MODified™ Histone
Peptide Array(version B3)
Catalog Nos. 13001 & 13005

Copyright 2014 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.
© 2014 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
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
MODified Histone Peptide Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Components and Storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Array Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols
Buffer Preparation and Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
MODified Histone Peptide Array Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Enzyme Interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Antibody Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Appendix
Section A. Sequential Antibody Incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Section B. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17

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Overview
The MODified™ Histone Peptide Array* is a valuable research tool that can be used to screen
antibodies, proteins and enzymes for interactions with histones and their post-translational
modifications. Each array contains 384 different histone modification combinations in duplicate
for acetylation, methylation, phosphorylation and citrullination modifications on the N-terminal
tails of histones H2A, H2B, H3 and H4.
This unique histone array contains up to four separate modifications per 19mer peptide to allow
researchers to study not only individual sites, but also the effects of neighboring modifications onrecognition and binding. The MODified Histone Peptide Array can be used to screen antibodies for cross-reactivity or to study protein and enzyme interactions. The array itself contains
peptide-cellulose-conjugates spotted onto the planar surface of a standard microscope slide in athree-dimensional layer. This high peptide density enables even protein-interaction sites with low
binding constants to be detected.
The simple array protocol works like a Western blot. Either ECL-based or colorimetric detection
systems can be used. The image is then captured using film or a CCD camera; no special equipment is needed. Active Motif’s free Array Analyze Software can be used to analyze the intensityof the spots and generate a graphical analysis of the histone peptide modification interactions.
Information about spot intensity, averages and errors can be saved in Excel-compatible files. For
added convenience, up to three individual modifications can be displayed in superposition to theexperimental data enabling better visualization of the effects of neighboring modifications.

product

formatcatalog no.

MODified™ Histone Peptide Array

1 array
5 arrays

13001
13005

MODified™ Array Labeling Kit

5 rxns13006

MODified™ Protein Domain Binding Kit

5 rxns13007

Related Products
Active Motif’s MODified™ Array Labeling Kit and MODified™ Protein Domain Binding Kit can bepurchased in conjunction with the MODified Histone Peptide Arrays. The MODified Array Labeling Kit (Catalog No. 13006) contains buffers, control antibody, secondary antibodies and ECL
reagents for chemiluminescent detection of five MODified Histone Peptide Arrays.
The MODified Protein Domain Binding Kit (Catalog No. 13007) is designed to screen protein
domains for interactions with histones and their post-translational modifications and contains
buffers and reagents for chemiluminescent detection of five MODified Histone Peptide Arrays. A
positive control G9a tudor domain protein is also included.
*CelluSpots™ arrays are manufactured under license by INTAVIS Bioanalytical Instruments AG
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1Flow chart of process(Optional)
Incubation with:
Enzymes

Blocked slide
Blocking(Optional)
Incubation with:Proteins

MODified™ Histone Peptide Array

Wash
Incubation with:
Antibodies

Wash
Detection by:
ECL orcolorimetricwww.activemotif.com

Wash

Incubation with:peroxidase (HRP) or
alkaline phosphatase (AP)2

Kit Performance and Benefits
The MODified Histone Peptide Array can be used to screen antibodies, proteins and enzymes forinteractions with histones and their post-translational modifications.
The array contains 59 different post-translational modifications for histone acetylation, methylation, phosphorylation and citrullination modifications on the N-terminal tails of histones H2A,
H2B, H3 and H4. Each 19mer peptide may contain up to four modifications each. Five control
spots are included on each array (locations P20-P24): biotin peptide, c-Myc tag, no histone
peptide and two background spots containing a mixture of modifications that are present on thearray.

MODified Histone Peptide Array

MODified Histone Peptide Array and reference grid for histone peptide locations.
Histone peptides containing 384 different modification combinations are spotted in duplicate onto the glass slide. One spot
is located on the left side of the slide and the duplicate spot is on the right side of the slide. The reference Excel file with thecorresponding histone peptide content can be downloaded from Active Motif’s website at www.activemotif.com/modified.

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Example Analysis of Histone H3 trimethyl Lys9 pAb (Catalog No. 39161)

Image of ECL detection of Histone H3 trimethyl Lys9 (H3K9me3) pAb.
Active Motif’s Histone H3 trimethyl Lys9 pAb (Catalog No. 39161) was used at 1:2,000 dilution on the MODified Histone
Peptide Array. Anti-rabbit HRP secondary antibody was used at 1:2500 dilution, followed by ECL detection and image capture
with a CCD camera.

pecificity factor

Histone H tri ethyl ys pAb

HR

H

H

H

H

HR

HR

HR

H

H

me

me

me

me

ac

me

me

me

a

s

a

s

me

me

Modification
Graphical analysis of Histone H3 trimethyl Lys9 (H3K9me3) pAb cross-reactivity.
Active Motif’s Array Analyze Software was used to analyze spot intensity from the ECL camera image. The results weregraphed as a specificity factor, which is the ratio of the average intensity of all spots containing the mark divided by the average intensity of all spots not containing the mark. The results show Active Motif’s Histone H3 trimethyl Lys9 pAb (Catalog No.
39161) has very little cross-reactivity with other histone modifications.

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Example Analysis of Histone Methyltransferase G9a and G9a mut (Catalog Nos. 31327 &
31328)A

B

C

Images of ECL detection of histone methyltransferase G9a treated arrays.
A) Twenty-five µM of Active Motif’s G9a methyltransferase (Catalog No. 31327) was incubated with the MODified Histone
Peptide Array overnight in the presence of 50 mM Tris, pH 9.0, 5 mM MgCl2, 4 mM DTT and 1 mM AdoMet.
B) Twenty-five µM of Active Motif’s G9a mutant H904K methyltransferase (Catalog No. 31328) was incubated with the MODified Histone Peptide Array overnight in the presence of 50 mM Tris, pH 9.0, 5 mM MgCl2, 4 mM DTT and 1 mM AdoMet.
C) No enzyme control. The methylation status for all three arrays (A-C) was then detected using Histone H3 dimethyl Lys9pAb at 1:2,000 dilution. Anti-rabbit HRP secondary antibody was used at 1:2500 dilution, followed by ECL detection and image capture with a CCD camera. Novel methylation sites were observed on array A, which was treated with the wild-type G9amethyltransferase, showing the ability of the G9a histone methyltransferase to use the peptide sequences on the array as
substrates for histone methylation. Example analysis of the spot intensity from the array incubated with histone methyltransferase G9a are shown on the next page.

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Example Analysis of Histone Methyltransferase G9a
Peptide No. Intensity avg.

Peptide sequence

Mod1

A1

0.3320ARTKQTARKSTGGKAPRKQ

unmod

Mod2

A13

0.6268A R T K Q T A R Kme1 S T G G K A P R K Q

K9me1

A14

0.8333

A R T K Q T A R Kme2 S T G G K A P R K Q

K9me2

A15

0.0564

A R T K Q T A R Kme3 S T G G K A P R K Q

K9me3

B23

0.3735A R T K Q T A Rme2a Kme1 S T G G K A P R K Q

R8me2s K9me1

B24

0.7933

A R T K Q T A Rme2a Kme2 S T G G K A P R K Q

R8me2s K9me2

C1

0.0644

A R T K Q T A Rme2a Kme3 S T G G K A P R K Q

R8me2s K9me3

C5

0.3431A R T K Q T A Rme2a Kme1 S T G G K A P R K Q

R8me2a K9me1

C6

0.6484

A R T K Q T A Rme2a Kme2 S T G G K A P R K Q

R8me2a K9me2

C7

0.1093

A R T K Q T A Rme2a Kme3 S T G G K A P R K Q

R8me2a K9me3

C110.2053

A R T K Q T A Cit Kme1 S T G G K A P R K Q

R8Citr

K9me1

C12

0.3189A R T K Q T A Cit Kme2 S T G G K A P R K Q

R8Citr

K9me2

C13

0.0819A R T K Q T A Cit Kme3 S T G G K A P R K Q

R8Citr

K9me3

C17

0.0440A R T K Q T A R Kme1 pS T G G K A P R K Q

K9me1

S10P

C18

0.0879A R T K Q T A R Kme1 S pT G G K A P R K Q

K9me1

T11P

C19

0.8930A R T K Q T A R Kme1 S T G G Kac A P R K Q

K9me1

K14ac

C20

0.0797

A R T K Q T A R Kme2 pS T G G K A P R K Q

K9me2

S10P

C21

0.1319A R T K Q T A R Kme2 S pT G G K A P R K Q

K9me2

T11P

C22

0.8570A R T K Q T A R Kme2 S T G G Kac A P R K Q

K9me2

K14ac
S10P

Mod3

Mod4

C23

0.0733

A R T K Q T A R Kme3 pS T G G K A P R K Q

K9me3

C24

0.0529A R T K Q T A R Kme3 S pT G G K A P R K Q

K9me3

T11P

D1

0.0568A R T K Q T A R Kme3 S T G G Kac A P R K Q

K9me3

K14ac

G20

0.4413A Rme2s T Kme1 Q T A Rme2s Kme2 S T G G K A P R K Q

R2me2s K4me1 R8me2s K9me2

H4

0.0668A Rme2s T Kme1 Q T A Rme2s Kme3 S T G G K A P R K Q

R2me2s K4me1 R8me2s K9me3

Intensity analysis of histone methyltransferase G9a incubation with the MODified Histone Peptide Array.
The MODified Histone Peptide array was incubated in the presence of histone methyltransferase G9a (Catalog No. 31327) using the conditions stated on the previous page and detected with Histone H3 dimethyl Lys9 pAb. The average spot intensity
values for selected modification combinations are shown. The trimethyl Lys9 modifications have very low intensity as they
are not suitable substrates for G9a methylation, nor are they detected by the dimethyl antibody. However, the unmodified,mono- and dimethyl Lys9 sites show good intensity values since the G9a histone methyltransferase was able to use each ofthese as substrates and the dimethyl antibody detected this new methylation as well as the dimethyl peptides that were notmodified. The impact of neighboring modifications is apparent when S10P or T11P exist on the same peptide as monomethyl
or dimethyl Lys9, as these phosphorylation sites greatly reduce the intensity values.

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Kit Components and Storage
MODified Histone Peptide Arrays are for research use only. Not for use in diagnostic procedures.
All components are guaranteed stable for 6 months from date of receipt when stored properly.
Reagents

Quantity

Storage

MODified™ Histone Peptide Array

1 array
5 arrays

4°C for up to 3 months or-20°C for up to 6 months

Additional materials required• TTBS Buffer (10 mM Tris-HCl, pH 7.4, 0.05% Tween 20 and 150 mM NaCl)• Non-fat dried milk
• Primary antibody, protein or enzyme for analysis• Appropriate enzyme activity buffer or protein binding buffer if the arrays are to be used forprotein or enzyme studies and a related primary antibody for recognition• Secondary antibody conjugated to horseradish peroxidase or alkaline phosphatase• Detection reagents (ECL or colorimetric)• Luminescent or colorimetric imaging system with CCD camera or X-ray film
• Optional: Active Motif’s free Array Analyze Software program capable of analyzing MODified Histone Peptide Arrays (available for download at www.activemotif.com/modified)
Array specifications• Standard microscope slides (26x76 mm, white coating)• 768 spots per slide (384 peptide-conjugate spots printed in duplicate)• Spot-to-spot distance 1.2 mm• Peptides are covalently bound to cellulose via C-terminus• Arrays contain control peptides and location marks

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Protocols
Buffer Preparation and Recommendations
TTBS Buffer
Prepare 100 ml TTBS Buffer containing 10 mM Tris-HCl pH 7.4, 0.05% Tween 20 and 150 mM
NaCl. This buffer will be used to prepare the Blocking Solution and for all the wash steps. Thisbuffer can be prepared in advance and stored at room temperature.
Blocking Solution
Prepare 10 ml TTBS Buffer containing 5% non-fat dried milk for blocking and antibody dilutions.
Blocking solution should be prepared just before use, or prepared and stored at 4°C for up to
24 hours.
Sample Enzymes
Prepare 3 ml of appropriate enzyme activity buffer. Dilute the enzyme for analysis in the activity
buffer, making sure to include any appropriate co-factors (e.g. include methyl donor AdoMet(also known as SAM) with a histone methyltransferase enzyme). Use the appropriate antibody(e.g. anti-H3 Lys9 dimethyl antibody) and corresponding species specific conjugated secondary
antibody for detection. Follow the recommendations below for preparing antibody dilutions.
Sample Proteins
Prepare 3 ml of appropriate protein binding buffer. Dilute the protein for analysis in the binding
buffer. Use the appropriate antibody and corresponding species specific conjugated secondary
antibody for detection. Follow the recommendations below for preparing antibody dilutions.
Sample Antibodies
Antibodies should be diluted in 3 ml Blocking Solution. As a general guideline we suggest using
monoclonal antibodies at a 1:2000 dilution. For polyclonal antibodies, a 2-fold dilution over theamount recommended by the antibody provider for Western blot is usually sufficient.
Use a corresponding species specific secondary antibody conjugated to either horseradish
peroxidase (HRP) or alkaline phosphatase (AP). Dilute conjugated secondary antibody as recommended by the antibody provider for Western blot in 3 ml Blocking Solution (1:2,500 may be asuitable dilution for most secondary antibodies).
Detection Reagents
Follow the recommendations of the manufacturer for the appropriate detection conditions. The
Detection Reagent should be added drop wise onto the array while tilting the array to ensurecomplete coverage of Detection Reagent over the full array.

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Control Peptides
The MODified Histone Peptide Array contains control spots (locations P20-P24), as referenced
on the Excel file (www.activemotif.com/modified). The control biotin spot can be detected witheither an anti-biotin antibody or a streptavidin-conjugated antibody, while the c-myc control canbe detected with an anti-c-myc antibody. The desired control antibody can be combined with theprimary antibody being tested, or added sequentially as described in Appendix A. The array also
contains a negative control peptide and two spots for background levels.
Note:

The non-fat dried milk used to generate the Blocking Solution may containtrace biotin molecules which can sequester the biotin or streptavidin detecting
antibody and reduce signal intensity. For detection of the biotin control peptide
on the MODified Histone Peptide Array, we recommend diluting the anti-biotin
or streptavidin-conjugated antibody in TTBS Buffer instead of Blocking Solution.

Quick Chart for Preparing Buffers*
Sample Type

Reagents to prepare

Components

Enzyme or Protein

Activity or Binding Buffer

Antibody

1 array

5 arrays

Provided by the customer

3 ml

15 ml

Blocking Solution

TOTAL REQUIRED

10 ml

50 ml

TTBS Buffer

TOTAL REQUIRED

90 ml

450 mlBlocking Solution

TOTAL REQUIRED

10 ml

50 ml

TTBS Buffer

TOTAL REQUIRED

70 ml

350 ml*If performing sequential antibody addition of sample antibody and control antibodies, pleaserefer to the calculations listed in Appendix A for preparing buffer volumes.

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Array Protocol
Read the entire protocol before use.
MODified Histone Peptide Array Protocol
The protocol below is for screening antibody cross-reactivity or protein and enzyme interactions
with histones and histone modifications. Either an ECL or colorimetric detection method can beused. Prepare the TTBS Buffer and Blocking Solution as described above in the section Buffer
Preparation and Recommendations. The protocol below is for the analysis of a single array. If
screening multiple arrays at the same time, use the Quick Chart for Preparing Buffers to determine the total volumes needed. For enzyme analysis begin with Step 1; for protein studies startwith Step 2; for antibody studies proceed to Step 3.
It is very important to ensure that the entire surface of the MODified Histone Peptide Arrays arecompletely covered with liquid during all incubation and wash steps. Uneven distribution of liquid
will result in inconsistent results.

Step 1: Enzyme Interactions
1.

Prepare the appropriate activity buffer for the enzyme being studied.

2.

Immerse one array in 3 ml activity buffer.
Note:

If working with multiple arrays at the same time, “4 Well Rectangular Dishes”
from NUNC (Catalog No. 267061) can be used for the incubation and wash
steps. The NUNC dish does not contain a lid, so it is recommended to use a low
setting on an orbital shaker when performing incubation and wash steps. Other
suitable containers may also be used.

3.

Incubate on an orbital shaker for 20 minutes at room temperature.

4.

Add the enzyme being studied and any necessary co-factors to the array. Incubate for thedesired time.

5.

Carefully pour off the buffer. Perform a quick rinse (30 seconds) with 5 ml TTBS buffer. Then
wash three times for 5 minutes each on an orbital shaker using 5 ml TTBS buffer.

6.

Prepare 10 ml of Blocking Solution.

7.

Immerse one array in 3 ml Blocking Solution.

8.

Incubate on an orbital shaker for 1-4 hour(s) at room temperature, or overnight at 4°C.

9.

Perform a quick rinse (30 seconds) with 5 ml TTBS buffer. Then wash three times for 5
minutes each on an orbital shaker using 5 ml TTBS buffer.

10. During the last wash step, prepare appropriate antibody dilutions.
11.

Dilute the primary antibody in 3 ml Blocking Solution. See the dilution suggestions in the
Buffer Preparation and Recommendations section on page 8.

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10Note:
To detect the c-Myc control peptide on the array, combine a c-Myc antibody with thesample antibody in 3 ml Blocking Solution. Make sure to use appropriate species-specificsecondary antibody for detection of both the sample and control antibodies. Due to thepresence of biotin in the non-fat dried milk used to make the Blocking Solution, we do notrecommend performing anti-biotin or streptavidin-conjugated antibody incubations in Blocking Solution. For sequential detection of the control and sample antibodies, please refer toAppendix A.
12. Add the primary antibody dilution to the array and incubate for 1 hour at room temperature,
or overnight at 4°C on an orbital shaker. Set the orbital shaker to a low setting to prevent
any cross contamination of antibody solutions if working with more than one array.
13. Perform a quick rinse (30 seconds) with 5 ml TTBS buffer. Then wash three times for 5
minutes each on an orbital shaker using 5 ml TTBS buffer.
14. During the last wash step, prepare the secondary antibody dilution in 3 ml Blocking Solution. Proceed to Step 4.

Step 2: Protein Interactions
1.

Prepare 10 ml of Blocking Solution.

2.

Immerse one array in 3 ml Blocking Solution.
Note:

If working with multiple arrays at the same time, “4 Well Rectangular Dishes”
from NUNC (Catalog No. 267061) can be used for the incubation and wash
steps. The NUNC dish does not contain a lid, so it is recommended to use a low
setting on an orbital shaker when performing incubation and wash steps. Other
suitable containers may also be used.

3.

Incubate on an orbital shaker for 1-4 hour(s) at room temperature, or overnight at 4°C.

4.

Carefully pour off the buffer. Perform a quick rinse (30 seconds) with 5 ml TTBS buffer. Then
wash three times for 5 minutes each on an orbital shaker using 5 ml TTBS buffer.

5.

During the last wash step, prepare 3 ml of the appropriate protein binding buffer.

6.

Add the protein being studied and any necessary co-factors to the protein binding buffer.
Incubate with the blocked array for the desired time.

7.

Perform a quick rinse (30 seconds) with 5 ml TTBS buffer. Then wash three times for 5
minutes each on an orbital shaker using 5 ml TTBS buffer.

8.

During the last wash step, prepare appropriate antibody dilutions.

9.

Dilute the primary antibody in 3 ml Blocking Solution. See the dilution suggestions in the
Buffer Preparation and Recommendations section on page 8.
Note:

To detect the c-Myc control peptide on the array, combine a c-Myc antibody
with the sample antibody in 3 ml Blocking Solution. Make sure to use appropriate species-specific secondary antibody for detection of both the sample andcontrol antibodies. Due to the presence of biotin in the non-fat dried milk used
to make the Blocking Solution, we do not recommend performing anti-biotin or

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streptavidin-conjugated antibody incubations in Blocking Solution. For sequential detection of the control and sample antibodies, please refer to Appendix A.
10. Add the primary antibody dilution to the array and incubate for 1 hour at room temperature,
or overnight at 4°C on an orbital shaker. Set the orbital shaker to a low setting to prevent
any cross contamination of antibody solutions if working with more than one array.
11.

Perform a quick rinse (30 seconds) with 5 ml TTBS buffer. Then wash three times for 5
minutes each on an orbital shaker using 5 ml TTBS buffer.

12. During the last wash step, prepare the secondary antibody dilution in 3 ml Blocking Solution. Proceed to Step 4.

Step 3: Antibody Studies
1.

Prepare 10 ml of Blocking Solution.

2.

Immerse one array in 3 ml Blocking Solution.
Note:

If working with multiple arrays at the same time, “4 Well Rectangular Dishes”
from NUNC (Catalog No. 267061) can be used for the incubation and wash
steps. The NUNC dish does not contain a lid, so it is recommended to use a low
setting on an orbital shaker when performing incubation and wash steps. Other
suitable containers may also be used.

3.

Incubate on an orbital shaker for 1-4 hour(s) at room temperature, or overnight at 4°C.

4.

Carefully pour off the buffer. Perform a quick rinse (30 seconds) with 5 ml TTBS buffer. Then
wash three times for 5 minutes each on an orbital shaker using 5 ml TTBS buffer.

5.

During the last wash step, prepare appropriate antibody dilutions.

6.

Dilute the primary antibody in 3 ml Blocking Solution. See the dilution suggestions in the
Buffer Preparation and Recommendations section on page 8.
Note:

To detect the c-Myc control peptide on the array, combine a c-Myc antibody
with the sample antibody in 3 ml Blocking Solution. Make sure to use appropriate species-specific secondary antibody for detection of both the sample andcontrol antibodies. Due to the presence of biotin in the non-fat dried milk used
to make the Blocking Solution, we do not recommend performing anti-biotin or
streptavidin-conjugated antibody incubations in Blocking Solution. For sequential detection of the control and sample antibodies, please refer to Appendix A.

7.

Add the primary antibody dilution to the array and incubate for 1 hour at room temperature,
or overnight at 4°C on an orbital shaker. Set the orbital shaker to a low setting to prevent
any cross contamination of antibody solutions if working with more than one array.

8.

Perform a quick rinse (30 seconds) with 5 ml TTBS buffer. Then wash three times for 5
minutes each on an orbital shaker using 5 ml TTBS buffer.

9.

During the last wash step, prepare the secondary antibody dilution in 3 ml Blocking Solution. Proceed to Step 4.
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Step 4: Addition of Conjugated Secondary Antibody
1.

Add the conjugated secondary antibody dilution to the array and incubate for 1 hour at room
temperature on an orbital shaker. Set the orbital shaker to a low setting to prevent any
cross contamination of antibody solutions if working with more than one array.

2.

Perform a quick rinse (30 seconds) with 5 ml TTBS buffer. Then wash three times for 5
minutes each on an orbital shaker using 5 ml TTBS buffer.

3.

During the last wash step, prepare the detection solution according to the manufacturer’s
recommendations. Approximately 1.5 ml of detecting solution is sufficient to cover the array.

Step 5: Detection
1.

Add the detection solution to the array. The Detection Reagent should be added drop
wise onto the array while gently tilting the array to ensure complete coverage. Let sit for
5 minutes at room temperature. The array needs to remain wet during image capture. We
recommend covering the array with clear plastic wrap or an acetate sheet during image
capture to maintain the moisture.

2.

For ECL detection, use a CCD camera, or film, to capture images at multiple exposure
times (e.g. 10 sec., 30 sec., 1 min., 2.5 min., 5 min. and 10 minutes). For weak antibodies an
exposure of 10-15 minutes may be needed for sufficient detection. It is also recommended
to take a white light image of the array in order to obtain orientation information for the
analysis step. For colorimetric detection a standard camera or scanner can be used tocapture images.

Step 6: Analysis
1.

Save the image file as a .tif file and compare the image to the reference grid containing thehistone peptide content. The .tif file will need to be resized to specific dimensions, as statedin the Array Analyze manual, for use in Active Motif’s Array Analyze Software. Programs
such as Adobe Photoshop or the freeware program, GIMP2 (http://www.gimp.org/) will allow
you to open and resize .tif files.
The reference Excel file for the histone peptide content and associated grid location can be
downloaded from our website at www.activemotif.com/modified.

2.

Active Motif’s free Array Analyze software is available for analysis of the MODified Histone
Peptide Arrays. The software program will analyze the spot intensity of the interactions fromthe array and generate a graphical analysis of the histone peptide interactions. Information
about spot intensity, averages and errors can be saved in Excel-compatible files to allow
for individual analysis. The Array Analyze software is designed for use on PCs only. Please
download the Array Analyze manual for information about the installation and operation of
the program. Both the manual and the Array Analyze Software can be downloaded from our
website at www.activemotif.com/modified.

3.

(Optional) Use a spot densitometry program to determine the relative intensity of each peptide. Compare the image file to the reference grid containing the histone peptide content.
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13

Appendix
Section A: Sequential Antibody Incubation
This protocol is designed for researchers that prefer to perform separate incubation of sample
antibody and control antibody. This protocol will require the preparation of additional Blocking
Solution and TTBS Buffer than the Standard Protocol. Refer to the Quick Chart below for buffer
preparation.
1.

If using enzyme samples, follow Step 1, instructions # 1-11. For instruction # 11, add the appropriate antibody dilution for detection of the enzyme reaction in 3 ml Blocking Solution.
Do not combine the control antibody into the mixture.
If using protein samples, follow Step 2, instructions # 1-9. For instruction # 9, add the appropriate antibody dilution for detection of the protein interaction in 3 ml Blocking Solution.
Do not combine the control antibody into the mixture.
If using antibody samples, follow Step 3, instructions # 1-6. For instruction # 6, add theappropriate sample antibody dilution in 3 ml Blocking Solution. Do not combine the control
antibody into the mixture.

2.

Add the sample antibody dilution to the array and incubate for 1 hour at room temperature,
or overnight at 4°C on an orbital shaker. Set the orbital shaker to a low setting to prevent
any cross contamination of antibody solutions if working with more than one array.

3.

Perform a quick rinse (30 seconds) with 5 ml TTBS Buffer. Then wash three times for 5
minutes each on an orbital shaker using 5 ml TTBS Buffer.

4.

During the last wash step, prepare the control antibody dilution in 3 ml Blocking Solution.

5.

Incubate for 1 hour at room temperature on an orbital shaker. Set the orbital shaker to a low
setting to prevent any cross contamination of antibody solutions if working with more than
one array.

6.

Perform a quick rinse (30 seconds) with 5 ml TTBS Buffer. Then wash three times for 5
minutes each on an orbital shaker using 5 ml TTBS Buffer.

7.

During the last wash step, prepare the secondary antibody dilution in 3 ml Blocking Solution. Add the species specific HRP-conjugated secondary antibody needed for detection of
the sample and control antibodies. If using secondary antibodies of different species, they
may be combined together into a single incubation step, or also incubated sequentially withwashing between addition of each antibody.

8.

Incubate for 1 hour at room temperature on an orbital shaker. Set the orbital shaker to a low
setting to prevent any cross contamination of antibody solutions if working with more than
one array.

9.

Perform a quick rinse (30 seconds) with 5 ml TTBS Buffer. Then wash three times for 5
minutes each on an orbital shaker using 5 ml TTBS Buffer.

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1410. During the last wash step, prepare the Detecting Reagent as stated in the Buffer Preparation and Recommendations section on page 8. Approximately 1.5 ml of Detecting Reagent issufficient to cover the entire array.
11.

Continue on with Step 5 and Step 6 of the Standard Protocol for detection and analysis of
the arrays.

Quick Chart for Preparing Buffers for Sequential Incubations
Sample Type

Reagents to prepare

Components

1 array

5 arrays

Enzyme or Protein

Activity or Binding Buffer

Provided by the customer

3 ml

15 ml

Blocking Solution

TOTAL REQUIRED

18 ml

80 ml

TTBS Buffer

TOTAL REQUIRED

150 ml650 mlBlocking Solution

TOTAL REQUIRED

18 ml

80 ml

TTBS Buffer

TOTAL REQUIRED

110 ml550 mlAntibody

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15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 to the array in thecorrect order.

Substrate is no longer
activeTest conjugate and substrate for activity by mixing a small
amount of HRP or AP and detecting solution together.

Enzyme inhibitor
present

Sodium azide will inhibit peroxidase reactions (HRP-conjugates).
Follow our recommendations to prepare buffers.

Concentration of antibodies is too low

Increase the amount of antibody used with the array.

CCD camera settings
not optimal

Verify the filter settings on the CCD camera and make sure they
are set to detect luminescence or colorimetric readouts basedon the type of detection being used.

High background

Concentration of
antibodies is too highFor monoclonal antibodies we suggest a 1:2000 dilution. For
polyclonal antibody, we suggest a 2-fold dilution over therecommendations of the antibody provider for Western blot
detection. If these recommendations were followed and there
is still high background, we suggest diluting the antibody 5 to
10-fold for future testing.

Uneven detection orinconsistent replicatesNot enough solution
used for incubation andwash steps

It is very important to ensure that the entire surface of the MODified Histone Peptide Arrays are completely covered with liquid
during all incubation and wash steps. Uneven distribution ofliquid will result in inconsistent results. If using large containers
for incubation and wash steps it may be necessary to increase
the volume of solution used.

Incubation steps performed on an unevensurface

It is very important to ensure that the entire surface of the
MODified Histone Peptide Arrays are completely covered withliquid during all incubation and wash steps. Uneven distributionof liquid will result in inconsistent results. Ensure that the arrayis placed on a flat, level surface during all incubation and wash
steps.

Array dried out during
image capture

During long exposure times it is possible for the Detection
Solution to be completely absorbed by the array, causing thearray to dry out. Place a sheet of clear plastic wrap or an acetate
sheet over the array in order to keep the array wet during image
capture.

Can the MODified Histone
Peptide Arrays be stripped
and re-used?

No. The MODified Histone Peptide arrays are suitable for one
use only. Standard stripping procedures do not adequately
remove the existing bound antibody and harsh conditions cancleave the peptide bond to the array.

What are the chemical stability limits of the arrays?

The arrays are stable at the physiological pH values. They aresensitive to mechanical stress and should not be touched or
wiped. Furthermore, the arrays should not be treated withstrong acids and bases, since the peptide bond and/or the linker
between the peptide and cellulose can be cleaved. The treatment with organic solvents like ethanol should also be avoided.
Several salt concentrations have been tested for blocking andwash buffers, and the arrays are robust in this respect.

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16Section 4: Technical Services
If you need assistance at any time, please call or send an email to Active Motif Technical Service
at one of the locations listed below.

Active Motif North America
Toll free:Direct:Fax:
E-mail:Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
0800/169 31 47
0800/90 99 79
0800/181 99 10+32 (0)2 653 0001
+32 (0)2 653 0050
eurotech@activemotif.com

Active Motif Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com
Active Motif China
Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

Visit Active Motif online at activemotif.com

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17