DNMT Activity / Inhibition Assay(version B2)
Catalog No. 55006

Copyright 2012 Active Motif, Inc.

Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of
any kind, either explicit or implied. Information may be changed or updated in this manual at any
time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation is proprietary information and protected by the copyright laws of the United States and international
treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2012 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights
reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective
companies.

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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Introduction
DNMT Activity / Inhibition Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
DNA Methyltransferase Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Components and Storage
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols
Buffer Preparation and Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Assay Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Calculation of DNMT Activity or Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Appendix
Section A. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

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Overview
DNA methylation is a major epigenetic modification in the genome of higher eukaryotes. ThisDNA covalent modification is catalyzed by DNA methyltransferase enzymes (DNMTs) and consistsin the addition of a methyl group from S-adenosyl-L-methionine (AdoMet) to the fifth carbon position of cytosine (C5), mostly within CpG dinucleotides. This methylation, together with histone
modifications, plays an important role in modulating chromatin structure, thus controlling gene
expression and many other chromatin-dependent processes.
Active Motif’s DNMT Activity / Inhibition Assay is a time-saving, non-radioactive assay to measure
DNA methyltransferase activity and/or inhibition from recombinant DNMT enzymes (DNMT1,
DNMT3a & DNMT3b) or nuclear extract samples. This sensitive ELISA-based method uses theability of methyl CpG binding domain (MBD) proteins to bind methylated DNA with high affinity. In the DNMT assay method, a universal CpG-enriched DNA substrate has been immobilized
on a 96-stripwell plate. Purified DNMTs or DNMT activities from nuclear extracts will catalyze
the transfer of methyl groups from the provided AdoMet reagent to the coated DNA substrate.
The resulting methylated DNA will be recognized by the His-tagged recombinant MBD2b in an
amount proportional to the enzyme activity. Addition of a polyHistidine antibody conjugated tohorseradish peroxidase (HRP) provides a sensitive colorimetric readout that is easily quantified byspectrophotometry. The assay includes a CpG methyltransferase enzyme as a positive control.

product

formatcatalog no.

DNMT Activity / Inhibition Assay

1 x 96 rxns

55006

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1

Introduction
DNMT Activity / Inhibition Assay
One of the earliest identified forms of epigenetic regulation of gene expression is DNA methylation. In vertebrates, DNA methylation occurs predominantly on the CpG dinucleotide andapproximately 60 to 90% of these dinucleotides are modified. Distinct DNA methylation patterns,
which can vary between different tissues and developmental stages, exist on specific loci. Since
CpG sequences are frequently clustered in proximal promoters of genes in higher eukaryotes,
modification at these sequences can have profound effects on transcription. DNA methylation
is associated with inhibition of transcription and a repressed chromatin state1,2, as well as X
chromosome inactivation3,4 and maintenance of imprinted genes5. DNA methylation can cause
gene silencing by interference with recognition and binding of positively acting transcription factors or by promotion of interaction and recruitment of negative factors. These last factors include
methyl-CpG-binding domain (MBD) proteins which recruit the enzymatic machinery to establish
silent chromatin6.
In mammals and other vertebrates, DNA methylation occurs at the C5 position of cytosine (5mC),
mostly within CpG dinucleotides. In this process, the enzymes use a conserved mechanism thathas been best studied in the bacterial 5mC methyltransferase, M.HhaI7,8. Briefly, the target cytosine is flipped out of the double helix (base flipping) and the recipient C5 position is activated bya transient, covalent complex formation with the enzyme at the C6 position. After methyl grouptransfer, the enzyme is released by beta-elimination together with the proton at position C5.
DNA methylation is accomplished by three enzymes: DNMT1, DNMT3a and DNMT3b. DNMT3aand DNMT3b fall in the group of de novo methyltransferases (MTases)9, enzymes that are able
to methylate previously unmethylated CpG sequences, while DNMT1 functions as a maintenancemethylase, copying pre-existing methylation marks onto the new DNA strand during replication10.
Although generally thought of as a maintenance enzyme that acts on hemimethylated DNA,DNMT1 has also been shown to function as a de novo DNA methyltransferase11,12,13. In addition,
two non-canonical family members, DNMT2 and DNMT3L, have been discovered14,15: DNMT2
methylates cytosine 38 in the anticodon loop of tRNA16, while DNMT3L is a catalytically inactive
DNMT. DNMT3L is known to associate with both DNMT3a and DNMT3b and may be responsible
for the recruitment of histone deacetylases to direct repression onto newly established imprints17.
It is still not clear how methyl groups are removed from DNA. Several studies recently suggestthat active DNA demethylation might be accomplished through a process of DNA repair18,19 that involves nucleotide exchange20,21, replacing 5-methylcytosine with unmodified cytosine. This could
be the physiological mechanism that operated during normal development in vivo22.
Although DNA methylation and histone modification are carried out by different chemical reactions and require different sets of enzymes, there seems to be a biological relationship between
the two systems that plays a part in modulating gene repression programming in the organism23,24. This relationship between DNA methylation and histone modification might be partiallymediated through methylcytosine-binding proteins, such as MECP2 or MBD2, that are capableof recruiting histone deacetylases to the methylated regions25,26. It is probable that the presenceof DNA methylation also directs H3K9 dimethylation, which is a mark of repressive chromatin,
perhaps through the interaction of G9a and DNMT1 with the replication complex27. There is also
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2

evidence that DNA methylation inhibits H3K4 methylation28,29 and, in plants, excludes the histone
variant H2AZ from nucleosomes30; both of these marks are associated with active transcription.
Understanding the relationship between DNA methylation and certain histone modifications
is also providing insights into the aberrant gene expression patterns observed in cancer31. De
novo DNA methylation plays a critical role in the development of certain cancers, e.g. renal cell
carcinoma, colon carcinoma, lung cancer and many others. More specifically, hypermethylationof tumor suppressor genes is one of the most consistent hallmarks of human cancers. DNA
methyltransferase inhibitors, therefore, represent promising new drugs for cancer therapies32,33,34.
Some of these compounds (nucleoside analogs or DNMT1 antisense molecules) have recently
been approved as antitumor agents and others are presently in various stages of their pre-clinicalor clinical development35,36.

Traditional Methods to Study DNA Methyltransferase Activity
To date, there are several methods used to study DNA methyltransferase activity / inhibition.
1. Radioactively labeled AdoMet: In this method the methyltransferase activity is measured
through the transfer of radioactively labeled methyl groups from the cofactor AdoMet to
DNA substrates37,38,39,40.
2. Methylation-sensitive restriction enzyme analysis: Isoschizomers of bacterial restriction
endonucleases with different sensitivities for 5-methylcytosine can be used to determine
the methylation status of specific CpG-dinucleotides41. Methylation-sensitive restriction
enzymes have several limitations including that methylation-sensitive restriction merely
informs on the methylation status of the cytosine residues which are recognized by the
restriction enzymes used.
3. Mass spectrometry: Direct detection of methylated cytosine residues can be accomplishedby MALDI-TOF mass spectrometry42, or by monitoring the conversion of AdoMet toS-adenosyl-homocysteine by liquid chromatography and mass spectrometry43.
Active Motif’s DNMT Activity / Inhibition Assay is a fast, user-friendly assay to specifically detect
DNA methyltransferase activity from recombinant DNMT enzymes or nuclear extract samples
without the need for radioisotopes or expensive equipment. This innovative method utilizes theability of methyl CpG binding domain (MBD) proteins to bind methylated DNA with high affinity.
A universal CpG-enriched DNA substrate has been immobilized in the wells of a 96-stripwellplate. Purified DNMTs or DNMT activities from nuclear extracts will catalyze the transfer of methylgroups from the provided AdoMet reagent to the coated DNA substrate. The resulting methylated
DNA will be recognized by the His-tagged recombinant MBD2b in an amount proportional to the
enzyme activity. Addition of a polyHistidine antibody conjugated to horseradish peroxidase (HRP)
provides a sensitive colorimetric readout that is easily quantified by spectrophotometry.

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3Kit Performance and Benefits
Range of detection: This assay is able to detect DNMT activity from purified enzymes in therange of 0.5 to 100 ng per well. For nuclear extract samples, we recommend using 0.5 to 10 µg
per well. The assay is able to detect DNMT1, DNMT3a and DNMT3b activity, but will not distinguish between the different enzymes. In order to detect activity of a specific DNMT, we recommend using purified DNMT of the isoform of interest.
Assay time: 3 hours.

DNMT Activity / Inhibition Assay
CpG Methyltransferase Control

2.5

OD 450nm

2
1.5
1
0.5
0

Blank

0.125
0.25
0.5
1
CpG Methyltransferase (U/well)

2

DNMT activity using a CpG Methyltransferase.
The DNMT Activity / Inhibition Assay was used to generate a standard curve using the provided CpG Methyltransferase
control enzyme. The assay was able to detect DNMT activity from as little as 0.125 units of enzyme with a 1.5 hour incubation
and a 5 minute developing time. Data shown are the results from wells assayed in duplicate. These results are provided fordemonstration only.

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D M

t ty fromlear tra ts

0.
0.

OD 450nm

0.5
0.
0.
0.2
0.1
0

Blank

0. 25

1.25

MC

2.5

5

10

lear tra t ( /well)

DNMT activity from MCF-7 nuclear extracts.
The DNMT Activity / Inhibition Assay was used to screen MCF-7 nuclear extracts (0.625 - 10 µg) prepared using Active
Motif’s Nuclear Extract Kit (Catalog No. 40010). The assay was able to detect DNMT activity from as little as 0.625 µg of
extract with a 1.5 hour incubation and a 5 minute developing time. Data shown are the results from wells assayed in duplicate. These results are provided for demonstration only.

D M nh t on w th 5aeo y yt ne

0.
0.

0.5
0.
25

OD 450nm

0.

0.
0.2
0.1
0

Blank

MC

k

a

lear tra t (5/well) /

a

5 a

a

a treatment

DNMT inhibition with 5-Aza 2´deoxycytidine treatment.
The DNMT Activity / Inhibition Assay was used to screen for DNMT inhibition in MCF-7 cells that were either untreated, or
treated with 5-Aza 2´deoxycytidine for 3 - 5 days. Nuclear extracts were prepared using Active Motif’s Nuclear Extract Kit(Catalog No. 40010) and 5 µg of each treatment condition was tested in the assay with a 1.5 hour incubation time and 3
minute developing time. The 5-Aza 2´deoxycytidine treated extracts showed a 25% inhibition of DNMT activity as comparedto the mock treated sample for 5 day treatment. Data shown are the results from wells assayed in duplicate. These results
are provided for demonstration only.

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5Kit Components and Storage
DNMT Activity / Inhibition Assay Kits are for research use only. Not for use in diagnostic
procedures. All components are guaranteed stable for 6 months from date of receipt whenstored properly.
Reagents

Quantity

Storage

Anti-polyHis-HRP antibody

11 µl

-20°C

Enzymatic Buffer AM1

11 ml

-20°C

100X AdoMet

110 µl-20°C

CpG Methyltransferase Enzyme (2 U/µl)

8 µl

-20°C

His-MBD2b protein

220 µl-80°C

Binding Buffer AM11

11 ml

4°C

10X Wash Buffer AM1

15 ml

4°C

10X Wash Buffer AM3

7 ml

4°C

10X Antibody Binding Buffer AM3

1.5 ml4°CDeveloping Solution

11 ml

4°C

Stop Solution

11 ml

4°C

96-well Assay Plate

14°C

Plate sealer

1

RT

Additional materials required• Recombinant DNMT enzyme or nuclear extract samples containing DNMT activity. For yourconvenience Active Motif also offers a Nuclear Extract Kit which can be used for the preparation of nuclear extract samples (Cat. Nos. 40010 & 40410).
• Multi-channel pipettor• Multi-channel pipettor reservoirs• Microcentrifuge tubes• Rocking platform at RT• Rocking platform at 37°C
• Microplate spectrophotometer capable of reading at 450 nm (655 nm as optional reference
wavelength)www.activemotif.com

6

Protocols
Buffer Preparation and Recommendations
Preparation of Complete Enzymatic Buffer
Prepare the amount of Complete Enzymatic Buffer required for the assay by adding 1 µl of 100X
AdoMet per 99 µl of Enzymatic Buffer AM1 and keep on ice (see the Quick Chart for Preparing
Buffers in this section). We recommend adding AdoMet immediately prior to use. Any remaining
Complete Enzymatic Buffer should be discarded if not used in the same day.
Note:

The Enzymatic Buffer does contain EDTA. Therefore, researchers interested in studying
metal co-activators should use an alternate buffer as suggested in the Troubleshooting
Guide in Section A of the Appendix.

Preparation of 1X Wash Buffer AM1
Prepare the amount of 1X Wash Buffer AM1 required for the assay as follows: For every 10 ml of
1X Wash Buffer AM1 required, dilute 1 ml 10X Wash Buffer AM1 with 9 ml distilled water (see the
Quick Chart for Preparing Buffers in this section). Mix gently to avoid foaming. The 1X Wash Buffer
AM1 may be stored at 4°C for one week. The Tween 20 contained in the 10X Wash Buffer AM1
may form clumps, therefore it is necessary to completely resuspend any precipitates by incubating at 50°C for 2 minutes and mixing prior to use.
Preparation of His-MBD2b Dilution
Prepare the amount of His-MBD2b required for the assay by adding 2 µl of His-MBD2b protein
per 98 µl of Binding Buffer AM11 and mix thoroughly (see the Quick Chart for Preparing Buffers inthis section). We recommend diluting His-MBD2b protein immediately prior to use. Any remaining
His-MBD2b dilution should be discarded if not used in the same day. During the first use, we
recommend making small aliquots of the His-MBD2b stock protein and storing at -80°C to avoid
multiple freeze/thaw cycles.
Preparation of 1X Wash Buffer AM3
Prepare the amount of 1X Wash Buffer AM3 required for the assay as follows: For every 10 ml of
1X Wash Buffer AM3 required, dilute 1 ml 10X Wash Buffer AM3 with 9 ml distilled water (see the
Quick Chart for Preparing Buffers in this section). Mix gently to avoid foaming. The 1X Wash Buffer
AM3 may be stored at 4°C for one week. The BSA contained in the 10X Wash Buffer AM3 mayform clumps, therefore it is necessary to completely resuspend any precipitates by incubating at
37°C for 2 minutes and mixing prior to use.
Preparation of 1X Antibody Binding Buffer AM3
Prepare the amount of 1X Antibody Buffer AM3 required for the assay as follows: For every 10 mlof 1X Antibody Binding Buffer AM3 required, dilute 1 ml 10X Antibody Binding Buffer AM3 with
9 ml distilled water (see the Quick Chart for Preparing Buffers in this section). Mix gently to avoid
foaming. The BSA contained in the 10X Antibody Binding Buffer AM3 may form clumps, therefore
it is necessary to completely resuspend any precipitates by incubating at 37°C for 2 minutes andmixing prior to use. Discard remaining 1X Antibody Binding Buffer AM3 after use.

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7Preparation of Anti-polyHis-HRP antibody
Dilute the anti-polyHis-HRP antibody 1:1000 with 1X Antibody Binding Buffer AM3 and mix
thoroughly. Use 100 µl per well.
Developing Solution
The Developing Solution should be warmed to room temperature before use. The Developing
Solution is light sensitive, therefore, we recommend avoiding direct exposure to intense light during storage. The Developing Solution may develop a yellow hue over time. This does not affectproduct performance. However, a blue color present in the Developing Solution indicates thatit has been contaminated and must be discarded. Prior to use, place the Developing Solution atroom temperature for at least 1 hour. Transfer the amount of Developing Solution required for theassay into a secondary container before aliquoting into the wells (see the Quick Chart for Preparing Buffers in this section). After use, discard remaining Developing Solution.
Stop Solution
Prior to use, transfer the amount of Stop Solution required for the assay into a secondary
container (see the Quick Chart for Preparing Buffers in this section). After use, discard remaining
Stop Solution.
WARNING: The Stop Solution is corrosive. Wear personal protective equipment when handling, i.e.
safety glasses, gloves and labcoat.

CpG Methyltransferase enzyme
A small volume of CpG Methyltransferase enzyme is provided as a positive control for the assay.
Please centrifuge the enzyme vial before opening the cap to ensure complete recovery of thesmall volume of material inside the vial. There is enough positive control enzyme included in the
kit to generate two standard curves. The CpG Methyltransferase is provided at 2 U/µl.
Preparation of DNMT containing samples
Purified DNMT enzymes or nuclear extracts containing DNMT activity can be used in the assay.
For purified DNMT enzymes a range of 0.5 ng - 100 ng is recommended, while for nuclear extracts a range of 0.5 µg - 10 µg is suggested. It is recommended initially to use a range of sample
concentrations in order to determine the amount of sample necessary to fall within the linear
range of the assay. Once the appropriate amount of sample has been determined, perform therest of the assays within the linear range. It is recommended to work with fresh DNMT or nuclear
extract samples. If samples need to be stored, small aliquots should be prepared and stored at-80°C to avoid multiple freeze/thaw cycles. To prepare nuclear extracts, we recommend using
Active Motif’s Nuclear Extract Kit (Catalog No. 40010). The use of fresh cell or tissue samples for
extract preparation is suggested, as frozen samples can lose enzymatic activity.

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8Quick Chart for Preparing Buffers
Reagents to prepare

Components

For 1
well

For 1 strip(8 wells)

For 6 strips(48 wells)

For 12 strips(96 wells)

Complete Enzymatic
Buffer AM1

Enzymatic Buffer AM1
100X AdoMet
TOTAL REQUIRED

109 µl1.1 µl110 µl891 µl9 µl
900 µl5.35 ml
54 µl
5.4 ml10.7 ml
108 µl10.8 ml

1X Wash Buffer AM1

Distilled water
10X Wash Buffer AM1
TOTAL REQUIRED

1.35 ml
150 µl
1.5 ml10.8 ml
1.2 ml
12 ml

63 ml
7 ml
70 ml

117 ml13 ml
130 mlHis-MBD2b Dilution

Binding Buffer AM11
His-MBD2b protein
TOTAL REQUIRED

107.8 µl
2.2 µl110 µl882 µl18 µl
900 µl5.29 ml
108 µl5.4 ml10.58 ml
216 µl10.8 ml

1X Wash Buffer AM3

Distilled water
10X Wash Buffer AM3
TOTAL REQUIRED

630 µl70 µl700 µl4.95 ml
550 µl
5.5 ml29.7 ml
3.3 ml33 ml

58.5 ml
6.5 ml65 ml

1X Antibody Binding
Buffer AM3

Distilled water
10X Antibody Binding Buffer AM3
TOTAL REQUIRED

99 µl
11 µl
110 µl810 µl90 µl900 µl4.86 ml
540 µl5.4 ml9.72 ml
1.08 ml
10.8 ml

Anti-polyHis-HRP Ab

1X Antibody Binding Buffer AM3
Anti-polyHis-HRP Ab
TOTAL REQUIRED

110 µl0.11 µl
110 µl900 µl0.9 µl900 µl5.4 ml5.4 µl5.4 ml10.8 ml
10.8 µl
10.8 ml

Developing Solution

TOTAL REQUIRED

110 µl900 µl5.4 ml10.8 ml

Stop Solution

TOTAL REQUIRED

110 µl900 µl5.4 ml10.8 mlwww.activemotif.com

9Assay Protocol
Read the entire protocol before use.
Determine the appropriate number of microwell strips required for testing samples, controls andblanks in duplicate. Store the unused strips in the aluminum pouch at 4°C. If less than 8 wells in a
strip need to be used, cover the unused wells with a portion of the plate sealer while you performthe assay. The unused wells are stable at room temperature for the duration of the assay if kept
dry. Once the assay is finished, unused strips should be returned to the aluminum pouch andstored at 4°C for a separate assay. Use the strip holder while performing the assay.
Prepare all the buffers required as described above in the section Buffer Preparation and Recommendations. Multi-channel pipettor reservoirs may be used for dispensing the 1X Wash Buffer
AM1, His-MBD2b dilution, 1X Wash Buffer AM3, Anti-polyHis-HRP antibody dilution, Developing
Solution and Stop Solution into the wells being used.
All of the washes in the DNMT assay protocol can be performed by inverting the plate and“flicking” the fluid into a sink. The inverted plate is then tapped on clean absorbent paper toremove excess fluid.

Standard Curve Preparation (Optional)
Use this plate set-up example to prepare a standard curve for the included CpG Methyltransferase enzyme in duplicate. The positive control CpG Methyltransferase is tested from 0.125 - 2
U per well in a 2-fold serial dilution. Prepare 1.5 ml Complete Enzymatic Buffer for use in thestandard curve by adding 15 µl 100X AdoMet to 1.485 ml Enzymatic Buffer. Vortex to mix.
Positive Control
1

2

3

4

5

6

7

8

9

10

11

12

A

2U

2U

–

–

–

–

–

–

–

–

–

–

B

1U

1U

–

–

–

–

–

–

–

–

–

–

C

0.5 U

0.5 U

–

–

–

–

–

–

–

–

–

–

D

0.25 U

0.25 U

–

–

–

–

–

–

–

–

–

–

E

0.125 U

0.125 U

–

–

–

–

–

–

–

–

–

–

F

Blank

Blank

–

–

–

–

–

–

–

–

–

–

G

–

–

–

–

–

–

–

–

–

–

–

–

H

–

–

–

–

–

–

–

–

–

–

–

–

1.

The CpG Methyltransferase enzyme is provided at a 2 U/µl concentration. Keep the enzyme
on ice. Before using, quick spin the contents to the bottom of the tube.

2.

Add 200 µl of Complete Enzymatic Buffer to wells A1 and A2.
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3.

Add 100 µl of Complete Enzymatic Buffer to wells B1 through F2.

4.

Add 2 µl CpG Methyltransferase enzyme to wells A1 and A2. Pipet up and down to mix.

4.

Perform a two-fold serial dilution by transferring 100 µl of the contents in row A to the wells
in row B.

5.

Mix the contents of row B by pipetting up and down 3-5 times. Do not change pipette tips
between well transfers.

6.

Transfer 100 µl of the contents of row B to row C and mix, as previously described.

7.

Continue this process until row E is reached.

8.

When row E is reached, discard 100 µl of the well contents so that the final volume is 100 µl.

9.

Row F will serve as the blank wells.

Step 1: Enzymatic Reaction
1.

In duplicate, prepare sample reactions. It is recommended to initially try a range of concentrations in order to determine the amount of sample needed to fall within the linear rangeof the assay. Follow the chart below to add sample, optional co-activator/inhibitor andcomplete Enzymatic Buffer to reach a final volume of 100 µl per well.
Purified DNMT enzymes: Recommended range of 0.5 ng - 100 ng per well
Nuclear extracts: Recommended range of 0.5 µg - 10 µg per well
Blank Wells: Add 100 µl Complete Enzymatic Buffer to each well2.

Reagent

One Reaction (Double amounts for duplicates)

Purified DNMT or Nuclear Extract

See above recommendations

Co-activator or Inhibitor

X µl

Complete Enzymatic Buffer

Up to 100 µl final volume

Using the provided adhesive plate sealer, cover the plate. Incubate the plate containing theprotein standard curve (optional) and samples for 1-2 hour(s) at 37°C with mild agitation (100rpm on a rocking platform). The incubation time depends on the intrinsic DNMT enzymatic
activity of your samples, but some guidelines are suggested below:
1 hour incubation: Recommended for active DNMT1 enzymes and optional standard curve
2 hour incubation: Recommended for active DNMT3a/3b enzymes and nuclear extracts

3.

After the incubation, remove the binding reaction with a multichannel pipette. Use clean
tips for each well to avoid cross-contamination of samples.

4.

Wash the wells 3 times with 200 µl of 1X Wash Buffer AM1.

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Step 2: Binding of His-MBD2b protein
5.

Add 100 µl of diluted His-MBD2b protein (1:50 dilution in Binding Buffer AM11) to all of thewells.

6.

Incubate at room temperature for 45 minutes with mild agitation (100 rpm on a rocking
platform).

7.

After the incubation, wash the wells 3 times with 200 µl of 1X Wash Buffer AM3.

Step 3: Binding of poly-Histidine HRP Antibody
8.

Add 100 µl of diluted anti-polyHis-HRP antibody (1:1000 dilution in 1X Antibody Binding Buffer AM3) to all of the wells.

9.

Incubate at room temperature for 45 minutes with mild agitation (100 rpm on a rocking
platform).

10. During this incubation, place the Developing Solution at room temperature.
11.

After the incubation, wash the wells 3 times with 200 µl of 1X Wash Buffer AM1.

Step 4: Colorimetric Reaction
12. Remove as much of the final wash as possible by blotting the plate on paper towels.
13. Add 100 µl of room temperature Developing Solution to all wells being used.
14. Incubate under low light conditions from 30 seconds to 10 minutes at room temperature
protected from direct light. Monitor the blue color development in the wells containing thehigher concentrations of sample until they turn medium to dark blue. Do not overdevelop.
Please read the Certificate of Analysis supplied with this kit for optimal development timeassociated with the CpG Methyltransferase standard curve.
15. Add 100 µl of Stop Solution to all the wells. In presence of the acid, the blue color turns
yellow.
16. Read absorbance on a spectrophotometer within 5 minutes at 450nm with an optionalreference wavelength of 655 nm. Blank the plate reader according to the manufacturer’s
instructions using the blank wells.
Reading the reference wavelength is optional. Most microtiter plate readers are equippedto perform dual wavelength analysis and with the appropriate software, will automaticallysubtract the reference wavelength absorbance from the test wavelength absorbance. If
your plate reader does not have this capability, you may read the plate twice, once at 450
nm and once at 655 nm then manually subtract the 655 nm OD from the 450 nm OD values.

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Calculation of DNMT Activity or Inhibition
To calculate activity or inhibition of DNMTs, average the duplicate readings for sample andblank wells and follow the formulas below:
DNMT activity (OD/h/mg) =(Average Sample OD - Average Blank OD)
Protein amount (µg)* x hour**x 1000*Protein amount added into the reaction in Step 1.1**Incubation time used for the reaction in Step 1.2[

DNMT Inhibition (%) = 1 -www.activemotif.com(Average Inhibitor Sample OD - Average Blank OD)](Average No Inhibitor Sample OD - Average Blank OD)

13x 100%

References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.

Miranda T.B. and Jones P.A. (2007) J.Cell Physiol. 213: 384-390.
Prokhortchouk E. and Defossez P.A. (2008) Biochem. Biophys. Acta. 1783: 2167-2173.
Chang S.C. et al. (2006) Front Biosci. 11: 852-866.
Hellman A. and Chess A. (2007) Science 315 (5815): 1141-1143.
Li E. et al. (1993) Nature 366: 362-365.
Bogdanovic O. and Veenstra G.J. (2009) Chromosoma 118: 549-565.
Klimasauskas S. et al. (1994) Cell 76: 357-369.
Cheng X. and Roberts R.J. (2001) Nucleic Acids Res. 29: 3784-3795.
Okano M. et al. (1999) Cell 99: 247-257.
Jeltsch A. (2006) Curr. Top. Microbiol. Immunol. 301: 203-225.
Fatemi et al. (2002) Eur. J. Biochem. 269: 4981-4984.
Gowher H. et al. (2005) Biochemistry 44: 9899-9904.
Liang et al. (2002) Mol. Cell Biol. 22: 480-491.
Okano M. et al. (1998) Nucleic Acids Res. 26: 2536-2540.
Aaola U. et al. (2000) Genomics 65: 293-298.
Goll M.G. et al. (2006) Science 311: 395-398.
Deplus R. et al. (2002) Nucleic Acids Res. 30: 3831-3838.
Barreto G. et al. (2007) Nature 445: 671-675.
Rai K. et al. (2008) Cell 135: 1201-1212.
Weiss A. et al. (1996) Cell 86: 709-718.
Schmmitz K.M. et al. (2009) Mol. Cell 33: 344-353.
Ma D.K. et al. (2009) Science 323: 1074-1077.
Cedar H. and Bergman Y. (2009) Nat. Rev. Genet. 10: 295-304.
Ooi S. K. et al. (2009) J. Cell Sci. 122 (16): 2787-2791.
Nan X. et al. (1998) Nature 393: 386-389.
Jones P.L. et al. (1998) Nature Genet. 19: 187-191.
Esteve P.O. et al. (2006) Genes Dev. 20: 3089-3103.
Hashimshony T. et al. (2003) Nature Genet. 34: 187-192.
Lande-Diner L. et al. (2007) J. Biol. Chem. 282: 12194-12200.
Zilberman D. et al. (2008) Nature 456: 125-129.
Kurkjian C. et al. (2008) Curr. Probl. Cancer 32 (5): 187-235.
Lyko F. and Brown R. (2005) J. Natl. Cancer Inst. 97 (20): 1498-1506.
Stresemann et al. (2006) Cancer Res. 66 (5): 2794-2800.
Mund C. (2006) Epigenetics 1 (1): 7-13.
Kalpana G. and Shoumei B. (2007) Drugs Today 43: 395.
Svedruzic Z.M. (2008) Curr. Med. Chem. 15: 92-106.
Roth M. and Jeltsch A. (2000) Bio. Chem. 381: 269-272.
Fuks et al. (2000) Nature Genet. 24: 88-91.
Yokochi T. and Robertson K.D. (2004) Methods Mol. Biol. 287: 285-296.
Kim B.Y. et al. (2004) Anal. Biochem. 326: 21-24.
Woo Y. H. et al. (2005) Anal. Biochem. 340: 336-340.
Humeny A. et al. (2003) Anal. Biochem. 313: 160-166.
Salyan M. E. et al. (2006) Anal. Biochem. 349: 112-117.

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Appendix
Section A: Troubleshooting Guide
Problem/question

Possible cause

Recommendation

No signal or weak signal

Omission of key
reagent

Check that all reagents have been added in all wells in thecorrect orderToo many freeze/thaw
cycles or inadequate
storage of protein

It is recommended to work with fresh DNMT or nuclear extract
samples. If samples need to be stored, small aliquots should
be prepared and stored at -80°C to avoid multiple freeze/thaw
cycles. During the kit’s first use, aliquot the stock His-MBD2b
protein into small aliquots and store at -20°C to avoid multiple
freeze/thaws

Incorrect assay
temperature

Enzymatic reaction should be performed at 37°C. All otherincubations can be performed at room temperature. Bring
Developing Solution and Stop Solution to room temperature
before using

Substrate or conjugate
is no longer active

Test conjugate and substrate for activity by mixing a smallaliquot of polyHis-HRP and Developing Solution together

Enzyme inhibitor
present

Sodium azide will inhibit the peroxidase reaction. Follow ourrecommendations to prepare buffers

Plate reader settings
not optimal

Verify the wavelength and filter settings in the plate reader

Inadequate volume ofDeveloping Solution

Check to make sure that correct volume is delivered by pipette

Studying a metal coactivator

The Enzymatic buffer contains EDTA which can interfere withthe study of metal co-activators. We recommend preparing abuffer without EDTA as follows: 50 mM Tris base, 1 mM DTT, 5%
Glycerol, and 100 µg/ml BSA. The final solution should be pH 7.8.
This can be used to perform the enzyme reaction.

Not enough sample
per wellFollow the guidelines included in the protocol for the amount of
purified DNMT or nuclear extract to use. Titrate your sample todetermine the optimal amount to use in the assay.

Protein sample is notproperly extracted

Use Active Motif’s nuclear extract Kit (Catalog No. 40010) to
prepare nuclear extracts. It is recommended to use fresh cells
or tissues for extract preparation as frozen samples can lose
enzymatic activity.

Developing time too
long

Stop enzymatic reaction as soon as the positive wells turn
medium-dark blue

Inadequate washing

Ensure all wells are filled with Wash Buffer and follow washingrecommendations

Incomplete washingof wells

Ensure all wells are filled with Wash Buffer and follow washingrecommendations

Well cross-contamination

Follow washing recommendations

Too much sample
per wellDecrease amount of sample per well.

No signal or weak signal
in sample wells only

High background in all
wells

Uneven color
development

High background insample wells

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15

Technical Services
If you need assistance at any time, please call or send an e-mail to Active Motif Technical Service
at one of the locations listed below.

Active Motif North America
Toll free:Direct:Fax:
E-mail:Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
0800/169 31 47
0800/90 99 79
0800/181 99 10+32 (0)2 653 0001
+32 (0)2 653 0050
eurotech@activemotif.com

Active Motif Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com
Active Motif China
Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

Visit Active Motif online at activemotif.com

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