required for each assay well f About the MU4 DNMT Enzyme Control The MU4 DNMT Enzyme Control is an enzyme with activity of both maintenance and de novo DNMTs and is used as the positive control of this assay We do not recommend using this enzyme control to generate a standard curve for quantifying the activity of your samples as the amount of the enzyme i
8. demethylation and expression of silenced genes DNMT inhibitors are currently being developed as potential anticancer agents Conventional DNMT activity inhibition assay methods are time consuming labor intensive have low throughput and or produce radioactive waste The original EpiQuik DNMT Activity Inhibition Assay Kit addressed this issue by introducing a simple method with an ELISA like 96 well plate format The EpiQuik DNMT Activity Inhibition Assay Ultra Kit Colorimetric is a further refinement of its predecessor kit by enhancing sample signals and significantly minimizing background signals in addition to being five times more sensitive e Colorimetric assay with easy to follow steps for convenience and speed The entire procedure can be completed within 3 hours and 45 minutes e Safe and innovative colorimetric assay without radioactivity extraction and chromatography e The ultra sensitive detection limit can be as low as 0 5 ug of nuclear extract or 0 5 ng of purified enzymes which is five times better than the predecessor kit e Optimized antibody amp enhancer solutions allow high specificity to 5 mC without cross reactivity to unmethylated cytosine e 96 stripwell microplate format allows for either low or high throughput analysis PRINCIPLE amp PROCEDURE The EpiQuik DNMT Activity Inhibition Assay Ultra Kit Colorimetric contains all reagents necessary for the measurement of DNMT activity or inhibition In thi
9. 2 It is recommended to use 5 ug to 10 ug of nuclear extract per well or 10 ng to 100 ng of purified enzyme per well 3 The concentration of inhibitors 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 MU2 at a 1 10 ratio e g add 0 5 ul of inhibitor to 4 5 ul of MU2 so that the original solvent of the inhibitor can be reduced to 1 of the reaction solution or less Tightly cover the strip well microplate with Adhesive Covering Film to avoid evaporation and incubate at 37 C for 90 120 min Note 1 The incubation time may depend on intrinsic DNMT activity In general 90 min incubation is suitable for active purified DNMT enzymes and 120 min incubation is required for nuclear extracts 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 Diluted MU1 1X Wash Buffer each time This can be done by simply pipetting Diluted MU1 in and out of the wells 3 Antibody Binding amp Signal Enhancing a Add 50 ul of the Diluted MU5 to each well then carefully cover with Parafilm M or aluminium foil and incubate at room temperature for 60 min b Remove the Diluted MU5 solution from each well c Wash each well with 150 ul of the Diluted MU1 each time for three times
10. 450 nm OD450 nm 0 8 5 0 1 5 10 20 Nuclear extract ug Demonstration of high sensitivity and specificity of the DNMT activity assay achieved by using nuclear extracts with the EpiQuik DNMT Activity Inhibition Assay Ultra Kit Colorimetric Nuclear extracts were prepared from MCF 7 cells by using the EpiQuik Nuclear Extraction Kit Cat No OP 0002 1 4 1 2 0 8 0 6 0 4 0 2 0 10 20 30 40 50 Dnmti ng Demonstration of high sensitivity and specificity of the DNMT activity inhibition assay achieved by using recombinant DNMT1 with the EpiQuik DNMT Activity Inhibition Assay Ultra Kit Colorimetric Page 5 Printed 2014 09 22 P 3009 ASSAY PROTOCOL Starting Materials Input Amount The amount of nuclear extracts for each assay can be between 0 5 ug and 20 yg with an optimal range of 5 ug to 10 ug The amount of purified enzymes can be 0 5 ng to 200 ng depending on the purity and catalytic activity of the enzymes Nuclear Extraction You can use your own method of choice for preparing nuclear extracts Epigentek also offers a nuclear extraction kit Cat No OP 0002 optimized for use with this kit Nuclear Extract or Purified DNMT Storage Nuclear extract or purified DNMT enzymes should be stored at 80 C until use 1 Buffer Solution amp Preparation a Prepare Diluted MU1 1X Wash Buffer 48 Assay Kit Add 13 ml of MU1 10X Wash Buffer to 117 ml of distilled water and adjust pH to 7 2
11. 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 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3009 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 3009 48 Cat P 3009 96 Upon Receipt MU1 10X Wash Buffer 14 ml 28 ml 47 MU2 DNMT Assay Buffer 4ml 8 ml RT MU3 Adomet 50X 60 ul 120 ul 20 C MU4 DNMT Enzyme Control 50 ug ml 6 ul 12 ul 20 C MU5 Capture Antibody 1000 ug ml 5 ul 10 ul 4 MU6 Detection Antibody 400 ug ml 6 ul 12 ul 20 C MU7 Enhancer Solution 6 ul 12 ul 20 C MU8 Developer Solution 5 ml 10 ml 47 MUS Stop Solution 5 ml 10 ml RT 8 Well Assay Strips With Frame 6 12 47 Adhesive Covering Film 1 1 RT User Guide 1 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 MU3 MU4 MU6 and MU7 at 20 C away fr
12. e E Sample Sample Sample Sample Sample Sample F Sample Sample 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 Reagents are added incorrectly Check if reagents are added in the signal in both the proper order with the right amount and positive control and if any steps in the protocol may have sample wells been omitted by mistake 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 9 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 3009 The well is incorrectly washed before enzyme reaction Ensure the well is not washed prior to adding the standard control and sample 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 filter 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 capped after each opening or use No signal or weak signal in only the positive control wells The DNMT enzyme control is insufficiently added to the well in Step 2c Ensure a su
13. fficient amount of DNMT enzyme control is added The quality of the DNMT enzyme control has been degraded due to improper storage conditions Follow the Shipping amp Storage guidance for storage instructions of MU4 DNMT Enzyme Control 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 positive control Ensure the well is not contaminated from adding sample or positive control accidentally or from using contaminated tips Incubation time with detection antibody is too long The incubation time at Step 3d should not exceed 45 min Over development of color Decrease the development time in Step 4a before adding MU9 Stop Solution in Step 4b No signal or weak signal only in sample wells Protein sample is not properly extracted or purified Ensure your protocol is suitable for DNMT protein extraction For the best results it is advised to use Epigentek s Nuclear Extraction Kit Cat No OP 0002 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
14. g MU9 and absorbance should be read on a 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 the 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 manualy subtract the 655 nm ODs from 450 nm ODs 2 If the stripwell microplate frame does not fit in the microplate reader transfer the solution to a standard 96 well microplate 5 DNMT Activity Calculation 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 Calculate the average duplicate readings for sample wells and blank wells Calculate DNMT activity using the following formula Sample OD Blank OD DNMT Activity OD h mg _ x 1000 Protein amount ug x hour Protein amount added into the reaction at step 2d in yg Incubation time at step 2f Example calculation Average OD450 of sample is 0 55 Average OD450 of blank is 0 05 Protein amount is 5 pg Incubation time is 2 hours 120 min 0 55 0 05 DNMT activity X 1000 50 OD h mg 5 X 2 Page 8 Printed 2014
15. iming in between each addition of solutions Color reaction is not evenly stopped due to an inconsistent order of adding solutions Ensure all solutions particularly MU8 Developer Solution and MU9 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 especially those with small volumes e g 1 ul are completely added into the wells Solutions or antibodies were not actually added into the wells Do not allow pipette tip to touch the outer edges or inner sides of the wells to prevent solutions from sticking to the surface Did not sufficiently shake the solutions in the wells evenly after adding MU9 Stop Solution in Step 4b Gently and evenly shake the plate frame across a flat surface so that the solutions in the wells are better distributed Do not stir Did not use the same pipette device throughout the experiment Use the same multi channel pipette device throughout the entire experiment as different pipette devices may have slight variations in performance Capture Antibody vial appears to be empty or insufficient in volume Buffer evaporated due to the very small volumes resulting in a higher concentrated antibody
16. nity of gene promoters also affects DNA methylation and transcriptional activity Three DNMTs DNMT1 DNMT3A and DNMT3B are required for the establishment and maintenance of DNA methylation patterns Two additional enzymes DNMT2 and DNMT3L may also have more specialized but related functions DNMT1 appears to be responsible for the maintenance of established patterns of DNA methylation while DNMT3A and DNMTS3B seem to mediate the establishment of new or de novo DNA methylation patterns DNMT3L is found to be a catalytically inactive regulatory factor of DNA methyltransferases which is essential for the function of DNMT3A and DNMT3B Diseased cells such as cancer cells may be different in that DNMT1 alone is not responsible for maintaining abnormal gene hypermethylation and both DNMT1 and DNMT3B may be cooperative for this function The local chromatin structure also contributes to the control of DNA methylation HH N CH nay DNA methyltransferase ry 5 O E a o n fe AdoMet AdoHcy sme c Fig 1 Methylation of cytosine in DNA via DNA methyltransferase and S adenosylmethionine The importance of DNA methylation is emphasized by the growing number of human diseases that are known to occur when DNA methylation information is not properly established and or maintained Abnormal DNA methylation associated with increased expression or the activity of DNMTs has been found in many different diseases especially in cancer Inhibition of DNMTs may lead to
17. om light 2 Store MU1 MU5 MU8 and the 8 Well Assay Strips at 4 C away from light 3 Store all remaining components MU2 MUSQ and the 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 MU1 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 transfer the amount of MU8 required into a secondary container tube or vial before adding MU8 into the assay wells in order to avoid contamination Check if a blue color is present in MU8 Developer Solution before each use as this would indicate contamination of the solution and should not be used MATERIALS REQUIRED BUT NOT SUPPLIED 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 O Adjustable pipette or multiple channel pipette Multiple channel pipette reservoirs Aerosol resistant pipette tips Microplate reader capable of reading absorbance at 450 nm Oo OF OF 1 5 ml microcentrifuge tubes Page 2 Printed 2014 09 22 P 3009 Incubator for 37 C incubation Distilled water Nuclear extract or purified enzyme samples containing DNMT activity Dnmt inhibtors optional oO
18. s assay a universal DNMT substrate is stably coated onto microplate wells DNMT enzymes transfer methyl group to cytosine from Adomet to methylate DNA substrate and the methylated DNA can be recognized with an anti 5 methylcytosine antibody The ratio or amount of methylated DNA which is proportional to enzyme activity can then be measured through an ELISA like reaction by reading the absorbance in a microplate 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 com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 09 22 P 3009 spectrophotometer at a wavelength of 450 nm The activity of DNMT enzymes is proportional to the optical density intensity measured Prepare nuclear extracts or purified enzymes Incubate with substrate amp assay buffer for 90 min Wash wells then add capture antibody Wash wells then add detection antibody and enhancer solution Add color developing solution for color develop ment then measure absorbance Schematic procedure of the EpiQuik DNMT Activity Inhibition Assay Ultra Kit Colorimetric 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 OD
19. s by a covalent addition of a methyl group at the 5 carbon of the cytosine ring resulting in 5 methylcytosine These methyl groups project into the major grooves of DNA and inhibit transcription In human DNA 5 methylcytosine is found in approximately 1 5 of genomic DNA primarily at CpG sites There are clusters of CpG sites at 0 3 to 2 kb stretches of DNA known as CpG islands that are typically found in or near promoter regions of genes where transcription is initiated In the bulk of genomic DNA most CpG sites are heavily methylated However CpG islands in germ line tissue and promoters of normal somatic cells remain unmethylated allowing gene expression to occur When a CpG island in the promoter region of a gene is methylated the expression of the gene is repressed The repression can be caused by directly inhibiting the binding of specific transcription factors and indirectly by recruiting methyl CpG binding proteins and their associated repressive chromatin remodeling activity In addition to the effect on gene transcription DNA methylation is also involved in genomic imprinting which refers to a parental origin specific expression of a gene and the formation of a chromatin domain DNA methylation is controlled at several different levels in normal and diseased cells The addition of methyl groups is carried out by a family of enzymes DNA methyltransferases DNMTs Chromatin Page 3 Printed 2014 09 22 P 3009 structure in the vici
20. s limited and catalytic activity unit is different Note Keep each of the diluted solutions except Diluted MU1 1X Wash Buffer on ice until use Any remaining diluted solutions other than Diluted MU1 should be discarded if not used within the same day 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 rights reserved Products are for research use only Printed 2014 09 22 P 3009 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 Blank Wells Add 50 ul of Diluted MU3 per well Positive Control Wells Add 50 ul of Diluted MU3 and 1 ul of MU4 per well Sample Wells Without Inhibitor Add 45 ul to 49 ul of Diluted MU3 and 1 ul to 5 ul of nuclear extracts or 1 to 5 ul of purified DNMT enzymes per well The total volume should be 50 ul per well Sample Wells With Inhibitor Add 40 ul to 44 ul of Diluted MU3 1 to 5 ul of nuclear extracts or 1 to 5 ul of purified DNMT enzymes and 5 ul of inhibitor solution per well The total volume should be 50 ul per well Note 1 Follow suggested well setup diagrams
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