Total Histone H3 ELISA(version A2)
Catalog No. 53110

Copyright 2009 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.
© 2009 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights
reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective
companies.
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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Performance and Benefits
Total Histone H3 ELISA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Components and Storage
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Protocols
Buffer Preparation and Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
ELISA Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Appendix
Section A. Preparation of Acid Extract/Crude Histones . . . . . . . . . . . . . . . . . . . . . . . . . 10
Section B. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

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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 Total Histone H3 ELISA is a simple solution for screening endogenous levels of histone H3 inhuman, mouse and rat systems. Total Histone H3 is a sandwich ELISA that utilizes a Histone H3
monoclonal antibody to capture histone H3 from your samples and a C-terminal rabbit polyclonal
Histone H3 antibody for 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 with acid extracts
from tissue or cell samples and is able to detect total histone H3 levels, regardless of the modification state, in as little as 15 nanograms of core histone and 60 nanograms 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. The Total Histone
H3 ELISA includes an unmodified Recombinant Histone H3 protein which enables you to build areference standard curve to quantitate the total amount of histone H3 in your samples.

product

formatcatalog no.

Total Histone H3 ELISA

1 x 96 rxns

53110

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1

Introduction
Total Histone H3
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
the core 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 aminoacid residues 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 ofthese specific histone modifications are conserved throughout eukaryotes. While the biologicalsignificance of some histone modifications remains to be understood, some have been demonstrated to correlate very closely with specific cellular states like transcriptional activity1,2.
Active Motif’s Histone Modification ELISA Kits make it is easy to screen for changes in histone H3levels. The Total Histone H3 ELISA works with acid extracts from tissue or cell samples as wellas purified core histones, such as those isolated using Active Motif’s Histone Purification Kits
(Catalog Nos. 40025 & 40026). The sensitive assay can detect total histone H3 levels in less than
3.5 hours. As this assay is performed in a 96-stripwell plate, a large number of samples can behandled simultaneously, allowing for high-throughput automation.
The Total Histone H3 ELISA has many applications, including screening total histone H3 levels
in response to various compounds. The Total Histone H3 Kit can also be used to normalize themethylation level of your samples when run in parallel with Active Motif’s Histone Modification
ELISAs for lysine 9 or lysine 27 methylation (Catalog Nos. 53106, 53108 & 53109). For normalization, the Total Histone H3 Kit and Histone Modification ELISA should be run in parallel. The
methylation specific data is then normalized by dividing it by the total histone H3 values.

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Kit Performance and Benefits
Detection limit: > 15 ng/well of purified core histones. For acid extracts, > 60 ng/well is recommended.
Range of detection: This ELISA provides quantitative results from 15 ng to 1 µg of purified core
histones or from 60 ng to 1 µg of acid extract. The linear range of the provided RecombinantHistone H3 protein is approximately from 1.5 to 50 ng/well.
Cross-reactivity: Human, mouse, rat, yeast, and a wider range of species reactivity is predicted
due to the high degree of sequence homology of histone H3.
Assay time: 3.5 hours.

Total Histone H3 ELISA Kit
Total Histone H3 ELISA
1.2

Histone H3 (OD450nm)

1
0.8
0.6
0.4
0.2
0

0

1.5625

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

50

100

62.5
125
Purified Core
Histones

500 1,000
HeLa Acid
Extract

Sample (ng/well)

Total Histone H3 detection.
The Total Histone H3 ELISA was used to assay purified HeLa core histones (62.5 - 125 ng) made using Active Motif’s Histone
Purification Mini Kit (Catalog No. 40026) and HeLa acid extracts (500 ng - 1 µg) prepared as stated in Appendix Section A. The
provided Recombinant Histone H3 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.

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3Kit Components and Storage
Total Histone H3 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

Total Histone H3 antibody

6 µl

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

10 µg
(1 µg/µl)-80°C

Histone H3 Capture Plate

14°C

Plate sealer

1

RT

Recombinant Histone H3 protein

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

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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
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 (see the Quick Chartfor Preparing Buffers in this section). 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 Total Histone H3 antibody 1:1000 with Assay Dilution Buffer. Use 50 µl per well.
Dilute the HRP-conjugated anti-rabbit IgG antibody 1:2000 with Assay Dilution Buffer. Use 50 µl
per well.
Developing Solution
The Developing Solution should be warmed to room temperature before use. The Developing
Solution is light sensitive, therefore, we recommend avoiding direct exposure to intense light during storage. The Developing Solution may develop a yellow hue over time. This does not affectproduct performance. However, a blue color present in the Developing Solution indicates thatit has been contaminated and must be discarded. Prior to use, place the Developing Solution atroom temperature for at least 1 hour. Transfer the amount of Developing Solution required for theassay into a secondary container before aliquoting into the wells (see the Quick Chart for Preparing Buffers in this section). After use, discard remaining Developing Solution.
Stop Solution
Prior to use, transfer the amount of Stop Solution required for the assay into a secondary
container (see the Quick Chart for Preparing Buffers in this section). After use, discard remaining
Stop Solution.
WARNING: The Stop Solution is corrosive. Wear personal protective equipment whenhandling, i.e. safety glasses, gloves and labcoat.
Diluting Recombinant Histone H3 protein
The Recombinant Histone H3 protein is provided as a control for quantitating the amount ofhistone H3 in your samples. There is enough recombinant protein for at least 2 standard curves.
During the first use, we recommend making 3 µl aliquots of the stock protein and storing at -80°C
to avoid multiple freeze/thaw cycles.

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5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 determinedby gel 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 ofsample necessary to fall within the linear area of the reference curve. Once the protein concentration for the linear area of the reference curve has been determined, perform the rest of theassays within the linear range.

Quick Chart for Preparing Buffers
For 1
well

For 1 strip(8 wells)

For 6 strips(48 wells)

For 12 strips(96 wells)

Total Histone H3 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 ml

Reagents to prepare

Components

Primary Antibody

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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
Use this plate set-up example to prepare a standard curve for the Total Histone H3 Kit in duplicate.
Recomb. H3

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 to a starting concentration of 2 ng/µl by adding 2 µl of
Recombinant Histone H3 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.
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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.

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 curve. Add desired amount of sample in 50 µl volume to plate.
Purified core histones: Recommended range of 15 ng - 1 µg
Acid extracts: Recommended range of 60 ng - 1 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.

Dilute the Total Histone H3 antibody 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.

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.

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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 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
your plate reader does not have this capability, you may read the plate twice, once at 450
nm and once at 655 nm then manually subtract the 655 nm OD from the 450 nm OD values.
Calculation of results using the standard curve
To generate a standard curve using the included Recombinant Histone H3 protein, average
the duplicate readings for each standard, control, and sample and subtract the opticaldensity (OD) obtained from the zero standard (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 H3
protein has an approximate linear range from 1.5 to 50 ng. The data can be linearizedusing log/log paper and regression analysis may also be applied.
To quantify the amount of total histone H3 in the samples, find the absorbance value 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 the standard curve
must be multiplied by the dilution factor.
See the example standard curve on the next page.

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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 1.5 50 ng/well)
1.4

Histone H3 (OD450nm)

1.2

y = 0.0225x + 0.1994
R2 = 0.98

1
0.8
0.6
0.4
0.2
0

0

10

20

30

40

50

60

Recombinant Histone H3 (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.

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.
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11.

Collect the supernatant fraction containing acid soluble proteins, and discard the acidinsoluble 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
10 mM PMSF
Note:

DTT and PMSF must be added immediately prior to use.

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11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 100 ng/well and for purified core histone, dilute down
to 30 ng/well

Concentration of antibodies is too high

Perform antibody titration to determine optimal working concentration. Start using 1:2000 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 well

For purified core histones, increase to 500 ng per well. For extracts, make sure you are using an acid extract by following theprotocol recommended in Appendix Section A. Increase amountof acid extract to 1 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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12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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