Histone H3 phospho Ser10 ELISA
Catalog No. 53111

Histone H3 phospho Ser28 ELISA
Catalog No. 53100(version A)

Active Motif North America
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Telephone:+81 3 5225 3638
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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
Introduction  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Performance and Benefits  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 3Kit Components and Storage
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
ELISA Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Appendix
Section A. Preparation of Acid Extract/Crude Histones . . . . . . . . . . . . . . . . . . . . . . . . . . . .  10
Section B. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Section C. Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16

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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.
Phosphorylation of serine 10 and serine 28 in the tail of histone H3 occur early in mitosis and serveas excellent mitotic markers. Histone H3 is phosphorylated on serine 10 during late S phase or G2
phase, while the phosphorylation of serine 28 occurs during prophase. In contrast to serine 10,
serine 28 phosphorylation has never been observed in interphase.
The Histone H3 phospho Ser10 ELISA is a simple solution for screening purified core histones orextracts for levels of phosphorylated serine 10 on histone H3 in human and other (wide range
predicted) systems. These kits are sandwich ELISAs that utilize a C-terminal Histone H3 antibody
to capture histone H3 from your sample and a mouse monoclonal antibody phosphorylated atserine 10 on histone H3 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 on a standard plate reader. 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 phosphohistone H3 (Ser10) in as little as 156 nanograms of acid extract. For added convenience, negativeand positive control acid extract is also included.
The Histone H3 phospho Ser28 ELISA is a simple solution for screening purified core histones
or extracts for levels of phosphorylated serine 28 on histone H3 in human, mouse, hamster andbovine systems. These kits are sandwich ELISAs that utilize a C-terminal Histone H3 antibody tocapture histone H3 from your sample and a rat monoclonal antibody phosphorylated at serine 28
on histone H3 for specific detection. A secondary antibody conjugated to horseradish peroxidase(HRP) and developing solutions provide a sensitive colorimetric readout that is easily quantified byspectrophotometry on a standard plate reader. 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 phospho-histone H3(Ser28) in as little as 156 nanograms of acid extract. For added convenience, negative and positive
control acid extract is also included.

product

formatcatalog no.

Histone H3 phospho Ser10 ELISA

1 x 96 rxns

53111

Histone H3 phospho Ser28 ELISA

1 x 96 rxns

53100

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1

Introduction
Histone H3 phospho Serine 10 and Serine 28
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 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 mitosis.
Cell division is a complex, tightly regulated process that is marked by mitosis. Two significant
mitotic events include microtubule formation and chromosome condensation. Phosphorylation ofserine 10 and serine 28 in the tail of histone H3 occur early in mitosis when chromosomes begin tocondense and during premature chromosome condensation induced in S-phase cells. Histone H3 isphosphorylated on serine 10 during late S phase or G2 phase, while the phosphorylation of serine
28 occurs during prophase1,2. In contrast to serine 10, serine 28 phosphorylation has never been observed in interphase1. Both phosphorylations occur during mitotic chromatin condensation before
nuclear division occurs, which make phosphorylation of histone H3 at Ser10 or Ser28 important
markers for cells undergoing mitosis.
Phosphorylated serine 28 on histone H3 has recently been shown to associate with destabilized
nucleosomes in transcribed chromatin, making this an interesting indicator of both mitotic activity
and transcriptional activation3.

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2

Kit Performance and Benefits
Detection limit: < 5 µg acid extract/well.
Range of detection: Histone phospho Ser10 and Ser28 ELISAs provide quantitative results from
156 ng to 5 µg of histones isolated by acid extraction.
Cross-reactivity: Histone H3 phospho Ser10 Kits work with human and a wider range of species
reactivity is predicted due to the high degree of sequence homology of histone H3. Histone H3
phospho Ser28 Kits work with human, mouse, hamster and bovine systems.
Assay time: 3.5 hours.

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Histone H3 phospho Ser 10 levels in paclitaxel and untreated HeLa acid extracts.
78 ng to 5 µg of Paclitaxel treated (Catalog No. 36201) and untreated HeLa acid extract (Catalog No. 36200), prepared asstated in Appendix A, were assayed in the Histone H3 phospho Ser10 ELISA. Data shown are the results from wells assayedin duplicate. These results are provided for demonstration only.

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Histone H3 phospho Ser 28 levels in paclitaxel and untreated HeLa acid extracts.
78 ng to 5 µg of Paclitaxel treated (Catalog No. 36201) and untreated HeLa acid extract (Catalog No. 36200), prepared asstated in Appendix A, were assayed in the Histone H3 phospho Ser28 ELISA. Data shown are the results from wells assayedin duplicate. These results are provided for demonstration only.

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3Kit Components and Storage
Histone H3 phospho Ser10 Kits and Histone H3 phospho Ser28 Kits are for research use only. Not
for use in diagnostic procedures. All components are guaranteed stable for 6 months from date ofreceipt when stored properly.

Reagents

Quantity

Storage

Histone H3 phospho Ser10 Mouse mAb, or
Anti-phospho Histone H3 Ser28 Rat mAb

10 µl
11 µl

4°C
4°C

HRP-conjugated anti-mouse IgG, or
HRP-conjugated anti-rat IgG

10 µl
10 µl

4°C
4°C

Histone H3 Capture Plate

14°C

Plate sealer

1

4°C

Assay Dilution Buffer AM2

15 ml

4°C

20X Wash Buffer

25 ml

4°C

Developing Solution

10 ml

4°C

Stop Solution

10 ml

4°C

Positive control HeLa acid extract(Paclitaxel treated)

25 µg/50 µl-80°C

Negative control HeLa acid extract

25 µg/50 µl-80°C

Additional materials required• Multi-channel pipettor• Multi-channel pipettor reservoirs• Rocking platform• Acid extracted samples or purified core histones• Microplate spectrophotometer capable of reading at 450 nm (655 nm as optional reference
wavelength)www.activemotif.com

4

Protocols
Buffer Preparation and Recommendations
Assay Dilution Buffer AM2
Assay Dilution Buffer AM2 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 distilled water (see Quick Chart for
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
homogenize the buffer by incubating at 50°C for 2 minutes and mixing prior to use.
Preparation of antibodies (see Quick Chart for preparing buffers in this section)
Dilute the Histone H3 phospho Ser 10 mouse monoclonal antibody 1:2000 with Assay Dilution Buffer
AM2. Use 50 µl per well. Dilute the anti-mouse HRP-conjugated secondary 1:5000 with Assay Dilution Buffer AM2. Use 50 µl per well.
Dilute the Histone H3 phospho Ser28 rat monoclonal antibody 1:500 with Assay Dilution Buffer AM2.
Use 50 µl per well. Dilute the anti-rat HRP-conjugated secondary 1:1000 with Assay Dilution Buffer
AM2. Use 50 µl per well.
Developing Solution
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. A blue color present in the Developing Solution
indicates that it has been contaminated and must be discarded. Prior to use, place the Developing 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 (see Quick Chartfor preparing buffers in this section). After use, discard remaining Developing Solution.
Stop Solution
100 µl are needed per well. Prior to use, transfer the amount of Stop Solution required for theassay into a secondary container (see 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.

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5

Diluting HeLa acid extracts
The HeLa acid extracts (paclitaxel treated and untreated) are both provided as controls for comparing levels of phosphorylated Serine 10 or Serine 28 on Histone H3. Sufficient extract is supplied
for 1 control curve of each. The paclitaxel treated HeLa acid extract is optimized to give a strong
signal when used at 156 ng to 5 µg/well. The untreated HeLa acid extract is provided as a negative
control. Avoid multiple freeze/thaw cycles of the extract.
Preparation of sample extracts
Histones isolated by acid extraction of tissue or cell samples or purified core histones can be usedin the assay. Histone phospho Ser10 and Ser28 ELISAs provide quantitative results from 156 ng to
5 µg. It is recommended initially to use a range of sample concentrations to determine the amountof sample necessary to fall within the linear range of the assay. Once the appropriate amount ofsample has been determined, perform the rest of the assays within the linear range. To prepare
histones isolated by acid extraction, follow the protocol in Appendix Section A.

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)

Primary Antibody

Histone H3 phospho Ser10 Ab
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 mlHistone H3 phospho Ser28 Ab
Assay Dilution Buffer
TOTAL REQUIRED

0.1 µl51.9 µl
52 µl

0.9 µl450 µl450 µl5.4 µl
2.7 ml
2.7 ml10.8 µl
5.4 ml5.4 ml

HRP-conjugated anti-mouse 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 ml1.1 µl5.4 ml5.4 ml

HRP-conjugated anti-rat IgG
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 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 mlOR

Secondary Antibody
ORwww.activemotif.com

6

ELISA Protocol
Determine the appropriate number of microwell strips required for testing samples, controls andblanks in duplicate. If less than 8 wells in a strip need to be used, cover the unused wells with aportion of the plate sealer while you perform the assay. The content of these wells is stable atroom temperature if kept dry and, therefore, can be used later for a separate assay. Store theunused strips in the aluminum pouch at 4°C. Use the strip holder for 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.

Control Curve Preparation (Optional)
If a control curve is not desired, skip this portion and proceed with Step 1 below.
Untreated

Paclitaxel treated

1

2

3

4

5

6

7

8

9

10

11

12

A

5 µg

5 µg

5 µg

5 µg

–

–

–

–

–

–

–

–

B

2.5 µg2.5 µg2.5 µg2.5 µg

–

–

–

–

–

–

–

–

C

1.25 µg1.25 µg1.25 µg1.25 µg

–

–

–

–

–

–

–

–

D

0.625 µg 0.625 µg 0.625 µg 0.625 µg

–

–

–

–

–

–

–

–

E

312 ng

312 ng

312 ng

312 ng

–

–

–

–

–

–

–

–

F

156 ng156 ng156 ng156 ng

–

–

–

–

–

–

–

–

G

78 ng

78 ng

78 ng

78 ng

–

–

–

–

–

–

–

–

H

0 µg

0 µg

0 µg

0 µg

–

–

–

–

–

–

–

–

1.

Dilute the untreated and paclitaxel treated HeLa acid extracts to a starting concentrationof 0.1 µg/µl. The control extracts provided with the kit contain 25 µg/50 µl (0.5 µg/µl) per
vial. Using the reported concentration, add 200 µl Assay Dilution Buffer AM2 to the vial andpipette up and down to mix thoroughly. The result will be 25 µg HeLa acid extract in a total
volume of 250 µl = 0.1 µg/µl.

2.

Add 100 µl of the diluted untreated HeLa extracts to wells A1 and A2.

3.

Add 100 µl of the diluted paclitaxel treated HeLa extracts to wells A3 and A4.

4.

Add 50 µl of Assay Dilution Buffer to wells B1 through H4.

5.

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.

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7

6.

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

7.

Transfer 50 µl of the contents of row B to row C and mix, as previous.

8.

Continue this process until row G is reached.

9.

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

10. Row H will serve as the blank wells and receives no acid extracts.

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 ofsample concentrations in order to determine the amount of sample necessary for optimal
detection. Add desired amount of sample in 50 µl volume to plate.

2.

Incubate plate containing the optional control curve and samples for 1 hour at room
temperature with mild agitation (100 rpm on a 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 phospho Ser10: Dilute the Histone H3 phospho Ser10 antibody 1:2000 in Assay
Dilution Buffer AM2 and mix thoroughly.
Histone H3 phospho Ser28: Dilute the anti-phospho-Histone H3 Ser28 antibody 1:500 in
Assay Dilution Buffer AM2 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 phospho Ser10: Dilute the HRP-conj. anti-mouse IgG 1:5000 in Assay Dilution
Buffer AM2 and mix thoroughly.
Histone H3 phospho Ser28: Dilute the HRP-conj. anti-rat IgG 1:1000 in Assay Dilution Buffer
AM2 and mix thoroughly.

9.

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

10. Incubate at room temperature for 1 hour with agitation.
11.

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

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8

Step 4: Colorimetric Reaction
12.

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

13.

Add 100 μl of room temperature Developing Solution to each well.

14.

Incubate under low light conditions for several minutes while monitoring color development. Please read the Certificate of Analysis supplied with this kit for optimal development
time associated with this lot number. Monitor the blue color development until the samples
turn medium to dark blue. For the optional control curve, monitor the wells containing 5 µg
paclitaxel treated HeLa acid extract until they turn medium to dark blue.

15.

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

16.

Read absorbance on a spectrophotometer within 5 minutes at 450nm with an optionalreference wavelength of 655 nm. Blank the plate reader according to the manufacturer’s
instructions using the blank wells.
Reading the reference wavelength is optional. Most microtiter plate readers are equippedto perform dual wavelength analysis and with the appropriate software, will automaticallysubtract the reference wavelength absorbance from the test wavelength absorbance. If 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.

References
1.
2.
3.

Goto, H., et al. (1999) J. Biol. Chem. 274(36): 25543-25549
Hooser, A. V. et al. (1998) J. of Cell Science 111: 3497-3506
Sun, J-M., et al (2007) Nucleic Acids Res. 35(19): 6640-6647

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9

Appendix
Section A. Preparation of Acid Extract/Crude histone proteins
This procedure can be used for a 150 mm plate that is 70% confluent. The yield is approximately
0.15 mg of 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 sufficientfor use in the Histone Phospho Serine ELISA Kits. The quantity of acid extract tested in the
Histone Phospho Serine 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.

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10

Lysis Buffer:
0.4 M HCl
Neutralization Buffer:
1 M Sodium Phosphate, dibasic, pH 12.5. Use 5M NaOH to adjust 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 1-2 µg/well

Concentration of antibodies is too high

Perform antibody titration to determine optimal working
concentration. For Histone H3 phospho Ser10, start using 1:5000
for primary antibody and 1:5000 for the secondary antibody.
For Histone H3 phospho Ser28, start using 1:1000 for primary
antibody and 1:2000 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 extracts, make sure you are using an acid extract by following the protocol recommended in Appendix Section A. Increaseamount of acid extract to 10 µg/well

No signal or weak signal
in positive control curve
wells

Incomplete mixing ofcontrol extracts

Upon addition of Assay Dilution Buffer AM2 to the positive
and negative control HeLa acid extract vials, pipette up anddown to thoroughly mix the extract. Follow the protocol togenerate an optional control curveHigh background in all
wells

Uneven color
development

High background insample wells

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12Section C. Related Products
Histone ELISAs		FormatCatalog No.

Histone H3 dimethyl Lys9 ELISA		Histone H3 trimethyl Lys9 ELISA		Histone H3 trimethyl Lys27 ELISA		
Total Histone H3 ELISA		

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

53108
53109
53106
53110

Recombinant Methylated Histones		

FormatCatalog No.

Recombinant Histone H2A		
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

31251
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

DNA Methylation		

FormatCatalog No.

MethylDetector™		
MethylCollector™		
MethylCollector™ Ultra		
UnMethylCollector™		
Fully Methylated Jurkat DNA		

50 rxns
25 rxns
30 rxns
30 rxns
10 µg

55001
55002
55005
55004
55003

Histone Purification		

FormatCatalog No.

Histone Purification Kit		
Histone Purification Mini Kit		

10 rxns
20 rxns

40025
40026

www.activemotif.com

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Chromatin Assembly		

FormatCatalog No.

Chromatin Assembly Kit		
HeLa Core Histones		

10 rxns
36 µg

53500
53501

Histone Acetyltransferase and Deacetylase Activity

FormatCatalog No.

HAT Assay Kit (Fluorescent)		
Recombinant p300 protein, catalytic domain		
HDAC Assay Kit (Fluorescent)		
HDAC Assay Kit (Colorimetric)		

1 x 96 rxns
5 µg
1 x 96 rxns
1 x 96 rxns

56100
31205
56200
56210

Co-Immunoprecipitation		

FormatCatalog No.

Nuclear Complex Co-IP Kit		
Universal Magnetic Co-IP Kit		

50 rxns
25 rxns

54001
54002

SUMOylation		

FormatCatalog No.

SUMOlink™ SUMO-1 Kit		
SUMOlink™ SUMO-2/3 Kit		

20 rxns
20 rxns

40120
40220

ChIP-IT™ Kits		

FormatCatalog No.

ChIP-IT™ Express		ChIP-IT™ Express Enzymatic		
ChIP-IT™ Express HT		
ChIP-IT™ Protein G Magnetic Beads		
Re-ChIP-IT™		
ChIP-IT™		
ChIP-IT™ w/o controls		
ChIP-IT™ Shearing Kit		
ChIP-IT™ Enzymatic 		
ChIP-IT™ Enzymatic w/o controls		
Enzymatic Shearing Kit		
Salmon Sperm DNA/Protein G agarose		
ChIP-IT™ Control Kit – Human		
ChIP-IT™ Control Kit – Mouse		ChIP-IT™ Control Kit – Rat		
Ready-to-ChIP HeLa Chromatin		
Ready-to-ChIP Hep G2 Chromatin		
Ready-to-ChIP K-562 Chromatin		
Ready-to-ChIP NIH/3T3 Chromatin		

25 rxns
25 rxns
96 rxns
25 rxns
25 rxns
25 rxns
25 rxns
10 rxns
25 rxns
25 rxns
10 rxns
25 rxns
5 rxns
5 rxns
5 rxns
10 rxns
10 rxns
10 rxns
10 rxns

53008
53009
53018
53014
53016
53001
53004
53002
53006
53007
53005
53003
53010
53011
53012
53015
53019
53020
53021

Active Motif also offers a growing list of application validated antibodies, including antibodies
for histones and histone modifications, transcription factor antibodies, DNA methylation-related
antibodies and ChIP validated antibodies. For a complete list go to www.activemotif.com/abs

www.activemotif.com

14

Transcription Factor ELISAs		FormatCatalog No.

TransAM™ AP-1 Family		TransAM™ AP-1 c-Jun		
TransAM™ GR		TransAM™ HIF-1		
TransAM™ IRF-3 (Human)		
TransAM™ IRF-7		
TransAM™ NFATc1		
TransAM™ NFkB Family		TransAM™ NFkB p50		
TransAM™ NFkB p52		
TransAM™ NFkB p65		
TransAM™ p53		
TransAM™ STAT Family		

2 x 96-well plates
1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate2 x 96-well plates
1 x 96-well plate1 x 96-well plate1 x 96-well plate1 x 96-well plate2 x 96-well plates

44296
46096
45496
47096
48396
50196
40296
43296
41096
48196
40096
41196
42296

For a complete list of the over 40 TransAM Kits available, please visit www.activemotif.com/transam

www.activemotif.com

15

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
Active Motif Europe
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
Azuma Bldg, 7th Floor
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
Visit Active Motif on the worldwide web at http://www.activemotif.com
At this site:• Read about who we are, where we are, and what we do• Review data supporting our products and the latest updates• Enter your name into our mailing list to receive our catalog, MotifVations newsletter andnotification of our upcoming products• Share your ideas and results with us• View our job opportunities
Don’t forget to bookmark our site for easy reference!
www.activemotif.com

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