Histone H3 monomethyl Lys4 ELISA
Catalog No. 53101

Histone H3 dimethyl Lys4 ELISA
Catalog No. 53112

Histone H3 trimethyl Lys4 ELISA
Catalog No. 53113(version B2)
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, California 92008, USA
Toll free:877 222 9543
Telephone:
760 431 1263
Fax:
760 431 1351
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UK Free Phone: 0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:		+32 (0)2 653 0001
Fax:		
+32 (0)2 653 0050
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone:+81 3 5225 3638
Fax:
+81 3 5261 8733
Copyright 2010 Active Motif, Inc.
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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.
© 2010 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
Kit Performance and Benefits  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 4Histone H3 monomethyl Lys4 ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Histone H3 dimethyl Lys4 ELISA  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Histone H3 trimethyl Lys4 ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Kit Components and Storage
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
ELISA Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Appendix
Section A. Preparation of Acid Extract/Crude Histones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Section B. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  18
Section C. Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  19
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

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Overview
The addition or removal of modifications such as phospho-, methyl- and acetyl- functional groups
to histones can have a profound effect on nuclear signaling as these dynamic modifications are
critical in the regulation of transcription, chromosome packaging, DNA damage repair and functional genomics. Screening extracts for specific histone modifications is a simple way to assess cell
health and the effect of treatment compounds on cell division.
The Histone H3 methylated Lys4 ELISAs are a simple solution for screening levels of mono-, di- ortrimethyl lysine 4 on histone H3 in human (other wide range predicted based on evolutionary
conservation) systems. These kits are sandwich ELISAs that utilize a Histone H3 monoclonal antibody to capture histone H3 from your samples and a rabbit polyclonal Histone H3 mono-, di- ortrimethyl Lys4 antibody for specific detection. A secondary antibody conjugated to horseradish
peroxidase (HRP) and developing solutions provide a sensitive colorimetric readout that is easily
quantified by spectrophotometry. The assay is performed in a convenient 96-stripwell plate,enabling you to simultaneously screen from 1 to 96 samples in a single experiment. It works withacid extracts from tissue or cell samples and is able to detect monomethyl or dimethyl histone H3
on lysine 4 from 625 nanograms of core histone preparation and 2.5 micrograms of acid extract.
Trimethyl histone H3 on lysine 4 can be detected in as little as 250 nanograms of core histone
preparations and 10 micrograms of acid extract.
For added convenience and a more quantitative interpretation of results, the histone methylation
ELISA kits all include Active Motif’s recombinant methylated histone technology. Each methylated
histone ELISA kit is supplied with a 99% pure Histone H3 recombinant protein that has been specifically methylated at the desired lysine site. The included Recombinant Histone H3 mono-, di- ortrimethyl Lys4 protein enables you to build a reference standard curve to quantitate the amountof specifically methylated H3 Lys4 in your samples.

product

formatcatalog no.

Histone H3 monomethyl Lys4 ELISA

1 x 96 rxns

53101

Histone H3 dimethyl Lys4 ELISA

1 x 96 rxns

53112

Histone H3 trimethyl Lys4 ELISA

1 x 96 rxns

53113

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1Flow Chart of Process

1

5

4

3

2

7

6

8

9

10

11

12

A
B
C
D
E
F

Sample containing
core histones oracid extracted histones

G
H

Histone H3 antibody
coated plate

Add sample
to the plate
21

11

01

9

8

7

6

5

4

3

2

1
A
B

C
D
E
F
G
H

1 hr.

2

1

9

8

7

6

5

4

3

11

10

12

A
B
C
D
E
F
G

Add modification specificprimary antibody

H

1 hr.
21

11

01

9

8

7

6

5

4

3

2

1
A

Add anti-IgG
HRP conjugate

B
C
D
E
F
G

Add developing
and stop solution

H

1 hr.
2

1

5

4

3

7

6

A
B
C
D
E
F
G
H

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2

8

9

10

11

12

Introduction
Histone H3 methylated Lys4
The basic structural unit of chromatin is the nucleosome, which consists of 146 base pairs (bp) of
DNA wrapped around a histone octamer. The histone octamer consists of two copies each of thecore histone H2A-H2B dimers and a tetramer of H3-H4. A linker histone, histone H1, binds chromatin outside the nucleosome unit to regulate chromatin structure.
Histone modifications such as phosphorylation, acetylation and methylation at specific amino acidresidues on the histone tails that extend beyond the core nucleosome have been found to influence and regulate transcription, chromosome packaging and DNA damage repair. Many of these
specific histone modifications are conserved throughout eukaryotes. While the biological significance of some histone modifications remains to be understood, some have been demonstrated tocorrelate very closely with specific cellular states like transcriptional activity1,2.
The methylation of lysine 4 (K4) on histone H3 has been correlated with the activation of genetranscription3, making methylated lysine 4 on histone H3 a significant marker in studying the stateof transcription activity.
Active Motif’s Histone Modification ELISA Kits make it is easy to screen for changes in methylationlevels. Histone H3 mono-, di- or trimethyl Lys4 ELISA works with acid extracts from tissue or cellsamples as well as purified core histones, such as those isolated using Active Motif’s Histone Purification Kits (Catalog Nos. 40025 & 40026). The sensitive, specific assay is able to detect methylated
histone H3 lysine 4 in less than 3.5 hours. As this assay is performed in a 96-stripwell plate, a large
number of samples can be handled simultaneously, allowing for high-throughput automation.
Histone Modification ELISAs have many applications including screening the effects of compounds
on the methylation levels of histone H3 lysine residues.

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3Kit Performance and Benefits
Range of detection:
Histone H3 monomethyl Lys4: This ELISA provides quantitative results from 625 ng to 10 µgof purified core histones or from 2.5 µg to 20 µg of histones isolated by acid extraction.
The linear range of the provided Recombinant Histone H3 monomethyl Lys4 protein isapproximately from 0 to 125 ng/well.
Histone H3 dimethyl Lys4: This ELISA provides quantitative results from 625 ng to 10 µg of
purified core histones or from 2.5 µg to 20 µg of histones isolated by acid extraction.
The linear range of the provided Recombinant Histone H3 dimethyl Lys4 protein is approximately from 0 to 100 ng/well.
Histone H3 trimethyl Lys4: This ELISA provides quantitative results from 250 ng to 10 µg of
purified core histones or from 10 µg to 20 µg of histones isolated by acid extraction.
The linear range of the provided Recombinant Histone H3 trimethyl Lys4 protein isapproximately from 0 to 50 ng/well.
Cross-reactivity: Human and a wider range of species reactivity is predicted due to the highdegree of sequence homology of histone H3.
Assay time: 3.5 hours.

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4Histone H3 monomethyl Lys4 (OD 450nm)

Histone H3 monomethyl Lys4 ELISA Kit
Histone H3 monomethyl Lys4 ELISA

2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0

3.9

8.8

15.6

31.25

62.5

125

250

Recombinant Histone H3 monomethyl Lys4 protein(provided positive control)

10000

10000

Purified HeLa
Core
Acid
Histones Extract

Sample (ng/well)

Histone H3 monomethyl Lys4 detection.
The Histone H3 monomethyl Lys4 ELISA was used to assay purified HeLa core histones (10 µg) made using Active Motif’s
Histone Purification Mini Kit (Catalog No. 40026) and HeLa acid extracts (10 µg) prepared as stated in Appendix Section A.
The provided Recombinant Histone H3 monomethyl Lys4 protein was assayed from 3.9 - 250 ng/well as a reference standard curve. Data shown are the results from wells assayed in duplicate. These results are provided for demonstration only.

Histone H3 monomethyl Lys4 ELISA
Histone H3 monomethyl Lys4 (OD 450nm)

1.6
1.4

Recombinant Histone H3
Recombinant Histone H3 monomethyl Lys4
Recombinant Histone H3 dimethyl Lys4
Recombinant Histone H3 trimethyl Lys4

1.2
1
0.8
0.6
0.4
0.2
0

0

3.9

7.8

15.6

31.25

62.5

125

250

Recombinant protein (ng/well)

Histone H3 monomethyl Lys4 specificity.
Recombinant Histone H3, mono-, di- and trimethyl Lys4 proteins were assayed from 3.9 ng - 250 ng per well using theHistone H3 monomethyl Lys4 ELISA. These results indicate the specificity of the assay. There is extremely low backgroundfrom histone H3 and little cross-reactivity for di- or trimethyl Lys4. This means that small, specific changes in monomethyl
Lys4 levels can easily be detected with this kit.

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5

Histone H3 dimethyl Lys4 ELISA Kit

Histone H3 dimethyl Lys4 (OD 450nm)

Histone H3 dimethyl Lys4 ELISA
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

0

1.56

3.125
6.25
12.5
25
Recombinant Histone H3 monomethyl Lys4 protein(provided positive control)

50

100

10000
10000
Purified HeLa Acid
Core
Extract
Histones

Sample (ng/well)

Histone H3 dimethyl Lys4 detection.
The Histone H3 dimethyl Lys4 ELISA was used to assay purified HeLa core histones (10 µg) made using Active Motif’s Histone Purification Mini Kit (Catalog No. 40026) and HeLa acid extracts (10 µg) prepared as stated in Appendix Section A. The
provided Recombinant Histone H3 dimethyl Lys4 protein was assayed from 1.56 - 100 ng/well as a reference standard curve.
Data shown are the results from wells assayed in duplicate. These results are provided for demonstration only.

Histone H3 dimethyl Lys4 ELISA

Histone H3 dimethyl Lys4 (OD 450nm)

1.2

Recombinant Histone H3
Recombinant Histone H3 monomethyl Lys4

1

Recombinant Histone H3 dimethyl Lys4
Recombinant Histone H3 trimethyl Lys4

0.8
0.6
0.4
0.2
0

0

1.56

3.125

6.25

12.5

25

50

100

Recombinant protein (ng/well)

Histone H3 dimethyl Lys4 specificity.
Recombinant Histone H3, mono-, di- and trimethyl Lys4 proteins were assayed from 1.56 ng - 100 ng per well using theHistone H3 dimethyl Lys4 ELISA. These results indicate the specificity of the assay. There is extremely low backgroundfrom histone H3 and little cross-reactivity for trimethyl Lys4. Histone H3 monomethyl Lys4 has approximately 25% crossreactivity with the dimethyl antibody. This signal can be normalized if the monomethyl and dimethyl Lys4 ELISA kits are
run in parallel.

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6

Histone H3 trimethyl Lys4 ELISA Kit
Histone H3 trimethyl Lys4 ELISA
Histone H3 trimethyl Lys4 (OD 450nm)

2.5
2
1.5
1
0.5
0

0

1.56

3.125
6.25
12.5
25
50
Recombinant Histone H3 trimethyl Lys4 protein(provided positive control)

100

10000
PurifiedCore
Histones

Sample (ng/well)

10000
HeLa
Acid
Extract

Histone H3 trimethyl Lys4 detection.
The Histone H3 trimethyl Lys4 ELISA was used to assay purified HeLa core histones (10 µg) made using Active Motif’s Histone Purification Mini Kit (Catalog No. 40026) and HeLa acid extracts (10 µg) prepared as stated in Appendix Section A. The
provided Recombinant Histone H3 trimethyl Lys4 protein was assayed from 1.56 - 100 ng/well as a reference standard curve.
Data shown are the results from wells assayed in duplicate. These results are provided for demonstration only.

Histone H3 trimethyl Lys4 ELISA
1.8
Recombinant Histone H3

Histone H3 trimethyl Lys4 (OD 450nm)

1.6

Recombinant Histone H3 monomethyl Lys4

1.4

Recombinant Histone H3 dimethyl Lys4

1.2

Recombinant Histone H3 trimethyl Lys4

1
0.8
0.6
0.4
0.2
0

0

1.56

3.125

6.25

12.5

25

50

100

Recombinant protein (ng/well)

Histone H3 trimethyl Lys4 specificity.
Recombinant Histone H3, mono-, di- and trimethyl Lys4 proteins were assayed from 1.56 ng - 100 ng per well using theHistone H3 trimethyl Lys4 ELISA. These results indicate the specificity of the assay. There is extremely low background from
histone H3 and little cross-reactivity for mono- or dimethyl Lys4. This means that small, specific changes in trimethyl Lys4
levels can easily be detected with this kit.

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7

Kit Components and Storage
Histone H3 methylated Lys4 ELISA 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

Histone H3 monomethyl Lys4 antibody, orHistone H3 dimethyl Lys4 antibody, orHistone H3 trimethyl Lys4 antibody

11 µl
11 µl
11 µl

4°C
4°C
4°C

HRP-conjugated anti-rabbit IgG

6 µl

4°C

Assay Dilution Buffer

15 ml

4°C

20X Wash Buffer

25 ml

4°C

Developing Solution

11 ml

4°C

Stop Solution

11 ml

4°C

Recombinant Histone H3 monomethyl Lys4, or
10 µg
Recombinant Histone H3 dimethyl Lys4, or(1 µg/µl)
Recombinant Histone H3 trimethyl Lys4		

-80°C

Histone H3 Capture Plate

14°C

Plate sealer

1

RT

Additional materials required• Histone samples (recombinant, purified or acid extracted)• Multi-channel pipettor• Multi-channel pipettor reservoirs• Rocking platform/orbital shaker• Microplate spectrophotometer capable of reading at 450 nm (655 nm as optional reference
wavelength)www.activemotif.com

8

Protocols
Buffer Preparation and Recommendations
Assay Dilution Buffer
Assay Dilution Buffer is provided as a 1X solution and is ready for use once thawed.
20X Wash Buffer (See the Quick Chart for Preparing Buffers in this Section.)
Prepare the amount of 1X Wash Buffer required for the assay as follows: For every 100 ml of 1X
Wash Buffer required, dilute 5 ml 20X Wash Buffer with 95 ml sterile water. 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 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 antibodies (See the Quick Chart for Preparing Buffers in this Section.)
Dilute the Histone H3 monomethyl Lys4 antibody 1:1000 with Assay Dilution Buffer AM2. Use 50 µl per
well. Dilute the anti-rabbit HRP-conjugated secondary 1:2000 with Assay Dilution Buffer. Use 50 µl
per well.
Dilute the Histone H3 dimethyl Lys4 antibody 1:2000 with Assay Dilution Buffer AM2. Use 50 µl per well.
Dilute the anti-rabbit HRP-conjugated secondary 1:5000 with Assay Dilution Buffer. Use 50 µl per
well.
Dilute the Histone H3 trimethyl Lys4 antibody 1:1000 with Assay Dilution Buffer. Use 50 µl per well.
Dilute the anti-rabbit HRP-conjugated secondary 1:2000 with Assay Dilution Buffer. Use 50 µl per
well.
Developing Solution (See the Quick Chart for Preparing Buffers in this Section.)
100 µl are needed per well. 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 overtime. This does not affect product performance. However, a blue color present in the Developing
Solution indicates that it has been contaminated and must be discarded. Prior to use, place theDeveloping Solution at room temperature for at least 1 hour. Transfer the amount of Developing
Solution required for the assay into a secondary container before aliquoting into the wells. Afteruse, discard remaining Developing Solution.
Stop Solution (See the Quick Chart for Preparing Buffers in this Section.)
100 µl are needed per well. Prior to use, transfer the amount of Stop Solution required for theassay into a secondary container. After use, discard remaining Stop Solution.
WARNING: The Stop Solution is corrosive. Wear personal protective equipment whenhandling, i.e. safety glasses, gloves and labcoat.

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9

Diluting Recombinant Histone H3 methylated Lys4 protein
The Recombinant Histone H3 mono-, di- or trimethyl Lys4 protein is provided as a control for
quantitating the amount of methylated histone H3. There is enough recombinant protein for atleast 2 standard curves. During the first use, we recommend making aliquots of the stock protein
and storing at -80°C to avoid multiple freeze/thaw cycles.
Preparing histone samples
Histone samples can be prepared using several techniques:1.

A simple acid extraction (see Appendix Section A) is recommended instead of a nuclear
extraction as histones are soluble in acidic solutions and many nuclear extraction procedures often exclude histones from the final sample. The acid extraction will provide crude
histones.

2.

Purified core histones, such as those obtained from Active Motif’s Histone Purification Kits
(Catalog Nos. 40025 & 40026) produce distinct, clean core histone samples as determined bygel electrophoresis.

3.

More stringent purification techniques use a hydroxyapatite column to provide highly pure,core histone samples, such as Active Motif’s HeLa core Histones (Catalog No. 53501).

Regardless of the histone preparation technique, it is recommended initially to use a range ofsample concentrations (e.g. 5 ng, 50 ng, 500 ng, 5 µg) in order to determine the amount of sample
necessary to fall within the linear area of the reference curve. Once the protein concentration forthe linear area of the reference curve has been determined, perform the rest of the assays within
the linear range.

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10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)

H3K4 monomethyl pAb

Histone H3 monomethyl Lys4 Ab
Assay Dilution Buffer
TOTAL REQUIRED

0.05 µl
52 µl
52 µl

0.45 µl
450 µl450 µl

2.7 µl
2.7 ml
2.7 ml5.4 µl5.4 ml5.4 mlSecondary Antibody

HRP-conjugated anti-rabbit IgG
Assay Dilution Buffer
TOTAL REQUIRED

0.026 µl
52 µl
52 µl

0.23 µl
450 µl450 µl1.35 µl
2.7 ml
2.7 ml

2.7 µl5.4 ml5.4 ml

H3K4 dimethyl pAb

Histone H3 dimethyl Lys4 Ab
Assay Dilution Buffer
TOTAL REQUIRED

0.026 µl51.9 µl52 µl0.23 µl
450 µl450 µl1.35 µl
2.7 ml
2.7 ml

2.7 µl5.4 ml5.4 mlSecondary Antibody

HRP-conjugated anti-rabbit IgG
Assay Dilution Buffer
TOTAL REQUIRED

0.01 µl
52 µl
52 µl

0.09 µl
450 µl450 µl0.54 µl
2.7 ml
2.7 ml1 µl
5.4 ml5.4 ml

H3K4 trimethyl pAb

Histone H3 trimethyl Lys4 Ab
Assay Dilution Buffer
TOTAL REQUIRED

0.05 µl51.95 µl52 µl0.45 µl
450 µl450 µl

2.7 µl
2.7 ml
2.7 ml5.4 µl5.4 ml5.4 mlSecondary Antibody

HRP-conjugated anti-rabbit IgG
Assay Dilution Buffer
TOTAL REQUIRED

0.026 µl
52 µl
52 µl

0.23 µl
450 µl450 µl1.35 µl
2.7 ml
2.7 ml

2.7 µl5.4 ml5.4 ml

1X Wash Buffer

Distilled water
20X Wash Buffer
TOTAL REQUIRED

1.9 ml100 µl2 ml

17.1 ml
0.9 ml
18 ml

95 ml
5 ml
100 ml

190 ml
10 ml
200 mlDeveloping Solution

TOTAL REQUIRED

112.5 µl

900 µl5.4 ml10.8 ml

Stop Solution

TOTAL REQUIRED

112.5 µl

900 µl5.4 ml10.8 mlwww.activemotif.com

11

ELISA 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 the 1X Wash Buffer as described above in the section Buffer Preparation and Recommendations. Multi-channel pipettor reservoirs may be used for dispensing the Wash Buffer, Assay
Dilution Buffer, Developing Solution and Stop Solution into the wells being used.

Standard Curve Preparation for H3 monomethyl Lys4
Use this plate set-up example to prepare a standard curve for the Histone H3 monomethyl Lys4
(H3 K4me1) Kit in duplicate.
H3 K4me1

1.

1

2

3

4

5

6

7

8

9

10

11

12

A

250 ng

250 ng

–

–

–

–

–

–

–

–

–

–

B

125 ng

125 ng

–

–

–

–

–

–

–

–

–

–

C

62.5 ng62.5 ng

–

–

–

–

–

–

–

–

–

–

D

31.25 ng31.25 ng

–

–

–

–

–

–

–

–

–

–

E

15.6 ng

15.6 ng

–

–

–

–

–

–

–

–

–

–

F

7.8 ng

7.8 ng

–

–

–

–

–

–

–

–

–

–

G

3.9 ng

3.9 ng

–

–

–

–

–

–

–

–

–

–

H

0 ng

0 ng

–

–

–

–

–

–

–

–

–

–

Recombinant Histones are provided at a 1 µg/µl concentration. Thaw the protein on ice.
Before using, vortex to the tube for 10 seconds and quick spin the contents to the bottom of
the tube.
Dilute the Recombinant Histone H3 monomethyl Lys4 to a starting concentration of
5 ng/µl by adding 1.25 µl of Recombinant Histone H3 monomethyl Lys4 to 250 µl of Assay
Dilution Buffer. The result will be 1.25 µg/250 µl = 5 ng/µl. Mix well by vortexing.

2.

Add 100 µl of the diluted Recombinant Histone to wells A1 and A2. Discard any unused
diluted Recombinant Histone.

3.

Add 50 µl of Assay Dilution Buffer to wells B1 through H2.
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12

4.

Perform a serial two-fold dilution of the extracts by transferring 50 µl of the extracts 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 50 µl of the contents of row B to row C and mix, as previously described.

7.

Continue this process until row G is reached.

8.

When row G is reached, discard 50 µl of the well contents so that the final volume is 50 µl.

9.

Row H will serve as the blank wells.

Standard Curve Preparation for H3 di- or trimethyl Lys4
Use this plate set-up example to prepare a standard curve for the Histone H3 dimethyl Lys4 (H3
K4me2) or trimethyl Lys4 (H3 K4me3) Kit in duplicate.
H3 K4me2 orH3 K4me3

1.

1

2

3

4

5

6

7

8

9

10

11

12

A

100 ng

100 ng

–

–

–

–

–

–

–

–

–

–

B

50 ng

50 ng

–

–

–

–

–

–

–

–

–

–

C

25 ng

25 ng

–

–

–

–

–

–

–

–

–

–

D

12.5 ng

12.5 ng

–

–

–

–

–

–

–

–

–

–

E

6.25 ng

6.25 ng

–

–

–

–

–

–

–

–

–

–

F

3.125 ng

3.125 ng

–

–

–

–

–

–

–

–

–

–

G

1.56 ng

1.56 ng

–

–

–

–

–

–

–

–

–

–

H

0 ng

0 ng

–

–

–

–

–

–

–

–

–

–

Recombinant Histones are provided at a 1 µg/µl concentration. Thaw the protein on ice.
Before using, vortex to the tube for 10 seconds and quick spin the contents to the bottomof the tube. During the first use we recommend making 3 µl aliquots of the stock protein for
future standard curves and storing at -80°C to avoid multiple freeze/thaw cycles.
Dilute the Recombinant Histone H3 di- or trimethyl Lys4 to a starting concentration of
2 ng/µl by adding 2 µl of Recombinant Histone H3 di- or trimethyl Lys4 to 1 ml of Assay Dilution Buffer. The result will be 2 µg/1000 µl = 2 ng/µl. Mix well by vortexing.

2.

Add 100 µl of the diluted Recombinant Histone to wells A1 and A2. Discard any unused
diluted Recombinant Histone.

3.

Add 50 µl of Assay Dilution Buffer to wells B1 through H2.

4.

Perform a serial two-fold dilution of the extracts by transferring 50 µl of the extracts in row
www.activemotif.com

13

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

7.

Continue this process until row G is reached.

8.

When row G is reached, discard 50 µl of the well contents so that the final volume is 50 µl.

9.

Row H will serve as the blank wells.

Step 1: Binding of H3 to the Capture Plate
1.

In duplicate, prepare the amount of desired sample. It is recommended to try a range ofconcentrations in order to determine the amount of sample necessary to fall within thelinear range of the reference standard curve. Add desired amount of sample in 50 µl volume
to plate.
Purified core histones: For Histone H3 monomethyl Lys4, we recommend a range of 625 ng
to 10 µg. For Histone H3 dimethyl Lys4, we recommend a range of 625 ng - 10 µg. For Histone
H3 trimethyl Lys4, we recommend a range of 250 ng - 10 µg.
Acid extracts: Recommended range of 5 - 20 µg.

2.

Incubate plate containing the protein standard curve and samples for 1 hour at room
temperature with agitation on orbital shaker or rocking platform.

3.

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

Step 2: Binding of Primary Antibody
4.

Histone H3 monomethyl Lys4 antibody: Dilute 1:1000 in Assay Dilution Buffer and mix
thoroughly.
Histone H3 dimethyl Lys4 antibody: Dilute 1:2000 in Assay Dilution Buffer and mix
thoroughly.
Histone H3 trimethyl Lys4 antibody: Dilute 1:1000 in Assay Dilution Buffer and mix
thoroughly.

5.

Add 50 µl of diluted primary antibody to each well.

6.

Incubate at room temperature for 1 hour with agitation.

7.

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

Step 3: Binding of Secondary Antibody
8.

Histone H3 monomethyl Lys4 antibody: Dilute the HRP-conjugated anti-rabbit IgG antibody 1:2000 in Assay Dilution Buffer and mix thoroughly.
www.activemotif.com

14

Histone H3 dimethyl Lys4 antibody: Dilute the HRP-conjugated anti-rabbit IgG antibody
1:5000 in Assay Dilution Buffer and mix thoroughly.
Histone H3 trimethyl Lys4 antibody: Dilute the HRP-conjugated anti-rabbit IgG antibody
1:2000 in Assay Dilution Buffer and mix thoroughly.
9.

Add 50 µl of the diluted secondary antibody solution to each well.

10. Incubate at room temperature for 1 hour without agitation.
11.

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

12.

After the incubation, wash the wells 3 times with 200 µl of wash buffer.

Step 4: Colorimetric Reaction
13.

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

14.

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

15.

Incubate under low light conditions from 30 seconds to 5 minutes at room temperature
protected from direct light. Please read the Certificate of Analysis supplied with this kit for
optimal development time associated with this lot number. Monitor the blue color development in the protein standard curve wells containing the higher concentrations of Recombinant Histone H3 mono-, di- or trimethyl Lys4 protein until they turn medium to dark blue.
Do not overdevelop.

16.

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

17.

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 yourplate reader does not have this capability, you may read the plate twice, once at 450 nm andonce at 655 nm then manually subtract the 655 nm OD from the 450 nm OD values.
Calculation of results using the standard curve
To generate a standard curve using the included Recombinant Histone H3 monomethyl,
dimethyl or trimethyl Lys4 protein, average the duplicate readings for each standard,
control, and sample and subtract the optical density (OD) obtained from the zerostandard (Row H blank wells).
Plot the OD for the standards against the quantity (ng/well) of the standards and draw thebest fit curve. The best curve fit may vary depending on the developing times usedand should be calculated each time a standard curve is run. Recombinant Histone H3monomethyl Lys4 protein has an approximate linear range from 0 to 125 ng. Recombiwww.activemotif.com

15

nant Histone H3 dimethyl Lys4 protein has an approximate linear range from 0 to
100 ng. Recombinant Histone H3 trimethyl Lys4 protein has an approximate linear range
from 0 to 50 ng. The data can be linearized using log/log paper and regression analysis
may also be applied.
To quantify the amount of methyl Lys4 from histone H3 in the samples, find the absorbancevalue for the samples on the y-axis and extend a horizontal line to the standard curve.
At the intersection point extend a vertical line to the x-axis and read the corresponding standard value. Note: If the samples have been diluted, the value read from thestandard curve must be multiplied by the dilution factor.
Example curve:
The following standard curve is provided for demonstration only. A standard curveshould be made every time an experiment is performed.
Standard Curve (Linear Range 1.56-50 ng/well)
1.8
y = 0.0275x + 0.2486

1.6

R

2

= 0.9936

1.4

OD 450nm

1.2
1
0.8
0.6
0.4
0.2
0
0

10

20
30
40
Recombinant Histone H3 trimethyl Lys4 (ng/well)

References
1.
2.
3.

Kirmizis, A., et al. (2004) Genes & Dev. 18: 1592-1605.
Squazzo, S., et al. (2006) Genome Res. 16: 890-900.
Rinn, J., et al. (2007) Cell 129(7):1311-1323.

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16

50

60

Appendix
Section A. Preparation of Acid Extract/Crude histone proteins
This procedure can be used for a confluent cell layer of 150 mm plate. The yield is approximately
0.15 mg of acid soluble nuclear proteins from 9 x 106 cells.
1.

Grow HeLa cells to 70% confluency in DMEM with 10% FBS.

2.

Treat cells as desired.

3.

Wash cells with 1X PBS and aspirate.

4.

Add 3-5 ml of PBS per 150 mm plate.

5.

Scrape cells from the plate and transfer to a 50 ml conical tube.

6.

Pellet the cells by centrifugation in a pre-cooled 4°C rotor at 200 x g for 5-10 minutes.

7.

Aspirate as much of the PBS as possible without disturbing the cell pellet.

8.

Resuspend the cell pellet in 5 volumes of Lysis Buffer (see below).

9.

Incubate on ice for 30 minutes and occasionally invert the tube to mix.

10. Centrifuge the lysate at 11,000 x g for 10 minutes at 4°C.
11.

Collect the supernatant fraction containing acid soluble proteins, and discard the acid-insoluble pellet.

12.

Immediately neutralize the acid extracted proteins by adding 2/5 the total volume of Neutralization Buffer (see below).

13.

Quantify the protein concentration of your acid extraction using either gel electrophoresis
or a Bradford Assay.
Gel electrophoresis is a more sensitive technique to determine histone concentration ashistones are most effectively stained by Coomassie dye in a gel matrix. To determine theprotein concentration run a BSA or histone standard curve on the gel.
A Bradford Assay can be used to determine total protein concentration, not just the concentration of crude histone proteins. A total protein determination, however, is sufficient for
use in the Histone Modification ELISA Kits. The quantity of acid extract tested in the Histone
Modification ELISA Kits are based on total protein determination values.

14.

Immediately aliquot the extract in small volumes to avoid multiple freeze/thaws.

15.

Store the protein at -80°C for long-term stability.

Lysis Buffer:
0.4 M HCl
Neutralization Buffer:
1 M Sodium phosphate, dibasic, pH 12.5. Use 5 M NaOH to adjust the pH.
2.5 mM DTT (add just prior to use)
10 mM PMSF (add just prior to use)www.activemotif.com

17Section B: 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 order

Substrate or conjugate
is no longer active

Test conjugate and substrate for activity by mixing a smallaliquot of 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

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

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

Well cross-contamination

Follow washing recommendations

Too much sample
per wellDecrease amount of sample per well. For acid extracts, dilute
down to 1-2 µg/well and for purified core histone, dilute down
to 125 ng/well

Concentration of antibodies is too high

Perform antibody titration to determine optimal working
concentration. Start using 1:2000 - 1:5000 for primary antibody
and 1:5000 - 1:10,000 for the secondary antibody. The sensitivityof the assay will be decreased

No signal or weak signal
in sample wells

Not enough sample
per well

For purified core histones, increase to 500 ng per well. For
extracts, make sure you are using an acid extract by following
the protocol recommended in Appendix Section A. Increaseamount of acid extract to 20 µg/well

No signal or weak signal
in standard curve wells

Too many freeze/thaw
cycles of protein

During the kit’s first use, aliquot the stock recombinant protein
control into 3 µl aliquots and store at -80°C to avoid multiple
freeze/thaws

High background in all
wells

Uneven color
development

High background insample wells

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18Section C. Related Products
Histone ELISAs		FormatCatalog No.

Histone H3 monomethyl Lys4 ELISA		Histone H3 dimethyl Lys4 ELISA		Histone H3 trimethyl Lys4 ELISA		Histone H3 dimethyl Lys9 ELISA		Histone H3 trimethyl Lys9 ELISA		Histone H3 monomethyl Lys27 ELISA		Histone H3 trimethyl Lys27 ELISA		Histone H3 phospho Ser10 ELISA		Histone H3 phospho Ser28 ELISA		
Total Histone H3 ELISA		

1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns

53101
53112
53113
53108
53109
53104
53106
53111
53100
53110

Recombinant Methylated Histones		

FormatCatalog No.

Recombinant Histone H2A		
Recombinant Histone H2B		
Recombinant Histone H3 (C110A)		
Recombinant Histone H3 monomethyl Lys4		
Recombinant Histone H3 dimethyl Lys4		
Recombinant Histone H3 trimethyl Lys4		
Recombinant Histone H3 monomethyl Lys9		
Recombinant Histone H3 dimethyl Lys9		
Recombinant Histone H3 trimethyl Lys9		
Recombinant Histone H3 monomethyl Lys27		
Recombinant Histone H3 dimethyl Lys27		
Recombinant Histone H3 trimethyl Lys27		
Recombinant Histone H3 monomethyl Lys36		
Recombinant Histone H3 dimethyl Lys36		
Recombinant Histone H3 trimethyl Lys36		
Recombinant Histone H3 monomethyl Lys79		
Recombinant Histone H3 dimethyl Lys79		
Recombinant Histone H3 trimethyl Lys79		
Recombinant Histone H4		
Recombinant Histone H4 monomethyl Lys20		
Recombinant Histone H4 dimethyl Lys20		
Recombinant Histone H4 trimethyl Lys20		

50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg
50 µg

31251
31252
31207
31208
31209
31210
31211
31212
31213
31214
31215
31216
31217
31218
31219
31220
31221
31222
31223
31224
31225
31226

Control Acid Extracts		

FormatCatalog No.

HeLa acid extract		
HeLa acid extract (Paclitaxel treated)		
HeLa acid extract (Sodium Butyrate treated)		
HeLa acid extract (Etoposide treated)		
HeLa acid extract (Anacardic acid treated)		

100 µg100 µg100 µg100 µg100 µg

36200
36201
36202
36203
36204

Histone Purification		

FormatCatalog No.

Histone Purification Kit		
Histone Purification Mini Kit		

10 rxns
20 rxns

40025
40026

HeLa Core Histones		

36 µg

53501

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19Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USA
Toll Free:877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:tech_service@activemotif.com
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104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone:		
0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone: 		+32 (0)2 653 0001
Fax: 		
+32 (0)2 653 0050
E-mail: 		
eurotech@activemotif.com
Active Motif Japan
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2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:japantech@activemotif.com
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