Global 5-hmC DNA ELISA(version A2)
Catalog No. 55025

Copyright 2020 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.
© 2020 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
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols
Buffer Preparation and Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Assay Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Section A. MseI Digestion of Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Section B. Plate Set Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Section C. Plate Coating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Section D. Standard Curve Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section E. DNA Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section F. Binding to the Coated Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Section G. Binding of Primary Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section H. Binding of Secondary Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section I. Colorimetric Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Section J. Analysis of Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Appendix
Section K. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

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Overview
The Global 5-hmC DNA ELISA Kit is designed to detect and quantify 5-hydroxymethylcytosine(5-hmC) in DNA fragments. 5-hmC is an oxidative product of 5-methylcytosine (5-mC) that is
catalyzed by the ten eleven translocation (TET) family of enzymes. Research has shown that
5-hmC levels are independent of 5-mC levels and both DNA modifications appear to have distinct
biological functions. 5-hmC is known to play a role in transcriptional regulation and embryonicdevelopment. It may also serve as a prognostic indicator in certain cancers and neurodegenerative disorders.
In the Global 5-hmC DNA ELISA Kit, genomic DNA of interest is fragmented by enzymatic digestion and heat denatured to create single-stranded DNA (ssDNA). 96-stripwell plates are coatedwith a DNA binding agent to enhance the capture of DNA fragments to the plate. Following theaddition of ssDNA to the coated wells, unbound DNA fragments are washed away. A primary antibody specific for 5-hmC and a secondary antibody conjugated to horseradish peroxidase (HRP)
are used for detection of bound hydroxymethylated fragments. The colorimetric readout is easily
quantified by spectrophotometry using a microplate reader at 450nm.
Each kit contains an optimized protocol and reagents necessary to perform DNA fragmentation,plate coating, capture, and colorimetric detection of 5-hmC. For added convenience, a DNA standard containing a known percentage of 5-hmC modification is included in the kit. By generating astandard curve, the 5-hmC levels in each DNA sample can be determined.

product

formatcatalog no.

Global 5-hmC DNA ELISA Kit

1 x 96 rxns

55025

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1Flow Chart of Process

21

11

01

9

8

7

6

5

4

Coat 96-well assay
plate with DNA
Binding Agent

3

2

1
A
B

C
D
E
F
G
H

1 hour at RT(or overnight at 4˚C)

Heat denature
MseI-digested
DNA samples
10 min. at 98˚C

12

11

10

9

8

7

6

5

4

3

2

1
A
B

C
D
E
F
G
H

21

11

01

9

Add DNA Standards
and Samples tothe coated plate
2 hours at 37˚C
8

7

6

5

4

3

2

1
A
B

C

Wash to remove
unbound DNA

D
E
F
G
H

12

11

10

9

8

7

6

5

4

3

2

1
A
B

C
D
E
F
G

Add 5-hmC
antibody
1 hour at RT

H

21

11

01

9

8

7

6

5

4

3

2

1
A

Add anti-IgG
HRP conjugate

B
C
D
E
F
G
H

1 hour at RT
5

4

3

2

1

7

6

8

9

10

11

12

A
B
C
D
E
F
G
H

Add developing
and stop solution
5-20 min.

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Introduction
DNA Modification 5-Hydroxymethylcytosine (5-hmC)
In mammals and other vertebrates, DNA methylation usually occurs at the carbon 5 (C5) positionof cytosine (5-mC), mostly within CpG dinucleotides. In 2009, Kriaucionis and Heintz and Tahiliani
et al. discovered another DNA modification, 5-hydroxymethylcytosine (5-hmC), which wasobserved to be elevated in neurons and embryonic stem cells1,2. The 5-hmC modification resultsfrom the enzymatic conversion of 5-mC into 5-hmC by the TET family of cytosine oxygenases2,3.
While the precise function of 5-hmC has yet to be determined, it has been postulated that it
could represent a pathway to demethylate DNA, as 5-hmC is repaired as mismatched DNA andreplaced with unmethylated cytosine3. Alternatively, 5-hmC may be produced by the addition offormaldehyde to DNA cytosines by DNMT proteins4.
DNA base modifications are dynamic marks and their relative concentration at given loci is
influenced by multiple factors including cell and tissue type, the differentiation state of a cell, andthe balance between cellular homeostasis and stress response. 5-hmC has been shown to havedistinct biological functions and cellular localizations from 5-mC. Proteins that bind specifically to
5-hmC include DNA repair factors, splicing mediators and transcription regulators5,6. Experimental
evidence indicates that 5-hmC plays a role in transcriptional regulation and embryonic development7-17.
Loss of global hydroxymethylation is associated with malignant human cancers such as melanoma. Moreover, mutations in TET2 are commonly observed in a wide range of hematopoietic
malignancies and are associated with poor patient outcomes18-20. Hydroxymethylation is also
most abundant in the brain, where it is believed to play an important role in neurological development and memory formation10, 21-23. Therefore, 5-hmC may serve as a prognostic indicator incertain cancers and neurodegenerative disorders, such as Alzheimer’s disease, Freidrich’s ataxia
and Huntington’s disease.
Using Active Motif’s Global 5-hmC DNA ELISA Kit, genomic DNA from different sample types (e.g.
normal and diseased), treatment conditions, clinical outcomes, or environmental backgrounds
can be analyzed for differences in global 5-hmC levels. A DNA standard is included in the kit toprepare a standard curve for sample quantification. The assay can detect as little as 0.16% 5-hmC
in tissue derived DNA. Results obtained with the Global 5-hmC DNA ELISA Kit enable higher
throughput processing of samples, require only 5-10 ng of genomic DNA per well.

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References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.

Kriaucionis, S. and Heintz, N. (2009) Science, 324: 924-929.
Tahiliani, M. et al. (2009) Science, 324: 930-935.
Huang, Y. et al. (2010) PLoS One, 26: e8888.
Liutkeviciute, Z. et al. (2009) Nat. Chem. Biol., 5: 400-402.
Iurlaro, M. et al. (2013) Genome Biology, 14(10): R119.
Spruijt, C. et al. (2013) Cell, 152(5): 1146-1159.
Gan, H. et al. (2013) Nature Communications, 4: 1995.
Tsumagari, K. et al. (2013) Epigenetics, 8(3): 317-332.
Nestor, C. et al. (2012) Genome Research, 22: 467-477.
Rudenko, A. et al. (2013) Neuron, 79(6): 1109-1122.
Blaschke, K. et al. (2013) Nature, 500(7461): 222-226.
Klug, M. et al. (2013) Genome Biology, 14(5): R46.
Bocker, M. et al. (2012) Nature Communications, 3: 818.
Ficz, G. et al. (2013) Cell Stem Cell, 13(3): 351-359.
Gao, Y. et al. (2013) Cell Stem Cell, 12(4): 453-469.
Piccolo, F. M. et al. (2013) Molecular Cell, 49(6): 1023-1033.
Hackett, J. A. et al. (2013) Science, 339(6118): 448-452.
Lian, C. G. et al. (2012) Cell, 150(6): 1135-1146.
Song, S. J. et al. (2013) Cell, 154(2): 311-324.
Huang, H. et al. (2013) Proc Natl Acad Sci USA, 110(29): 11994-11999.
Colquitt, B. M. et al. (2013) Proc Natl Acad Sci USA, 110(36): 14682-14687.
Lister, R. et al. (2013) Science, 341(6146): doi: 10.1126/science.1237905.
Kaas, G. A. et al. (2013) Neuron, 79(6): 1086-1093.

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4Kit Performance and Benefits
The Global 5-hmC DNA ELISA Kit is used to quantify 5-hmC levels in genomic DNA when comparing the samples to serial dilutions of the provided DNA standard.
Range of detection: The dynamic range of the standard curve is 2.4 x 10-14 to 1.55 x 10-12 moles(0.484 nM – 31 nM) of 5-hmC. The assay has been shown to detect as little as 0.16% 5-hmC intissue derived genomic DNA.
Sample DNA can be used in the range of 5 ng - 30 ng per well. However, our data indicate thatthe best agreement between ELISA and mass spectrometry was obtained when 5-10 ng of input
DNA was used. For tissue samples containing high levels of 5-hmC (ex. brain) we recommend
starting with 5-10 ng/well. For tissue samples containing lower levels of 5-hmc, a concentrationof 10-30 ng/well is recommended, with 10 ng providing the best agreement with mass spectrometry. If the OD 450nm readings of the samples fall outside the limits of detection for the assay(as determined by the standard curve), we suggest performing a titration of the sample DNA to
determine the appropriate quantity to use within the assay. Then perform the rest of the assay atthe determined concentration.
Assay time: 6 hours

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Global 5-hmC DNA ELISA Kit Components and Storage
Global 5-hmC DNA ELISA Kits are for research use only. Not for use in diagnostic procedures. Allcomponents are guaranteed stable for 6 months from date of receipt when stored properly.
Reagents

Quantity

Storage

MseI enzyme (10 U/μl)

20 μl

-20°C

MseI Reaction Buffer (10X)

65 μl

-20°C

5-hmC DNA Standard (20X, 100 ng/μl)

4 μl

-20°C

DNA Binding Agent (lyophilized)

2 x 60 mg-20°C

5-Hydroxymethylcytosine pAb

10 μl

-20°C

Anti-rabbit HRP-conj. secondary Ab

10 μl

4°C

Antibody Dilution Buffer

10 ml

4°C

20X Wash Buffer

25 ml

4°C

Developing Solution

11 ml

4°C

Buffer W2 (2X)

70 ml

4°C

96-well plate

1 ea

4°C

Stop Solution

11 ml

RT

Plate sealer

4 ea

RT

0.2 ml PCR stripwell tubes

1 pk

RT

Additional materials required• Nanodrop™, Qubit™ or equivalent method to determine DNA concentration• Incubator set to 37°C
• Thermal cycler or water bath set to 98°C
• Ice bucket and ice• Orbital shaker (e.g. Multi-microplate Genie from Scientific Industries)• 1.5 or 2 ml microcentrifuge tubes and 15 ml conical tubes• Microcentrifuge with PCR tube and microcentrifuge tube adapters• Multichannel pipette, 10-200 µl pipette tips and plastic reservoirs• Vortex• DNase-free sterile water• Distilled water• Spectrophotometric microplate reader capable of reading at 450 nm with optional reference wavelength of 655 nm.
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6Buffer Preparation and Recommendations
MseI enzyme
The MseI enzyme is provided ready to use at 10 U/μl. Enough enzyme is provided to perform up
to 32 DNA digestions as described in Step A of the protocol.
10X MseI Reaction Buffer
The reaction buffer is provided at a 10X concentration.
5-hmC DNA Standard
The 5-hmC DNA standard for the Global 5-hmC DNA ELISA Kit is a synthetic oligo containing 9.5%5-hmC modification, provided at a concentration of 100 ng/μl. A standard curve will be preparedfrom the oligo stock by performing a seven point 1:2 serial dilution with a top input of 5 ng oligo(7.74 x 10-13 moles oligo, 1.55 x 10-12 moles 5-hmC). Sample results are then compared to the standard curve to extrapolate number of moles 5-hmC, % 5-hmC or molarity of 5-hmC in each sample.
DNA Binding AgentThe DNA Binding Agent is provided lyophilized and each vial is sufficient to coat 48-wells of
the assay plate. Before opening, quick spin the vial for 1 minute at room temperature to collectcontents to the bottom of the tube. Add 1 ml 1X Buffer W2 and vortex until fully dissolved. Transfer
the entire contents of the vial to a 15 ml conical tube. Add 5 ml 1X Buffer W2 to the conical tube
for a total volume of 6 ml and vortex to mix.
Antibody Dilution Buffer
The Antibody Dilution Buffer is provided ready to use. IMPORTANT: Bring to room temperature
before use.
1X Buffer W2 (See the Quick Chart for Preparing Buffers)
For every 10 ml of 1X Buffer W2 required, combine 5 ml 2X Buffer W2 with 5 ml distilled water.
Each half plate (48 wells) requires 43 ml of 1X Buffer W2. We recommend preparing 60 ml. Use
the 1X Buffer W2 at room temperature.
1X Wash Buffer (See the Quick Chart for Preparing Buffers)
For every 10 ml of 1X Wash Buffer required, combine 0.5 ml 20X Wash Buffer with 9.5 ml distilledwater. Each half plate (48 wells) will require 43 ml of 1X Wash Buffer. We recommend preparing
60 ml. Mix gently to avoid foaming. The 1X Wash Buffer may be stored at 4°C for one week. The
Tween 20 contained in the 20X Wash Buffer may form visible aggregates, therefore, homogenize
the buffer by incubating at 50°C for 2 minutes and mixing prior to use. Use the 1X Wash Buffer atroom temperature.
Developing Solution (See the Quick Chart for Preparing Buffers)
The bottle of Developing Solution should be placed at room temperature for 1 hour before use.
Transfer the amount of Developing Solution required for the assays into a secondary container
before aliquoting into the wells. After use, discard remaining Developing Solution.

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The Developing Solution is light sensitive, therefore, we recommend avoiding direct exposure tointense light during storage. The Developing Solution may develop a yellow hue over time. However, this does not affect product performance. A blue color in the Developing Solution indicatesthat it has been contaminated and must be discarded.
Stop Solution (See the Quick Chart for Preparing Buffers)
Prior to use, transfer the amount of Stop Solution required for the assay into a secondary container. After use, discard any remaining Stop Solution.
WARNING: The Stop Solution is corrosive. Wear personal protective equipment whenhandling, i.e. safety glasses, gloves and labcoat.

Quick Chart for Preparing Buffers
1X Bufer W2

1X Wash Buffer

Components

6 strips (48 wells)

12 strips (96 wells)

Distilled water

30 ml

60 ml

2X Buffer W2

30 ml

60 ml

Total Volume

60 ml

120 mlDistilled water

57 ml

114 ml

20X Wash Buffer

3 ml

6 ml

Total Volume

60 ml

120 mlDeveloping Solution

Total Volume

5.4 ml10.8 ml

Stop Solution

Total Volume

5.4 ml10.8 mlwww.activemotif.com

8Assay Protocol
Read the entire protocol before use.
Prior to starting the Global 5-hmC DNA ELISA Kit, genomic DNA is fragmented using restriction
digestion with the provided MseI enzyme. This enzymatic digestion will create the appropriate
size range of DNA fragments for optimal detection in the assay. The digested DNA can be stored
at -20°C and the assay performed on another day.

Section A: MseI Digestion of Genomic DNA
We recommend preparing high-quality genomic DNA using a commercially available kit or a standard established protocol. Determine DNA concentration by UV spectrophotometry or fluorescence-based quantification. The Global 5-hmC DNA ELISA can be performed on approximately
5-30 ng MseI fragmented genomic DNA per well. We suggest starting with 0.5 μg of genomic
DNA per sample type for the digestion and then performing the assay in duplicate.
1.

In microcentrifuge tubes, set up a restriction digest for each DNA sample to be tested.
Also, prepare a mock digestion reaction that will be used as a blank for DNA quantification
following the digestion reaction. A small loss of DNA is anticipated as part of the digestion
process, therefore quantification MUST be performed following MseI digestion to accurately
determine the DNA quantity for use in the assay. A single mock digestion reaction can beused as a blank for all the DNA digestions performed on the same day.
Reagents
Genomic DNA

DNA Sample

Mock Digestion

0.5 μg (vol. will vary)

0 μl

2 μl

2 μl

0.5 μl

0.5 μlUp to 20 μl

17.5 μl

20 µl

20 µl

MseI Reaction Buffer (10X)
MseI enzyme (10 U/μl)
DNase-free Sterile water
Total Volume

2.

Mix well by pipetting and incubate at 37°C for 2-4 hours.

3.

Heat-inactivate MseI by incubating the reaction mixture at 65°C for 20 minutes. Quick spintubes to collect any condensate.

4.

The Global 5-hmC DNA ELISA Kit is very sensitive to DNA concentration so DNA MUST bequantified following MseI digestion to account for small changes in concentration thatcan occur as a result of the incubation. Measure the DNA concentration via absorbance
spectrophotmetry (ex. NanoDrop™) of each sample using the mock digestion as a blank.
Alternatively you can use fluorescence-based quantitation (ex. Qubit™).

Note: If the assay will not be performed immediately, the digested DNA can be stored at -20°C.

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9Section B: Plate Set-Up
The 96 well plate included in the kit contains 12 sets of 8-well strips. To improve the binding
efficiency of the genomic DNA to the assay plate, a DNA Binding Agent is applied to each well.
The DNA Binding Agent is provided lyophilized and each vial is sufficient to coat 48 wells at atime or 6 strips (48 wells). Wells can be coated and used the same day as the assay or allowed toincubate overnight at 4°C with the DNA Binding Agent and used the next day.
Take out the 96 well plate from the foil pouch. Remove six of the 8-well strips from the strip
holder and place back into the pouch for long term storage at 4°C and later coating. Allow the
strip holder and remaining six strips to warm to room temperature.
Each sample, standard and blank should be conducted in duplicate. Plan your experiment accordingly with two columns for standards and blanks and the remaining four columns for up to 16
samples in duplicate.
An example plate layout is shown below.
DNA Standards

MseI-digested DNA

1

2

3

4

5

6

A

5 ng

5 ng

Sample 1

Sample 1

Sample 9

Sample 9B

2.5 ng

2.5 ngSample 2

Sample 2

Sample 10

Sample 10C1.25 ng1.25 ng

Sample 3

Sample 3

Sample 11

Sample 11D

0.625 ng

0.625 ng

Sample 4

Sample 4

Sample 12

Sample 12E0.3125 ng 0.3125 ng

Sample 5

Sample 5

Sample 13

Sample 13

F

0.156 ng

0.156 ng

Sample 6

Sample 6

Sample 14

Sample 14G

0.078 ng

0.078 ng

Sample 7

Sample 7

Sample 15

Sample 15

H

Blank

Blank

Sample 8

Sample 8

Sample 16

Sample 16

Section C: Plate Coating1.

Be sure the 6 strips (48 wells) and strip holder have been warmed to room temperature.

2.

Prepare 1X Buffer W2 according to the Quick Chart for Preparing Buffers, which will beenough for the rest of the protocol.

3.

Before opening, quick spin the vial of DNA Binding Agent for 1 minute at room temperature
to collect contents to the bottom of the tube. Add 1 ml 1X Buffer W2 and vortex until fully
dissolved. Transfer the entire contents of the vial to a 15 ml conical tube. Add 5 ml 1X Buffer
W2 to the conical tube and vortex to mix.

4.

Add 100 μl of the reconstituted DNA Binding Agent to each well. Cover the wells with theadhesive plate sealer. Incubate at room temperature for 1 hour or overnight at 4° C.

5.

Remove the plate sealer and discard the DNA Binding Agent from the wells of the plate bywww.activemotif.com

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inverting the plate over a liquid waste receptacle. Then tap the inverted plate on absorbent
paper towels untill the excess liquid is removed from the wells.
6.

To wash the wells add 150 μl 1X Buffer W2 to each well and immediately discard by inverting the plate over a liquid waste receptacle. Then tap the inverted plate on absorbent paper
towels untill the excess liquid is removed from the wells.

7.

Repeat the wash 2 more times for a total of three washes.

8.

Tap the inverted plate 3-5 times on absorbent paper towels to remove all the liquid. The
plate can be used immediately or allowed to air dry inverted for up to 4 hours. If it cannot
be used the same day seal and place at 4°C overnight for use the next day.

Section D: Standard Curve Preparation
A standard curve will be created by making serial dilutions of the 5-hmC DNA standard. Enoughdilution will be made for each in duplicate.
1.

In a new microcentrifuge tube, create a 5 ng/μl working stock of the 5-hmC DNA Standard
by performing a 1:20 dilution, combining 2 μl 5-hmC DNA Standard (20X, 100 ng/μl) with 38
μl Buffer W2. Pipet up and down 5-7 times to mix.

3.

Take out one strip of the 0.2 ml PCR Stripwell tubes. Pipet 245 μl Buffer W2 into the firsttube of the strip. Then pipet 110 μl Buffer W2 into the seven remaining tubes.

4.

To perform the standard curve serial dilution, transfer 5 μl of the 5 ng/μl working stock to
the first tube of the strip (contains 245 μl Buffer W2) to make a concentration of 0.1 ng/ul.
Pipet up and down 5-7 times to mix contents, then transfer 110 μl of this mixture to the next
tube. Pipet up and down 5-7 times to mix contents. Then transfer 110 μl to next well. Repeat
in this manner through the seventh tube. The last tube (tube 8) is buffer only and will serveas the assay blank.

Section E: DNA Sample Preparation
First be sure that the DNA concentration of each MseI digested sample was quantified as described at the end of Section A.
Digested DNA samples will be diluted with 1X Buffer W2 for a final concentration of 5-30 ng/wellwith a final volume of 50 µl per well. It is important that samples fall within the linear range of the
DNA Standards for accurate quantification. It may be necessary to test several DNA concentrations to determine the optimal amount for the sample type. For tissue samples containing highlevels of 5-hmC (ex. brain) we recommend starting with 5-10 ng/well. For tissue samples containing lower levels of 5-hmC, a concentration of 10-30 ng/well is recommended. Our data indicatethat the best agreement between ELISA and mass spectrometry was obtained for all tissue types
when 10 ng/well of input DNA was used.
DNA will be prepared in the 0.2 ml PCR stripwell tubes for heat denaturation in a thermal cycler.

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1.

Label a 0.2 ml stripwell PCR tube for each sample DNA. 16 samples will require four strips(16 tubes).

2.

Each sample assay requires 50 μl of sample. We recommend making up a total volume of
110 μl in order to make enough for each sample in duplicate plus extra to account for pipetting loss.
Example: If the sample assay is to to contain 5 ng/well add 11 ng MseI digested DNA to the
0.2 ml stripwell PCR tube and bring up final volume to 110 μl with Buffer W2. For 10 ng/well,
add 22 ng MseI disgested DNA and bring up final volume to 110 μl with Buffer W2. Pipet up
and down 5-7 times to mix.

3.

Cap tubes and briefly vortex to mix. Collect liquid to the bottom of tubes

4.

Place samples in a thermal cycler and incubate at 98° C for 10 minutes to denature DNA.

5.

Immediately plunge samples into an ice-water bath for at least 5 minutes. Keep samples on
ice until ready to add single stranded DNA to the coated plate in Section F.

Section F: Binding DNA to the Coated Plate
1.

Quick spin the heat denatured samples to collect any condensate.

2.

Add 50 μl of the standards, blanks, and denatured DNA samples in duplicate to the coatedassay wells according to the plate layout. Ensure the bottom of the wells are completely
covered.

3.

Cover the plate with adhesive plate sealer and incubate at 37°C for 2 hours.

4.

During this incubation, prepare the 5-Hydroxymethylcytosine pAb dilution as described
below. Be sure the Antibody Dilution Buffer is brought to room temperature before use.

Prepare a 1:2,000 dilution of the 5-hydroxymethylcytosine pAb into Antibody Dilution Buffer byperforming a two-step dilution. First, prepare a 1:10 dilution into Antibody Dilution Buffer. Then
prepare a 1:200 dilution into the Antibody Dilution Buffer.
Preparing 5-Hydroxymethylcytosine pAb

Initial 1:10 Dilution

Secondary 1:200 Dilution

Components

6 strips (48 wells)

5-hmC pAb

2 μl

Antibody Dilution Buffer

18 μl

Total Volume

20 μl

5-hmC pAb pre-diluted 1:10

13 μl

Antibody Dilution Buffer
Total Volume

2.587 ml
2.6 ml5.

Remove the plate sealer and discard liquid by quickly inverting the plate over a liquid waste
receptacle. Then, tap the inverted plate on absorbent paper towels to remove excess.

6.

Add 150 μl Buffer W2 to each well and immediately discard as described above.

7.

Repeat the wash 2 more times for a total of three washes.
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12Section G: Binding of Primary Antibody
1.

Add 50 μl of 1:2,000 diluted 5-Hydroxymethylcytosine pAb to each well being used, including blank wells. Cover the plate with plate sealer and incubate for 1 hour at room temperature with mild agitation (200-300 rpm on an orbital shaker).

2.

During this incubation, prepare the Anti-rabbit HRP-conj. secondary Ab dilution as described
below. Be sure the Antibody Dilution Buffer is brought to room temperature before use.

3.

Prepare the 1X Wash Buffer as specified in the Quick Chart for Preparing Buffers.

Prepare a 1:4,000 dilution of anti-rabbit HRP-conj. secondary antibody into Antibody Dilution
Buffer. We recommend performing a two-step dilution by first preparing a 1:10 dilution into Antibody Dilution Buffer. Then prepare a 1:400 dilution into Antibody Dilution Buffer for the required
amount of antibody needed for the experiment.
Preparing Anti-rabbit HRP-conj. secondary Ab
Components
Initial 1:10 Dilution

6 strips (48 wells)

Anti-rabbit HRP-conj. secondary Ab

2 μl

Antibody Dilution Buffer

18 μl

Total Volume

20 μl

Anti-rabbit HRP-conj. pre-diluted 1:10

6.5 μlSecondary 1:400 Dilution Antibody Dilution Buffer
Total Volume

2.594 ml
2.6 ml5.

Remove the plate sealer and discard liquid by quickly inverting the plate over a liquid waste
receptacle. Then, tap the inverted plate on absorbent paper towels to remove excess.

6.

Add 150 μl 1X Wash Buffer to each well and immediately discard as described above.

7.

Repeat the wash 2 more times for a total of three washes.

Section H: Binding of Secondary Antibody
1.

Add 50 μl of 1:4,000 diluted Anti-rabbit HRP-conj. secondary Ab to each well being used,
including blank wells. Cover the plate with plate sealer and incubate for 1 hour at room
temperature with mild agitation (200-300 rpm on an orbital shaker).

2.

During this incubation, place the Developing and Stop Solutions at room temperature.

3.

Remove the plate sealer and discard liquid by quickly inverting the plate over a liquid waste
receptacle. Then, tap the inverted plate on absorbent paper towels to remove excess.

4.

Add 150 μl 1X Wash Buffer to each well and immediately discard as described above.

5.

Repeat the wash 2 more times for a total of three washes.

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13Section I: Colorimetric Reaction
1.

Remove as much of the final wash as possible by blotting the plate on paper towels.

2.

Add 100 µl of room temperature Developing Solution to all wells being used.

3.

Please read the Certificate of Analysis supplied with this kit for optimal development timeassociated with this lot. Monitor the blue color development in the standard curve wells sothat the color gradient goes from light blue in the lowest concentration well to dark blue inthe highest concentration well. The Blank wells should remain clear. Do not overdevelop.
a. For high abundance samples, 5-7 minute development should be adequate.
b. For low abundance samples, development time should be 6-8 minutes. However, customers may choose to increase development time to as much as 20-22 minutes to increasesignal in the lower range of the standard curve. In this case one or more upper points of thestandard curve may need to be discarded.

4.

Add 100 µl of Stop Solution to all wells. In presence of the acid, the blue color turns yellow.

5.

Read absorbance on a spectrophotometer within 5 minutes at 450 nm with an optionalreference wavelength of 655 nm. Blank the plate reader according to the manufacturer’s
instructions using the blank wells.

Section J: Analysis of Results
Generation of Standard Curve
A standard curve can be generated by plotting the raw absorbance values of the 5-hmC Standard
serial dilutions. We recommend using curve fitting software to achieve the best fit line. For
longer development times, the 5 ng standard may need to be dropped as shown in the example
below.

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14

Calculation of % 5-hmC from Raw Absorbance Data
% 5-hmC of samples can be calculated from the standard curve as follows:First calculate mass of 5-hmC in ng in the sample:
ng 5-hmC =(Sample OD - Y-intercept)(Slope* 9.5†)

To obtain % 5-hmC:% 5-hmC =(ng 5-hmC)(ng DNA input)*100

9.5 is a normalization factor to normalize 5-hmC in the standard to 100%, since the oligo contains 9.5% 5-hmC†

Calculation of moles of 5-hmC from Raw Absorbance Data
Moles of 5-hmC in samples can be calculated from the standard curve by plotting the standards
as moles of 5-hmC against the raw absorbance values. See the table below for the conversion ofng 5-hmC Standard to moles 5-hmC. This assumes a 50 μl volume added per well.
ng 5-hmC

moles 5-hmC

Molarity, 5-hmC (in 50 ul standard volume)5

1.55E-12

3.10E-08

2.5

7.75E-13

1.55E-08

1.25

3.87E-13

7.75E-09

0.625

1.94E-13

3.87E-09

0.3125

9.68E-14

1.94E-09

0.15625

4.84E-14

9.68E-10

0.078125

2.42E-14

4.84E-10

Downloadable Data Analysis Template
For convenience we have created a data analysis Excel template where you can copy and paste
in your raw absorbance values and nanograms of DNA per well and it will calculate both absoluteand relative quantification of your unknown samples. It is available for download under theDocuments tab on the product page.

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Appendix
Section K: Troubleshooting Guide
Problem/question

Possible cause

Recommendation

No signal or weak signal in
all wells

Omission of key
reagent

Check that all reagents have been added in all wells in thecorrect order.

Substrate or conjugate
is no longer active

Test conjugate and substrate for activity by mixing a smallaliquot of Anti-rabbit HRP-conj. secondary Ab 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 absorbance wavelength (450 nm) of the plate reader

Incorrect assay
temperature

Bring Developing Solution and Stop Solution to room temperature before using

Inadequate volume ofDeveloping Solution

Check to make sure that correct volume is delivered by pipette

Developing time too
long

Stop enzymatic reaction as soon as the positive wells turn
medium-dark blue or the blank wells turn a faint blue color

Concentration of antibodies is too highIncrease antibody dilutions

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. Ensure no residual wash buffer remains priorto addition of developing solution

Well cross-contamination

Follow washing recommendations

Inaccurate pipetting

Verify that the same amount of sample, primary antibody,
secondary antibody, developing and stop solutions are added
to each well

Too much sample
per wellDecrease amount of sample per well. Titrate the amount ofgenomic DNA to use per well such that it falls within the rangeof the DNA standards. We suggest a range of 5-30 ng/well

Concentration of antibodies is too high

Perform antibody titration to determine optimal working concentration. Start using 1:4000 for primary antibody and 1:5000
for the secondary antibody. The sensitivity of the assay will bedecreased

No signal or weak signal
in sample wells

Not enough sample
per wellIncrease amount of sample per well. Titrate the amount ofgenomic DNA to use per well such that it falls within the rangeof the DNA standards. We suggest a range of 5-30 ng/well

No signal or weak signal
in standard curve wells

Omission of key
reagent

Ensure that all reagents have been added to the wells in thecorrect order. Verify that DNA standards were prepared as
instructed in Section D of the manual.

High background in all
wells

Uneven color
development between
replicates

Signal in sample well beyond the detectable range
for the microplate reader

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16

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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17