Enabling Epigenetics Research

MODified™ Array Labeling Kit
Catalog No. 13006(Version A2)

Copyright 2010 Active Motif, Inc.

Revision

Date

Description of Change

A2

September, 2023

C-Myc control monoclonal antibody was changed to AbFlex® Myc-Tag
antibody (rAb) (Cat# 91203). The dilution for use of this recombinant
has been updated.

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 inthis manual at any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated,
in whole 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 andinternational 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 theirrespective companies.

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Contents
Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
MODified™ Histone Peptide Array  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
Kit Components and Storage  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  5
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  5
Array Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  5
Protocols  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Quick Chart for Preparing Buffers  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8
MODified™ Histone Peptide Array Labeling Protocol  . . . . . . . . . . . . . . . . . . . . . . . .  8
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Section A: Sequential Antibody Incubation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

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Overview
The MODified™ Array Labeling Kit contains the buffers and reagents needed for easy labelingand chemiluminescent detection of the MODified™ Histone Peptide Arrays. The MODified™
Array Labeling Kit contains blocking buffer, wash buffer, rabbit and mouse HRP-conjugatedsecondary antibodies and ECL reagents for chemiluminescent detection. For added
convenience, a positive control Myc-Tag recombinant antibody is included to recognize thearrays’ control Myc-Tag.
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 induplicate 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 on recognition and binding. The MODified™ Histone Peptide Array can beused 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 astandard microscope slide in a three-dimensional layer. This high peptide density enables
even protein-interaction sites with low binding constants to be detected.
Following labeling and detection, the image is then captured using film or a CCD camera;no special equipment is needed. Active Motif’s free Array Analyse Software can be used toanalyze the intensity of 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 canbe displayed in superposition to the experimental data enabling better visualization of theeffects of neighboring modifications.
The MODified™ Histone Peptide Arrays are available individually, or in packs of five. The
arrays are not included in the MODified Array Labeling Kit and must be purchased separately.

Product
MODified™ Histone Peptide Array
MODified™ Array Labeling Kit

FormatCatalog No.

1 array13001

5 arrays13005

5 rxns13006*CelluSpots™ arrays are manufactured under license by INTAVIS Bioanalytical Instruments AG

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1Flow Chart of Process
Blocked slide(Optional)
Incubation with:
Enzymes

Blocking(Optional)
Incubation with:Proteins

MODified™ Histone Peptide Array

Wash
Incubation with:
Antibodies

Detection by:
ECL orcolorimetric

Wash

Wash

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

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Kit Performance and Benefits
The MODified™ Histone Peptide Array can be used to screen antibodies, proteins andenzymes for interactions 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 ofhistones 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, MycTag, no histone peptide and two background spots containing a mixture of modifications thatare present on the array.
The MODified™ Array Labeling Kit includes an antibody to recognize the Myc-Tag control spot
as well as anti-rabbit and anti-mouse HRP-conjugated secondary antibodies for detection.

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 rightside of the slide. The reference Excel file with the corresponding histone peptide content can be
downloaded from Active Motif’s website at activemotif.com/MODified.

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Example Analysis of Histone H3 trimethyl Lys9 pAb, Cat. No. 39161

Image of ECL detection of Histone H3 trimethyl Lys9 (H3K9me3) pAb.
Active Motif’s Histone H3 trimethyl Lys9 pAb (Cat. 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.

Histone H3 trimethyl Lys9 pAb

40

Specificity factor

35
30
25
20
15
10
5
0

e1

e2a

R2m

H3

e2

e3

K4m

H3

K4m

H3

c

e2s

e2a

K4m

H3

K4a

H3

R8m

H3

e2s

R8m

H3

3

me

R2m

H3

3

me

K27

H3

K9

H3

Modification
Graphical analysis of Histone H3 trimethyl Lys9 (H3K9me3) pAb cross-reactivity.
Active Motif’s Array Analyse Software was used to analyze spot intensity from the ECL camera
image. The results were graphed as a specificity factor, which is the ratio of the average intensity ofall 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 (Cat. No. 39161) has very little crossreactivity with other histone modifications.

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4Kit Components and Storage
MODified™ Histone Peptide Arrays and the MODified™ Array Labeling Kit are for research use
only. Not for use in diagnostic procedures. Please store each component at the temperature
indicated in the table below. Kit and components are guaranteed for 6 months after receipt
when stored properly.
Reagents

Quantity

Storage

Blocking Buffer AM2

100 ml-20°C

10X Wash Buffer AM4

100 ml4°C

AbFlex® Myc-Tag rAb

5 µl

-20°C

Anti-mouse HRP-conjugated secondary antibody

10 µl

4°C

Anti-rabbit HRP-conjugated secondary antibody

10 µl

4°C

ECL Reagent A

50 µl

4°C

ECL Reagent B

2 x 13 ml

4°C

Additional Materials Required• MODified™ Histone Peptide Arrays (these are not included in the MODified™ Array
Labeling kit)
• Primary antibody, protein or enzyme for analysis• Appropriate enzyme activity buffer or protein binding buffer if the arrays are to be used
for protein or enzyme studies and a related primary antibody for recognition• Secondary antibody conjugated to horseradish peroxidase, only if using a sample
antibody from a species other than mouse or rabbit• Luminescent imaging system with CCD camera or X-ray film
• NUNC Well Rectangular Dishes (Cat. No. 267061) or suitable chamber forarray incubation• Optional: Active Motif’s free Array Analyse Software program capable of analyzing
MODified™ Histone Peptide Arrays (available for download at activemotif.com/MODified)

Array Specifications (not included in the MODified™ Array Labeling Kit)
• 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
Blocking Buffer AM2
Thaw the blocking buffer prior to starting the assay. Once thawed, the buffer should be
stored at 4°C and used within 24 hours. Otherwise, the buffer should be re-frozen at -20°C.
Preparation of 1 X Wash Buffer
Prepare the amount of 1X Wash Buffer required for the labeling assay as follows: For every
100 ml of 1X Wash Buffer required, dilute 10 ml 10X Wash Buffer AM4 with 90 ml distilledwater (see the Quick Chart for Preparing Buffers). Mix gently to avoid foaming. The 1X Wash
Buffer may be stored at 4°C for one week. The Tween 20 contained in the 10X Wash Buffer
AM4 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.
Sample Enzymes
Prepare 3 ml of appropriate enzyme activity buffer. Dilute the enzyme for analysis in theactivity 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 conjugatedsecondary 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 thebinding buffer. Use the appropriate antibody and corresponding species specific conjugatedsecondary antibody for detection. Follow the recommendations below for preparing
antibody dilutions.
Sample Antibodies
Antibodies should be diluted in 3 ml Blocking Buffer AM2. As a general guideline we suggest
using monoclonal antibodies at a 1:2,000 dilution. For polyclonal antibodies, the amountrecommended by the antibody provider for Western blot is usually sufficient.
Use a corresponding species specific secondary antibody conjugated to horseradish
peroxidase (HRP). Dilute conjugated secondary antibody, as recommended for Western blotby the antibody provider, in 3 ml Blocking Buffer AM2 (1:2,500 is a suitable dilution for mostsecondary antibodies).
HRP-conjugated secondary antibodies
HRP-conjugated secondary anti-rabbit and anti-mouse antibodies are included in the kit.
Sufficient antibody is provided for the detection of five arrays. Both the anti-rabbit and antimouse HRP-conjugated antibodies should be diluted 1:2,500 in Blocking Buffer AM2 (i.e. use
1.2 µl HRP-conjugated antibody per 3 ml Blocking Buffer AM2).

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Control Peptides
The MODified™ Histone Peptide Array contains control spots (locations P20-P24), as
referenced on the spreadsheet (activemotif.com/MODified). The control Myc-Tag peptide
can be detected with the included Myc-Tag antibody as explained below. The control biotin
peptide can be detected with either an anti-biotin antibody or a streptavidin-conjugatedantibody (not included). The array also contains a no histone negative control peptide andtwo spots for background levels.
Note:

Blocking Buffer AM2 may contain trace biotin molecules which can sequester
the anti-biotin or streptavidin-conjugated detecting antibody and reducesignal intensity. For detection of the biotin control peptide on the MODified™
Histone Peptide Array we recommend diluting the anti-biotin or streptavidinconjugated antibody in 1 X Wash Buffer instead of using Blocking Buffer AM2.

Control Myc-Tag Antibody
The control Myc-Tag antibody is provided for the detection of the Myc-Tag peptide at location
P21 on the MODified™ Histone Peptide Array. The Myc-Tag antibody can be combined directlywith the sample antibody and applied to the array. (For sequential detection of control andsample antibodies, please refer to Appendix A).
The Myc-Tag antibody should be used at a 1:3,000 dilution. Add 1 µl of Myc-Tag antibody to
3 ml Blocking Buffer AM2 also containing the appropriate dilution of sample antibody.
For conjugated secondary antibody detection of the Myc-Tag control antibody, the antimouse HRP-conjugated secondary antibody should be diluted 1:2,500 in Blocking Buffer AM2(i.e. use 1.2 µl HRP-conjugated antibody per 3 ml Blocking Buffer AM2).
Detecting Solution
To prepare the Detecting Solution, dilute ECL Reagent A into ECL Reagent B in a 15 ml
conical tube. For a single array, add 1.5 µl of ECL Reagent A to 5 ml of ECL Reagent B. Vortex
briefly to mix. Detecting Solution should be prepared just before use and kept protected fromlight. The Detecting Solution should be added drop wise onto the array while gently tilting
the array to ensure complete coverage of Detecting Solution over the full array. The arrayneeds 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.

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7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 Buffer AM2

TOTAL REQUIRED

10 ml

50 ml

1X Wash Buffer

10X Wash Buffer AM4

9 ml

45 ml

Distilled Water

81 ml

405 mlTOTAL REQUIRED

90 ml

450 mlECL Reagent A

TOTAL REQUIRED

1.5 µl7.5 µlECL Reagent B

TOTAL REQUIRED

5 ml

25 ml

Blocking Buffer AM2

TOTAL REQUIRED

10 ml

50 ml

1X Wash Buffer

10X Wash Buffer AM4

7 ml

35 ml

Distilled water

63 ml

315 mlTOTAL REQUIRED

70 ml

350 mlECL Reagent A

TOTAL REQUIRED

1.5 µl7.5 µlECL Reagent B

TOTAL REQUIRED

5 ml

25 ml

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

MODified™ Histone Peptide Array Labeling Protocol
Please read the entire protocol before use.
The following protocol is for screening antibody cross-reactivity or protein and enzyme
interactions with histones and histone modifications using a chemiluminescent detection
method. Prepare the Blocking Buffer and 1X Wash Buffer as described in the previous 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 start with 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
are completely covered with liquid during all incubation and wash steps. Uneven distributionof liquid will result in inconsistent results.

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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 alow 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 forthe desired time.

5.

Carefully pour off the buffer. Perform a quick rinse (30 seconds) with 5 ml 1X Wash
Buffer. Then wash three times for 5 minutes each on an orbital shaker using 5 ml 1X
Wash Buffer.

6.

Transfer 10 ml of thawed Blocking Buffer AM2 into 15 ml conical tube. Blocking Buffer
AM2 can be kept at 4°C for up to 24 hours.

7.

Immerse one array in 3 ml Blocking Buffer AM2.

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 1X Wash Buffer. Then wash three times
for 5 minutes each on an orbital shaker using 5 ml 1X Wash Buffer.

10. During the last wash step, prepare appropriate antibody dilutions.
11. Dilute the primary antibody in 3 ml Blocking Buffer AM2. See the dilution suggestionsin the Buffer Preparation and Recommendations section on page 8.
Note:

To detect the Myc-tag control peptide on the array, combine the included
Myc-tag antibody at a 1:3,000 dilution with the diluted sample antibody in 3
ml Blocking Buffer AM2. For sequential detection of the control and sample
antibodies, please refer to Appendix 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 morethan one array.
13. Perform a quick rinse (30 seconds) with 5 ml 1X Wash Buffer. Then wash three times
for 5 minutes each on an orbital shaker using 5 ml 1X Wash Buffer.
14. During the last wash step, prepare the secondary antibody dilution.
Note:

To detect the Myc-tag control antibody, combine the included anti-mouse
HRP-conjugated antibody at a 1:2,500 dilution with the diluted species specificsecondary antibody for the sample antibody in 3 ml Blocking Buffer AM2.

15. Proceed to Step 4.

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Step 2: Protein Interactions
1.

Transfer 10 ml of thawed Blocking Buffer AM2 into 15 ml conical tube. Blocking Buffer
AM2 can be kept at 4°C for up to 24 hours.

2.

Immerse one array in 3 ml Blocking Buffer AM2.

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 alow 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 1X Wash
Buffer. Then wash three times for 5 minutes each on an orbital shaker using 5 ml 1X
Wash 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 3 ml of protein
binding buffer. Incubate with the blocked array for the desired time.

7.

Perform a quick rinse (30 seconds) with 5 ml 1X Wash Buffer. Then wash three times
for 5 minutes each on an orbital shaker using 5 ml 1X Wash Buffer.

8.

During the last wash step, prepare appropriate antibody dilutions.

9.

Dilute the primary antibody in 3 ml Blocking Buffer AM2. See the dilution suggestionsin the Buffer Preparation and Recommendations section on page 8.

Note:

To detect the Myc-tag control peptide on the array, combine the included
Myc-tag antibody at a 1:3,000 dilution with the diluted sample antibody in 3
ml Blocking Buffer AM2. 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 morethan one array.
11. Perform a quick rinse (30 seconds) with 5 ml 1X Wash Buffer. Then wash three times
for 5 minutes each on an orbital shaker using 5 ml 1X Wash Buffer.
12. During the last wash step, prepare the secondary antibody dilution.
Note:

To detect the Myc-tag control antibody, combine the included anti-mouse
HRP-conjugated antibody at a 1:2,500 dilution with the diluted species specificsecondary antibody for the sample in 3 ml Blocking Buffer AM2.

13. Proceed to Step 4.

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10Step 3: Antibody Studies
1.

Transfer 10 ml of thawed Blocking Buffer AM2 into 15 ml conical tube. Blocking Buffer
AM2 can be kept at 4°C for up to 24 hours.

2.

Immerse one array in 3 ml Blocking Buffer AM2.

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 alow 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 1X Wash
Buffer. Then wash three times for 5 minutes each on an orbital shaker using 5 ml 1X
Wash Buffer.

5.

During the last wash step, prepare appropriate antibody dilutions.

6.

Dilute the primary antibody in 3 ml Blocking Buffer AM2. See the dilution suggestionsin the Buffer Preparation and Recommendations section on page 8.

Note:

To detect the Myc-tag control peptide on the array, combine the included
Myc-tag antibody at a 1:3,000 dilution with the diluted sample antibody in 3
ml Blocking Buffer AM2. 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 morethan one array.

8.

Perform a quick rinse (30 seconds) with 5 ml 1X Wash Buffer. Then wash three times
for 5 minutes each on an orbital shaker using 5 ml 1X Wash Buffer.

9.

During the last wash step, prepare the secondary antibody dilution.

Note:

To detect the Myc-tag control antibody, combine the included anti-mouse
HRP-conjugated antibody at a 1:2,500 dilution with the diluted species specificsecondary antibody for the sample in 3 ml Blocking Buffer AM2.

10. 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 hourat room temperature on an orbital shaker. Set the orbital shaker to a low setting toprevent any cross contamination of antibody solutions if working with more than
one array.

2.

Perform a quick rinse (30 seconds) with 5 ml 1X Wash Buffer. Then wash three times
for 5 minutes each on an orbital shaker using 5 ml 1X Wash Buffer.

3.

During the last wash step, prepare the Detecting Solution as stated in the Buffer
Preparation and Recommendations section on page 8.

Step 5: Detection
1.

The Detecting Solution 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 thearray with clear plastic wrap or an acetate sheet during image capture to maintainthe moisture.

Note:

2.

If working with multiple arrays at the same time, we recommend going
through the entire detection process and image capture with a single arrayand then moving on to the second array and so forth. Additional arrays canremain in the final wash from the secondary antibody until they are ready to
be processed.

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 exposureof 10-15 minutes may be needed for sufficient detection. It is also recommended totake a white light image of the array in order to obtain orientation information for the
analysis step.

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Step 6: Analysis
1.

Save the image file as a .tif file and compare the image to the reference gridcontaining the histone peptide content. The .tif file will need to be resized to specificdimensions, as stated in the Array Analyse manual, for use in Active Motif’s Array
Analyse Software. Programs such as Adobe Photoshop or the freeware program,
GIMP2 (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 activemotif.com/MODified™.

2.

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

Note:

The Array Analysis Software designates the control as c-Myc. Please notethat c-Myc and Myc-tag are used interchangeably. We use a Myc-tag peptide
present in the array.

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

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Appendix
Section A: Sequential Antibody Incubation
This protocol is designed for researchers that prefer to perform separate incubation ofsample antibody and control antibody. This protocol will require the preparation of additional
Blocking Buffer and 1X Wash 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 theappropriate antibody dilution for detection of the enzyme reaction in 3 ml Blocking
Buffer AM2. Do not combine the control antibody into the mixture.
If using protein samples, follow Step 2, instructions # 1-9. For instruction # 9, add theappropriate antibody dilution for detection of the protein interaction in 3 ml Blocking
Buffer AM2. Do not combine the control antibody into the mixture.
If using antibody samples, follow Step 3, instructions # 1-6. For instruction # 6,
add the appropriate sample antibody dilution in 3 ml Blocking Buffer AM2. 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 morethan one array.

3.

Perform a quick rinse (30 seconds) with 5 ml 1X Wash Buffer. Then wash three times
for 5 minutes each on an orbital shaker using 5 ml 1X Wash Buffer.

4.

During the last wash step, prepare the Myc-tag control antibody dilution in 3 ml
Blocking Buffer AM2. The Myc-tag antibody should be used at a 1:3,000 dilution.

5.

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

6.

Perform a quick rinse (30 seconds) with 5 ml 1X Wash Buffer. Then wash three times
for 5 minutes each on an orbital shaker using 5 ml 1X Wash Buffer.

7.

During the last wash step, prepare the secondary antibody dilution in 3 ml Blocking
Buffer AM2. Add the species specific HRP-conjugated secondary antibody neededfor detection of the sample antibody (the kit includes anti-rabbit and anti-mouse
HRP-conjugated antibodies). The control Myc-tag antibody requires the addition of a
1:2,500 dilution of anti-mouse HRP-conjugated antibody for the secondary antibody
dilution. If using secondary antibodies of different species, they may be combined
together into a single incubation step, or also incubated sequentially with washing
between addition of each antibody.

8.

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

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9.

Perform a quick rinse (30 seconds) with 5 ml 1X Wash Buffer. Then wash three times
for 5 minutes each on an orbital shaker using 5 ml 1X Wash Buffer.

10. During the last wash step, prepare the Detecting Solution as stated in the Buffer
Preparation and Recommendations section on page 8. Approximately 1.5 ml ofDetecting Solution is sufficient to cover the entire array.
11. Continue on with Step 5 and Step 6 of the Standard Protocol for detection andanalysis of the arrays.
Quick Chart for Preparing Buffers for Sequential Incubations
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 Buffer AM2

TOTAL REQUIRED

18 ml

80 ml

1X Wash Buffer

10X Wash Buffer AM4

15 ml

65 ml

Distilled Water

135 ml585 mlTOTAL REQUIRED

150 ml650 mlECL Reagent A

TOTAL REQUIRED

1.5 µl7.5 µlECL Reagent B

TOTAL REQUIRED

5 ml

25 ml

Blocking Buffer AM2

TOTAL REQUIRED

18 ml

80 ml

1X Wash Buffer

10X Wash Buffer AM4

11 ml

55 ml

Distilled water

99 ml

495 mlTOTAL REQUIRED

110 ml550 mlECL Reagent A

TOTAL REQUIRED

1.5 µl7.5 µlECL Reagent B

TOTAL REQUIRED

5 ml

25 ml

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15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 arrayin the correct order.

Substrate is nolonger active

Test conjugate and substrate for activity by mixing
a small amount of HRP or AP and detecting
solution together.

Enzymeinhibitor 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 andmake sure they are set to detect luminescence orcolorimetric readouts based on 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, use the amountrecommended by the antibody provider for Western
blot detection. If these recommendations were
followed and there is still high background, we suggest
diluting the antibody 2 to 5-fold for future testing.

Uneven detection orinconsistent replicatesNot enough solution
used for incubation
and wash steps

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

Incubation steps
performed on an
uneven surface

It is very important to ensure that the entire surface ofthe MODified™ Histone Peptide Arrays are completely
covered with liquid during all incubation and wash
steps. Uneven distribution of liquid will result ininconsistent results. Ensure that the array is placedon a flat, level surface during all incubation andwash steps.

Array dried out during
image capture

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

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Problem/Question

Possible Cause

Recommendation

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

No. The MODified™ Histone Peptide arrays are suitablefor one use only. Standard stripping procedures do
not adequately remove the existing bound antibody
and harsh conditions can cleave 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 are sensitive to mechanical stress and should not
be touched or wiped. Furthermore, the arrays should
not be treated with strong acids and bases, since thepeptide bond and/or the linker between the peptide
and cellulose can be cleaved. The treatment withorganic solvents like ethanol should also be avoided.
Several salt concentrations have been tested forblocking and wash buffers, and the arrays are robust in
this respect.

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17Technical Services

China

Japan

EuropeNorth America

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.

Toll free:877 222 9543

Direct:

760 431 1263

Fax:

760 431 1351

E-mail:tech_service@activemotif.com

UK Free Phone:

0800/169 31 47

France Free Phone:

0800/90 99 79

Germany Free Phone:

0800/181 99 10

Direct:+32 (0)2 653 0001

Fax:

+32 (0)2 653 0050

E-mail:eurotech@activemotif.com

Direct:+81 (0)3 5225 3638

Fax:

+81 (0)3 5261 8733

E-mail:japantech@activemotif.com

Direct:(86)-21-20926090

Cell Phone:
E-mail:18521362870
techchina@activemotif.com

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