DNA Methylation

DNA Methylation
5-hmC Analysis
Methylated DNA Enrichment
DNMT Assays
Bisulfite Conversion
Whole Genome Amplification

innovative technologies for DNA methylation analysis

Active Motif offers an expansive line of products to provide
researchers with the necessary tools for achieving successwhen analyzing DNA methylation.
Whether you are performing bisulfite conversion or evaluating
the methylation status of CpG islands, Active Motif makes it
simpler than ever to get faster and more reliable results.

Methylated DNA Standards
DNA Methylation Services
DNA Methylation Antibodies
Enabling Epigenetics ResearchDNA Methylation – technologies that streamline analysis
DNA Methylation is a heritable epigenetic event that plays a pivotal role in gene regulation, especially during
development and in carcinogenesis. Methylation occurs via the enzymatic transfer of methyl groups by DNA
methyltransferase enzymes to the C-5 position of cytosine in CpG dinucleotides to produce 5-methylcytosine(5-mC). However, alternate forms of cytosine methylation have been identified, such as 5-hydroxymethylcytosine(5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Methylation at the C-3 position (3-methylcytosine(3-mC)) has also been shown to occur. Due to the important regulatory roles of DNA methylation in gene silencing,
imprinting and chromosomal inactivation, and because aberrant methylation has been linked to developmental
defects and cancers, Active Motif has generated a variety of products to streamline DNA methylation analysis.
5-Hydroxymethylcytosine (5-hmC)
5-Hydroxymethylcytosine (5-hmC), derived from the oxidation of 5-Methylcytosine (5-mC) by TET enzymes, is a novel epigenetic marker
that is also an important regulator of development and carcinogenesis. 5-hmC is often associated with actively transcribed genes, supportinga complementary role to 5-mC or an intermediary function during demethylation. High levels of 5-hmC are observed in the central nervous
system of higher organisms and are also linked to pluripotency of stem cells.

Hydroxymethyl Collector™ for fast, efficient enrichment of 5-hydroxymethylcytosine
The Hydroxymethyl Collector™ Kit was
designed for the highly specific capture of
DNA fragments that contain 5-hmC residues.
The method takes advantage of an efficientchemical labeling procedure that enables theenriched samples to be collected as doublestranded DNA fragments. This makes it easy
to prepare libraries for various downstream
applications, including Next-Generation
sequencing (Figure 1).

Figure 1: Human tiling array using DNA enriched with Hydroxymethyl Collector.
Human brain DNA that was enriched using the Hydroxymethyl Collector Kit was amplified by whole-genome amplification and hybridized to an Affymetrix Human Tiling 2.0R Array A containing chromosomes 1 and 6. This image shows
a 1.2 million base pair view of chromosome 6 where there is a clear enrichment of 5-hmC across the entire length of
the JARID2 gene.

How it works: β-glucosyltransferase enzyme
is used to transfer a modified glucose moiety
to 5-hmC that is then chemically labeledwith a biotin conjugate for capture using
streptavidin magnetic beads (Diagram 1,
next page). This unique chemistry ensures nocross-reactivity with 5-mC, and it is not limited by the specific properties or consensus
sequences that constrain traditional methods, such as glucosyl-sensitive restriction
enzyme digestion. Furthermore, modificationof 5-hmC occurs independent of sequence
context, enabling the detection of both
CpG and non-CpG methylated DNA. The useof streptavidin magnetic beads allows forquick and efficient recovery of samples, andthe specificity of the streptavidin captureenables more stringent binding and wash
conditions. The result is a reduction in background and increased sensitivity, allowing
enrichment of DNA fragments containing as
few as two 5-hmC residues.

*Patent Pending.

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OH

Biotin

NH2

N3
N

N
H

HO
HO

N3

O
NH2

O
OH

β-Glucosyltransferase+ UDP-Azide-Glucose(1 hr at 37°C)

2. Modified
Glucosyl-5-hmC

O

N
H

Biotin Conjugate
Solution(1 hr at 37°C)

SA

Enriched DNA
containing
5-hmC

N3
HO
HO

O

OH

Elute the DNA
from the Biotin(30 min at RT)N

5. Eluted DNA is purified using
the included reagents.

N
H

O

O

N
H

Magnetic
Streptavidin Beads(30 min at RT)

BiotinN3ON

3. Biotinylated
Glucosyl-5-hmC

O
NH2

NH2

OH

N

O

1. Fragmented DNA
containing 5-hmC

HO
HO

O

HO
HO

O
O

NH2

OH

N

4. DNA is captured forwashing and elution.

N
H

O

Diagram 1: Hydroxymethyl Collector method.
Fragmented dsDNA (100-500 bp) is combined with β-Glucosyltransferase enzyme in the presence of a UDP-Azide-Glucose donor. The enzyme adds this modified glucose onto the
5-hmC residues. A biotin conjugate is then attached and the complex is captured using streptavidin magnetic beads and a magnet. Elution Buffer is added, which releases the 5-hmC
enriched DNA fragments from the biotin linker. Finally, the included purification reagents are utilized to clean up the DNA prior to its use in downstream applications.

Active Motif’s hMeDIP Kit was designed forenrichment of hydroxymethylated DNA.
Using a highly selective 5-hmC antibody
for specific immunocapture of hydroxymethylated genomic DNA fragments, thehydroxymethylated DNA immunoprecipitation, or hMeDIP, method allows genomewide targeted enrichment of hydroxymethylated DNA sequences. The high level of
specificity of the 5-hmC antibody usedin our hMeDIP Kit offers several advantages, including the ability to efficiently
immunoprecipitate both single-stranded
and double-stranded DNA, to distinguishbetween 5-hmC and 5-mC, and to detectboth CpG and non-CpG methylated DNA.
Furthermore, the use of magnetic beads for
capture ensures rapid processing of samples.
The hMeDIP Kit can be run in parallel to our% of Input

Specific immunocapture of 5-hmC DNA using antibody-based enrichment
20
18
16
14
12
10
8
6
4
2
0

Sample 1
Sample 2

unDNA

meDNA

hmeDNArabbit IgG

Figure 2: Specificity of the hMeDIP Kit.
Mse I digested human genomic DNA (500 ng) was spiked with 25 pg of either methylated (meDNA), hydroxymethylated (hmeDNA) or unmethylated APC DNA (unDNA). Samples were then processed using the hMeDIP Kit with purified 5-hydroxymethylcytosine pAb. Eluted DNA was purified and tested using real-time PCR with the included APC
PCR primer mix. The 5-hydroxymethylcytosine antibody specifically enriched the IP sample containing the hydroxymethylated APC DNA, but did not enrich for methylated or unmethylated DNA. The APC locus analyzed in this experiment is not methylated in human genomic DNA and therefore should not amplify. This experiment was performed todetect the presence of the spiked control DNA only. Samples were assayed in duplicate.

MeDIP Kit for comparative analysis of DNA
methylation patterns. The hMeDIP Kit also
includes unmethylated, 5-methylcytosine

and 5-hydroxymethylcytosine control DNAsand PCR primers to ensure the specificity of
the 5-hmC immunocapture (Figure 2).

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3

DNA Methylation – technologies that streamline analysis
5-Hydroxymethylcytosine-specific enzymes to distinguish between 5-mC and 5-hmC
Active Motif offers two different enzymes
for use in distinguishing between the 5-mC
and 5-hmC forms of DNA methylation,
the PvuRts1I restriction enzyme and the
β-Glucosyltransferase enzyme.

The PvuRts1I restriction enzyme is the only
enzyme to directly differentiate between the
5-mC and 5-hmC forms of DNA methylation.
Therefore, it serves as a valuable tool for
analyzing DNA methylation patterns within
the genome. The enzyme is not only able
to directly distinguish between 5-mC and5-hmC, but is also able to cleave bothglucosylated and non-glucosylated 5-hmC
DNA, thus eliminating the need toglucosylate 5-hmC residues prior todigestion analysis (Figure 3).

The β-Glucosyltransferase enzyme labels
5-hmC DNA by transferring a glucose moiety
from UDP-Glucose to the 5-hmC residue indouble-stranded DNA to create glucosyl5-hmC DNA (Diagram 2). The 5-hmC DNA canbe quantified directly by radioactive labelingof 5-hmC with a [14C] UDP-Glucose donor.
Alternatively, 5-hmC DNA methylation patterns can be analyzed using glucosyl-sensitive
restriction enzyme digestion.

N3
NH2

OH

β-Glucosyltransferase

N

Figure 3: PvuRts1 I enzyme digestion of 5-hmC DNA.
One µg of unmethylated (Un), 5-methylcytosine (mC)or 5-hydroxymethylcytosine (hmC) Methylated DNA
Standard (Catalog No. 55008) was incubated in theabsence or presence of 1 unit PvuRts1 I enzyme for
30 minutes at 22°C. Each reaction was run on a 2.5%
agarose gel alongside a 1 kb DNA ladder.

N
H

O

HO
HO

O
O

NH2

OH

N

UDPG

hmC

Glucosyl-hmC

N
H

O

Diagram 2: Depiction of the β-Glucosyltransferase enzyme reaction.

Recombinant Tet1 for 5-hmC conversion assays
The Tet1 protein is a member of the TET
family of cytosine oxygenases that convert
5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). For researchersinterested in studying the mechanism of how
5-hmC is generated, Active Motif offers arecombinant Tet1 protein for use in 5-hmC
conversion assays (Figure 4).

Figure 4: Recombinant Tet1 activity assay.
Double-stranded DNA containing 5-methylcytosine
was incubated with 5 µg of recombinant Tet1 enzyme(+Tet1) or without Tet1 enzyme (-Tet1). These samples
and an unmethylated DNA control (DNA) were thenspotted onto a nylon membrane and incubated with
5-Hydroxymethylcytosine antibody (Catalog No.
39769) to detect the conversion of 5-methylcytosine
into 5-hydroxymethylcytosine.

+Tet1
-Tet1
DNA

Ordering information for 5-hmC products
For more information and complete product details, please call or visit us at www.activemotif.com/dnamt.
Product

FormatCat. No.

Hydroxymethyl Collector™

25 rxns

55013

hMeDIP

10 rxns

55010

PvuRts1I restriction enzyme

50 units

55011

β-Glucosyltransferase enzyme

500 units

55012

Recombinant Tet1 protein, active

25 µg

31363

Active Motif also offers custom services (page 9) and antibodies (page 11) for the analysis of 5-hydroxymethylcytosine. For a complete up-to-date
list of all of our available products to study 5-hmC, please call or visit us at www.activemotif.com/hmc.

4

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5-Methylcytosine (5-mC)
5-Methylcytosine (5-mC), also known as the “5th base,” is formed when DNA methyltransferases catalyze the transfer of a methyl groupfrom S-adenosyl-L-methionine to cytosine. 5-mC is found in CpG-rich regions, and its function is associated with transcriptional repression,
particularly as it relates to genomic imprinting, repression of transposable elements and gene silencing.

MethylCollector Ultra™ for rapid isolation of CpG methylated DNA

Advantages• Improved efficiency – high-affinity
binding provides greater enrichment
than other MBD capture methods• Faster procedure – magnetic protocol
can be completed in less than 3 hours

Break open cells and shear
DNA by Sonication or
Enzymatic Shearing.

• Versatility – eluted DNA is suitablefor use in various downstream
applications such as endpoint orreal-time PCR, bisulfite conversion,or Next-Generation sequencing*Technology covered under U.S. Patent No. 7,425,415.

Capture the methylated
protein-DNA complexes withnickel-coated magnetic beads.

methylatedDNA

The purified, methylated
DNA is readyfor PCR analysis.

Wash to remove theunmethylated
DNA fragments.

Elute the bound, methylatedDNA, while simultaneously
degrading the protein complexes.

Diagram 3: Flow chart of the MethylCollector Ultra process.
In MethylCollector Ultra, genomic DNA is sheared by either sonication or enzymatic digestion, then incubatedwith the recombinant His-MBD2b/MBD3L1 protein complex. Magnetic beads capture the protein-DNA complexes.
Optimized buffers ensure that fragments with little or no methylation are removed. Methylated DNA is then eluted
from the beads. Following clean up, the eluted DNA is ready for use in various downstream applications.

Higher enrichment capability than other methods
A direct comparison of Active Motif’s
MethylCollector Ultra Kit versus a competitor’s MBD-Biotin capture method illustrates
the specificity of MethylCollector Ultra.
Enriched methylated DNA was analyzed
by qPCR using primers for two different
promoters (Figure 5), methylated SNRPN
promoter (red), and unmethylated FOXD2(blue). The MethylCollector Ultra Kit showsearly amplification of the SNRPN locus at 28
cycles, while the unmethylated FOXD2 amplifies at 36 cycles. This validates the increasedenrichment capability of the MBD2b/MBD3L
capture protein and the specificity of theassay for CpG methylated DNA. Competitor
MM’s technique showed no enrichment ofmethylated DNA, as both methylated andunmethylated DNA amplified at 28 cycles.

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• Uses minimal sample material – works
with as little as 1 ng (~200 cells) of
fragmented DNA

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The MethylCollector™ Ultra Kit is a rapid
magnetic assay for targeted enrichmentof CpG methylated DNA from genomic
DNA fragments that have been preparedby sonication or enzymatic digestion. Our
magnetic bead based protocol enables CpG
enrichment to be completed in less than
3 hours, much faster than antibody immunoprecipitation, which typically requires
overnight incubation. MethylCollector Ultra
is based on the Methylated CpG Island
Recovery Assay (MIRA) which uses a MBD2b/
MBD3L1 protein complex that binds with highaffinity to methylated CpG dinucleotides(Diagram 3). This methodology provides
better enrichment than assays that utilize
methyl-binding protein (MBD) alone, enabling
recovery of DNA fragments containing as few
as 5 methylated CpGs, or as little as 1 ng ofDNA (an equivalent of ~200 cells).

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Figure 5: Real-time PCR analysis reveals the specificity of MethylCollector Ultra vs competing technologies.
100 ng of human, male genomic DNA was digested with Mse I and tested using MethylCollector Ultra and competitor
MM’s kit. Eluted DNA was analyzed using PCR primers for both methylated, SNRPN (red), and unmethylated, FOXD2(blue), promoters. Only MethylCollector Ultra showed specific enrichment for CpG methylated DNA as shown by theclear separation in amplification cycles.

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5

DNA Methylation – technologies that streamline analysis
HypoMethylCollector™ for positive identification of unmethylated CpGs
HypoMethylCollector™ is the first commercially available kit for the specificisolation and enrichment of unmethylated
CpG dinucleotides. HypoMethylCollector
utilizes the specificity of the CXXC binding domain towards unmethylated CpGs tocapture and enrich for DNA fragments thatlack methylation. This makes it possible toidentify hypomethylated promoters and to
study the effects of compounds that inhibit
methylation. Instead of relying on negative
data from methyl-specific binding techniques
to identify hypomethylated promoters,
HypoMethylCollector offers a reliable technique that provides positive identification of
unmethylated CpG regions (Figure 6).

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DO#;IEbeYkic[j^obWj[ZFigure 6: Endpoint PCR results confirm the specificity of HypoMethylCollector across multiple loci.
The human, male genomic DNA (200 ng) provided in HypoMethylCollector was tested. Both unboundand eluted fractions were collected and analyzed in PCR for 36 cycles across multiple loci. The twounmethylated promoters (APC and p48) showed strong bands in the eluted fractions, while themethylated promoters (Xist and NY-ESO) were primarily found in the unbound fraction.

Optimized reagents ensure specific isolation
Active Motif’s HypoMethylCollector Kit uses
a recombinant His-CXXC protein to specifically bind unmethylated DNA fragments containing as few as one CpG dinucleotide. The
kit provides two binding buffers, a low-salt
buffer for use with samples containing lessthan 5 CpGs per fragment and a higher salt
buffer for efficient binding of fragments withmore than 5 CpGs. Nickel-coated magnetic
beads capture the protein-DNA complexes

and the unmethylated DNA is eluted fromthe beads. Following clean up, the eluted
DNA is ready for use.
A side-by-side comparison of fractions
obtained from both HypoMethylCollector
and MethylCollector Ultra Kits illustrate thespecificity of each technique at binding andenriching for the appropriate methylation
status across multiple loci (Figure 7).

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advantages• Sensitivity – detects unmethylated
CpGs from 10 ng - 1 µg of DNA
fragmented by sonication or
enzymatic digestion

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Additionally, DNA collected from
HypoMethylCollector was bisulfite treatedusing Active Motif’s Bisulfite Conversion Kit
and analyzed by sequencing. Of the 8 clones
sequenced for the unmethylated APC locus,
only one clone contained a single methylated CpG of the 19 CpG sites within thesequenced region (Figure 9, next page).

Xist

APC

NY-ESO

Figure 7: Direct comparison of HypoMethylCollector and MethylCollector Ultra illustrates specificity.
Both HypoMethylCollector and MethylCollector Ultra were run according to the protocols using either 200 ng or
100 ng, respectively, of the provided Mse I digested human, male genomic DNA. Real-time PCR analysis was run acrossmultiple loci on both the unbound and eluted fractions. HypoMethylCollector clearly captures the unmethylated loci
(APC and p48) while MethylCollector Ultra enriches for methylated promoters (Xist and NY-ESO).
APC – contains 29 CpGs and is unmethylated in healthy tissues
p48 – contains 22 CpGs and is unmethylated in healthy tissues
Xist – contains 8 CpGs and is methylated in males (it is unmethylated in females)
NY-ESO – contains 6 CpGs and is methylated in human DNA• Faster procedure – magnetic protocol
can be completed in less than 3 hours• Controls ensure success – includes
positive control DNA and PCR primers• Versatility – eluted DNA is suitable foruse in various downstream applications
such as PCR, sequencing, or amplification and labeling for microarray analysis*Patent Pending

6

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MeDIP for highly selective enrichment of methylated DNA
In addition to its hMeDIP Kit, Active Motifalso offers a MeDIP Kit for selectiveenrichment of single-stranded DNA
fragments containing 5-mC from genomic
DNA. The MeDIP Kit utilizes a highly specific monoclonal 5-mC capture antibody
for methylated DNA immunoprecipitation(MeDIP). The antibody is able to distinguishbetween 5-mC and 5-hmC, making thisapproach more selective than conventional
bisulfite conversion or enzymatic methods.
A one-step immunoprecipitation and theuse of Protein G magnetic beads reduces
the protocol time to ensure rapid recovery
of methylated DNA. The MeDIP Kit also
includes a bridging antibody to optimize
enrichment, as well as human genomic DNA
and PCR primers for use as controls to confirm the efficiency of the immunocapture.
(Figure 8).

45
40

5-Methylcytidine

35

Mouse IgG

% Input

30
25
20
15
10
5
0

ZC3H13GAPDH
PCR Primer Pair

Figure 8: Real-time PCR results of the control DNA with ZC3H13 and GAPDH PCR primer sets.
Mse I digested human genomic DNA (500 ng) was processed using the MeDIP Kit with the 5-methylcytosine mAb or negative control mouse IgG. Eluted DNA was column purified with Active Motif’s
Chromatin IP DNA Purification Kit (Catalog No. 58002) and tested using real-time PCR with the included
ZC3H13 PCR primer mix and a negative control GAPDH PCR primer mix. The 5-methylcytosine antibody
specifically enriched methylated DNA with the ZC3H13 locus but did not enrich for DNA with either the
GAPDH locus or the mouse IgG. Data shown are the results from samples assayed in duplicate.

Bisulfite conversion made simpler
Bisulfite conversion and subsequent DNA
sequencing provides detailed information on
the methylation pattern of individual DNA
molecules with single-base-pair resolution.
Active Motif’s Bisulfite Conversion Kit simplifies the analysis of DNA methylation by
providing optimized reagents, an easy-to-use
protocol and a positive control conversion-

specific PCR primer pair that enables you
to validate the success of the conversion
procedure before spending extra time andmoney on sequencing (Figure 10). The conversion reaction can be performed in as little as
1.5 hours with a 99% conversion efficiency ofunmethylated cytosines to uracils.

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Figure 9: The Bisulfite Conversion Kit was used to confirm the specificity of the HypoMethylCollector™ Kit.
HypoMethylCollector was used to enrich for unmethylated DNA fragments. This DNA was bisulfite treated using
the Bisulfite Conversion Kit. Converted DNA was amplified by PCR. The gel extracted PCR product was cloned and8 colonies were selected for sequencing using the unmethylated APC promoter region. Only one clone contained
a single methylated CpG of the 19 CpG sites within the sequenced region. This validates that HypoMethylCollector
specifically enriches unmethylated DNA fragments.

Figure 10: Agarose gel analysis of PCR products
generated with the conversion-specific PCR primer
pair from the Bisulfite Conversion Kit.
PCR results of human (A) and mouse (B) genomic DNA
that was either bisulfite converted (Bis.) according
to the Bisulfite Conversion Kit or untreated (Untr.)
and amplified using the included positive control
conversion-specific PCR primer pair. The primer pairis specific towards bisulfite converted DNA in humanand mouse as is shown with the 220 bp amplicon in
the converted DNA and not in the untreated DNA.

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DNA Methylation – technologies that streamline analysis
DNMT Activity / Inhibition Assay
Active Motif’s DNMT Activity / Inhibition
Assay is a rapid, non-radioactive method
for screening the DNA methyltransferase(DNMT) activity of recombinant DNMT
enzymes or nuclear extract samples. The

96-stripwell format also allows for both
high and low throughput screening. Using
the affinity of methyl-CpG-binding domain(MBD) protein toward methylated DNA,
the assay targets DNMT activity from yourDNMT1 Inhibition Assay

Advantages

1.2
1

OD 450nm

0.8• Non-radioactive – colorimetric assay iseasily quantified by spectrophotometry
on a microplate reader at 450 nm

-43%

0.6

• Sensitive – unique methyl CpG binding domain (MBD) protein approachenhances the sensitivity of detection
from either purified DNMT proteins or
nuclear extracts

0.4
0.2
0

Blank

Mock

0.5 mM Procaine

2 mM Procaine

sample to catalyze the transfer of methylgroups to a universal CpG-enriched DNA
substrate that is immobilized on the plate.
DNMT activity can then easily be quantified
by spectrophotometry (Figure 11).

5 mM Procaine

MCF-7 cells +/- 96 hour Procaine treatment

Figure 11: DNMT inhibition in MCF-7 cells with procaine treatment.
The DNMT Activity / Inhibition Assay was used to screen for DNMT inhibition in MCF-7 cells that were either
untreated or treated with procaine for 96 hours. Nuclear extracts were prepared using Active Motif’s Nuclear Extract
Kit (Catalog No. 40010). Five µg of each treatment condition were tested in the assay with a 1.5 hour incubation
time and a 3 minute developing time. The 5 mM procaine treatment showed a 43% inhibition of DNMT activity ascompared to the mock treated sample. Data shown are the results from wells assayed in duplicate.

• Fast – assay can be completed inless than 3 hours• Flexible – stripwell plate format enables
screening in low or high throughput

DNA Standards for analyzing different types of methylation
To help ensure the accuracy of your results,
Active Motif offers the Methylated DNA
Standard Kit for use as a control when performing DNA methylation analysis. The kit
provides 3 recombinant DNA standards for
unmethylated DNA, 5-mC methylated DNA,and 5-hmC methylated DNA (Figure 12).

Figure 12: Dot blot analysis of Methylated DNA
Standard Kit samples.
Single- and double-stranded DNA (50 ng) derived fromeach of the three different Methylated DNA Standards
were spotted onto positively charged nylon membrane, then probed with 5-mC or 5-hmC antibody.
Top panel: 5-Methylcytosine antibody (Clone 33D3)(Cat. No. 39649, 1:1,000 dilution), detects only ssDNA.
Bottom panel: 5-Hydroxymethylcytosine antibody(Cat. No. 39769, 1:5,000 dilution).
Lane 1: Single-stranded DNA.
Lane 2: Double-stranded DNA.

The kit also includes a PCR primer mix for
both endpoint and real-time amplification.

Ordering information for DNA methylation products
For more information and a complete list of our DNA methylation products, please call or visit us at www.activemotif.com/dnamt.

8

Product

FormatCat. No.

MethylCollector™ Ultra

30 rxns

55005

HypoMethylCollector™

30 rxns

55004

MeDIP

10 rxns

55009

Bisulfite Conversion Kit

50 rxns

55016

DNMT Activity / Inhibition Assay

1 x 96 rxns

55006

Methylated DNA Standard Kit

3 x 2.5 µg

55008

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Genome-wide analysis services for DNA methylation
The reliable identification of differential DNA
methylation is important for researchersinterested in biomarker identification, as well
as for those trying to understand the basisof disease, drug mechanism of action orenvironmental influences on epigenetics. To
help speed your research, Active Motif offers
end-to-end, genome-wide DNA methylation
services to identify differentially methylated
regions. These services are based on theenrichment of DNA methylation using antiMeDIP

bodies to 5-methylcytosine (Figure 13). As analternative to antibody-based enrichment ofmethylated regions, we also offer a methodthat enriches methylated DNA using a
recombinant methylated DNA binding complex of MBD2b and MBD3L1 (also known as
MIRA-Seq) (Figure 14). Following enrichment,
Next-Gen sequencing libraries are made,DNA is sequenced and alignment of the tags
across the genome reveals regions where
DNA methylation resides.

CpG Density

Active Motif’s Epigenetic Services teamhas expanded its DNA analysis portfolio toinclude genome-wide hydroxymethylcytosine (hmC), formylcytosine (fC) and carboxylcytosine (caC) profiling. The services are
based on DNA immunoprecipitation using
antibodies that specifically recognize each
modification. Following enrichment, DNA issequenced using Illumina Next-Gen sequencing technology, resulting in genome-wide
enrichment profiles.
Genome-wide DNA methylation andDNA variant services offered• MeDIP-Seq – enrichment of methylated
DNA with a highly specific 5-methylcytosine antibody

Figure 13: Next-Gen sequencing data correlates with CpG density.
DNA was enriched by MeDIP from 1 µg of adaptor-ligated, human PBMC DNA. Next-Gen sequencing was performed
and tags were mapped to generate a whole-genome DNA methylation profile. The image above shows that theenriched regions correlate well with CpG density.

MethylCollector™ Ultra-Seq
CpG Density• MethylCollector™ Ultra-Seq – based on
the patented MIRA (Methylated CpG
Island Recovery Assay) technology• hMeDIP-Seq – enrichment using
our most specific and highly cited
5-hydroxymethylcytosine antibody• fC & caC genome-wide profiling –enrichment using a 5-formylcytosine
or a 5-carboxylcytosine antibody,followed by Next-Gen sequencing

CpG island

Figure 14: Next-Gen sequencing data detects methylation at CpG shores.
Next-Gen sequencing was performed on DNA enriched from 1 µg of human PBMC DNA using MethylCollector™ Ultra,
and tags were mapped to generate a whole-genome DNA methylation profile. The image above shows an example of
DNA methylation detected at CpG shores rather than in the CpG island itself. This data agrees with recent findings
showing that methylation frequently occurs in the regions adjacent to CpG islands.

• Next-Gen Bisulfite Sequencing –determination of the locations andmethylation status of individual
CpG dinucleotides
For more information on any of the DNA
methylation or DNA variant services offered,please visit www.activemotif.com/services.

Choose the DNA methylation kits that best suits your specific research needs
Active Motif’s extensive portfolio of kits forstudying DNA methylation enables you toselect from a variety of enrichment options,
including whether to enrich for non-CpG or
CpG methylated DNA, if ssDNA or dsDNA
is enriched, as well as the capture method
used. This allows you to choose the features
you need in order to achieve the results you
want in your DNA methylation research.

Methylation at:
CpG non-CpG
Hydroxymethyl Collector™
MethylCollector™ Ultra
HypoMethylCollector™
hMeDIP
MeDIP

✓
✓
✓
✓
✓

✓

DNA Enriched:
ssDNA dsDNA
–
✓

–

–

–

–

✓
✓

✓
✓

✓
✓
✓
–

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Magnetic Capture
Beads Method
Chemical
✓

✓
✓
✓
✓

ProteinProtein
Antibody
Antibody

9

DNA Methylation – technologies that streamline analysis
5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC)
5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) are novel DNA modifications found to exist in many vertebrate cell types, including
embryonic stem cells. The TET family of cytosine oxygenase enzymes, which convert 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine(5-hmC), further oxidize 5-hmC into 5-fC and 5-caC. 5-fC and 5-caC appear in the paternal pronucleus after fertilization (Figure 16A), concomitant with the disappearance of 5-methylcytosine (5-mC). The levels of 5-fC and 5-caC are gradually diluted out by DNA replication, rather thanbeing enzymatically removed (Figure 15). While this pathway represents a mechanism by which DNA methylation (5-mC) is removed, thesenovel modifications may also be serving unique functions in pre-implantation development.

5-fC
5-mC
DAPI

5-caC
5-mC
DAPI

Figure 15: Replication-dependent loss of 5-fC and 5-caC revealed in a 2-cell metaphase embryo.
Shown are representative immunofluorescent images of mitotic chromosome spreads that have been co-stained with Active Motif’s 5-Formylcytosine (5-fC) or 5-Carboxylcytosine(5-caC) antibodies (red, Catalog Nos. 61223 and 61225, respectively), a 5-methylcytosine (5-mC) antibody (green) and DAPI (blue) at the two-cell stage of mouse preimplantation
development. The 5-fC and 5-caC antibodies were used at a 1:2000 dilution. The images reveal that at the two-cell stage, only one of the two sister chromatids is enriched for
5-fC and 5-caC, consistent with findings that 5-fC and 5-caC levels are diminished by half in blastomeres with each round of DNA replication (Inoue et al. (2011)*).

B
5-fC
pAb

5-caC
pAb

5-c
aC
5-f
C
5-h
mC
5-m
C
5-C

A

10 ng

5-fC
pAb

3 ng
1 ng
0.3 ng0.1 ng

5-mC
pAb

5-c
aC
5-f
C
5-h
mC
5-m
C
5-C

5-mC
pAb

10 ng

Merge

Merge

5-caC
pAb

3 ng
1 ng
0.3 ng0.1 ngFigure 16: Characterization of 5-fC and 5-caC antibodies by whole mount staining and dot blot.
(A) Shown are representative whole mount confocal images of fertilized oocytes co-stained with Active Motif’s 5-Formylcytosine (5-fC) and 5-Carboxylcytosine (5-caC) antibodies(red, Catalog Nos. 61223 and 61225, respectively), and a 5-methylcytosine (5-mC) antibody (green). The 5-fC antibody was used at a 1:4000 dilution and the 5-caC antibody was used
at a 1:2000 dilution (Inoue et al.*). (B) Dot blot analysis was used to confirm the specificity of the 5-fC antibody for 5-formylcytosine and the 5-caC antibody for 5-carboxylcytosine.
Varying amounts of single-stranded DNA oligonucleotides corresponding to the immunogen and related sequences were spotted onto nitrocellulose and probed with the
5-fC antibody (upper image, 1:5,000 dilution) and the 5-caC antibody (lower image, 1:2000 dilution). Lane 1: oligomer containing 5-carboxylcytosine. Lane 2: oligomer containing
5-formylcytosine. Lane 3: oligomer containing 5-hydroxymethylcytosine. Lane 4: oligomer containing 5-methylcytosine. Lane 5: oligomer containing unmodified cytosine.
* Images were kindly provided by the laboratory of Yi Zhang, HHMI Investigator at the University of North Carolina at Chapel Hill. The shown images reference experimental datathat is described in detail in Inoue et al. (2011) Cell Research 21: 1670-1676.

10

www.activemotif.com

Antibodies for DNA methylation research
For your convenience, Active Motif offers avariety of DNA methylation-related antibodies. Active Motif is committed to providing
the highest quality antibodies for studying
TargetProduct

3-mC

5-caC

5-fC

5-hmC

the biology of the nucleus. Each antibody wemake is rigorously tested. Many of the DNA
methylation antibodies have been validated
for use in ChIP and immunofluorescence (IF).

The table below is a partial list. For more
information, and to see a complete list of
DNA methylation antibodies, please visit our
website at www.activemotif.com/methylabs.

Reactivity

Application

FormatCatalog No.

3-Methylcytosine polyclonal antibody (Serum)

Human, Mouse, Wide Range

DB

100 µl
10 µl

61179
61180

3-Methylcytosine polyclonal antibody (IgG)

Human, Mouse, Wide Range

DB

100 µg
10 µg

61111
61112

5-Carboxylcytosine polyclonal antibody (Serum)

Not Species Specific

DB, IF100 µl
10 µl

61225
61226

5-Carboxylcytosine polyclonal antibody (IgG)

Not Species SpecificDB

100 µg
10 µg

61229
61230

5-Formylcytosine polyclonal antibody (Serum)

Not Species Specific

DB, IF100 µl
10 µl

61223
61224

5-Formylcytosine polyclonal antibody (IgG)

Not Species SpecificDB

100 µg
10 µg

61227
61228

5-Hydroxymethylcytosine monoclonal antibody

Human, Mouse, Wide Range

DB, MeDIP

100 µg
10 µg

39999
40000

5-Hydroxymethylcytosine polyclonal antibody (Serum)

Human, Mouse, Wide Range

DB, IF, IHC, MeDIP

100 µl
10 µl

39769
39770

5-Hydroxymethylcytosine polyclonal antibody (IgG)

Human, Mouse, Wide Range

DB, MeDIP

100 µg
10 µg

39791
39792

5-mC

5-Methylcytosine monoclonal antibody

Not Species SpecificDB, FC, IHC, IP, MeDIP

50 µg

39649

DNMT1

DNMT1 monoclonal antibody

Human, Mouse

ChIP, IHC, IP, WB

100 µg

39204

DNMT2

DNMT2 polyclonal antibody

Human, Mouse

WB

100 µg

39205

DNMT3ADNMT3A monoclonal antibody

Human, Mouse

ChIP, IF, IHC, WB

100 µg

39206

DNMT3BDNMT3B monoclonal antibody

Human, Mouse

ChIP, IF, IP, WB

100 µg

39207

DNMT3LDNMT3L polyclonal antibody

Human

WB

100 µl
10 µl

39907
39908

HP1

HP1a monoclonal antibody

Mouse

ChIP, ELISA, ICC, IF, IHC

100 µg
10 µg

39977
39978

MBD1

MBD1 monoclonal antibody

Human

WB

100 µg

39215

MBD2

MBD2 polyclonal antibody

Human

WB

200 µl
10 µl

39547
39548

MBD3

MBD3 monoclonal antibody

Human

WB

100 µg

39216

MBD4

MBD4 polyclonal antibody

Human

WB

100 µg

39217

MeCP2

MeCP2 monoclonal antibody

Human, Mouse, Rat

ChIP, IF, IHC, IP, WB

100 µg
10 µg

61285
61286

Tet

Tet1 polyclonal antibody

Human, Mouse

ChIP, ChIP-Seq, WB

100 µl
10 µl

61443
61444

Tet2 monoclonal antibody

HumanIP, WB

100 µg
10 µg

61389
61390

Tet3 polyclonal antibody

Mouse

WB

100 µl
10 µl

61395
61396

ChIP = Chromatin Immunoprecipitation; DB = Dot Blot; FC = Flow Cytometry; IF = Immunofluorescence; ICC = Immunocytochemistry;
IHC = Immunohistochemistry; IP = Immunoprecipitation; MeDIP = Methylated DNA Immunoprecipitation; WB = Western Blot

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11

DNA Methylation

innovative technologies for DNA methylation analysis• CGBP Antibody• Methylated DNA Standard Kit

Decarboxylase

DNMT• DNMT Activity / Inhibition Assay• DNMT Antibodies• HP1 Antibodies• MBD Antibodies• MeCP2 Antibodies• Uhrf1 Antibody

Tet• MethylCollector Ultra™
• HypoMethylCollector™
• MeDIP• Bisulfite Conversion• 5-mC Antibodies• Methylated DNA Standard Kit• Tet Antibodies• Recombinant Tet1 Protein• Hydroxymethyl Collector™
• hMeDIP• PvuRts1I• b-Glucosyltransferase• 5-hmC Antibodies• Methylated DNA Standard Kit

Tet• 5-caC Antibodies• 5-Carboxylcytosine
DNA Standard Kit

Tet• Tet Antibodies• Recombinant Tet1 Protein• Tet Antibodies• Recombinant Tet1 Protein• 5-fC Antibodies

Enabling Epigenetics ResearchNorth America 877 222 9543 Europe +32 (0)2 653 0001 Japan +81 (0)3 5225 3638

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