Tag-ChIP-IT®
(version A3)
Catalog No. 53022

Copyright 2017 Active Motif, Inc.

Please Read Before Starting:
The Tag-ChIP-IT® Kit contains an optimized protocol and buffer system to ensure the highest
level of sensitivity and lowest background when working with low abundance transcription factor
targets. It is important to follow our protocol and recommendations throughout the entire manualas many steps may differ from traditional ChIP protocols. Please pay particular attention to ournotes regarding the processing of input chromatin for agarose gel analysis prior to the chromatin
immunoprecipitation reaction.

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.
© 2017 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
Protocol Overview and Time Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Components and Storage
Tag-ChIP-IT® Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols – Experimental Set Up
Chromatin Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Buffer Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Protocols – Preparation of Sheared Chromatin
Section A. Cell Fixation Starting with Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Section B. Chromatin Sonication of Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Protocols – Chromatin Immunoprecipitation
Section C. Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Protocols – ChIP DNA Purification
Section D. Reversal of Cross-links and DNA Purification . . . . . . . . . . . . . . . . . . . . . . . 16
Protocols – ChIP DNA Analysis
Section E. Quantitative PCR (qPCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Section F. ChIP-Seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Appendix
Section G. Prepare AM-tag Fusion Protein Construct . . . . . . . . . . . . . . . . . . . . . . . . . 20
Section H. Transfection of Cells with AM-tag Construct. . . . . . . . . . . . . . . . . . . . . . . . 20
Section I. qPCR Primer Design and Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Section J. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

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Overview
Chromatin Immunoprecipitation (ChIP) is a powerful tool for studying protein/DNA interactions,
including transcription factors, co-regulatory proteins, modified histones, chromatin-modifying
enzymes and polymerases because it enables identification of the localization of proteins boundto specific DNA loci. When used in combination with whole-genome analysis such as ChIP-Seq or
ChIP-chip, insights are possible into gene regulation, gene expression, mechanisms of chromatin
modification and pathway analysis.
However, ChIP can be technically demanding. The method requires high-quality antibodies torecognize the fixed, target-bound proteins of interest. For many transcription factors, suitable
ChIP-seq validated antibodies are not available or have low binding affinity. Additionally, ChIP
antibodies are often unable to distinguish between protein isoforms, thereby making it difficult to
study the regulatory effects of sequence variants and truncations.
Active Motif’s Tag-ChIP-IT® Kit is designed to overcome these obstacles enabling chromatin
immunoprecipitation of transcription factors, sequence variants and truncated proteins thathave been expressed using Active Motif’s unique AM-tag. The AM-tag is specifically designed tominimize cross-reactivity with mammalian samples, thereby reducing background signal. Anotherbenefit of the AM-tag is that the sequence is unstructured which allows the tag to protrude fromthe protein of interest for maximum exposure during the immunoprecipitation. This increases enrichment efficiency by allowing even low abundance transcription factors to be detected. Simply
clone the AM-tag in frame with the C-terminus of your protein of interest in the expression vectorof your choice. Following transfection and expression of your tagged protein, the Tag-ChIP-IT Kit
can be used to isolate chromatin and perform immunoprecipitation using an antibody directedagainst the AM-tag.
The Tag-ChIP-IT Kit contains sufficient reagents to perform 16 chromatin preparation and immunoprecipitation reactions. A chromatin preparation is defined as one 100 mm cell culture
dish. We recommend the use of Active Motif’s ChIP-IT® qPCR Analysis Kit in combination with the
Tag-ChIP-IT Kit for complete data analysis. Active Motif also offers the pAM_1C_JunD vector for
use as a control. To learn about available qPCR primer sets or Active Motif’s EpiShear™ sonication
devices, please visit our website at www.activemotif.com/chip.

product

formatcatalog no.

Tag-ChIP-IT®

16 rxns53022

pAM_1C Empty Vector

20 µg

53023

pAM_1C_JunD Vector

50 µg

53044

ChIP-IT® qPCR Analysis Kit

10 rxns

53029

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1Flow Chart of Process
Clone gene of interest in framewith C-terminus AM-tag.

Transfect cells for expressionof AM-tagged fusion protein.

Incubate cells 24-72 hours post-transfection.
If desired, treat cells with compounds.

Add AM-Tag Antibody.

Break open cells andshear DNA by sonication.

Cross-link protein to DNA inliving cells with formaldehyde.

Elute chromatin fromcolumn and save flow-through.

Reverse cross-links, digest
with Proteinase K, and DNA purify.
DNA is now ready for analysis.

Capture antibody-boundprotein/DNA complexes and wash
using ChIP Filtration Columns.

Flow Chart of the Tag-ChIP-IT Assay.
Prior to using the Tag-ChIP-IT Kit, the protein of interest is cloned into an expression vector containing the
C-terminal AM-tag, such as Active Motif’s pAM_1C Empty Vector (Catalog No. 53023). Following sequence
verification of the cloned insert, cells are transfected for expression of the AM-tagged fusion protein. Oncetransfection and protein expression conditions have been optimized, chromatin is prepared from the transfected cells using the Tag-ChIP-IT Kit. Intact cells are fixed with a specially formulated formaldehyde buffer,
which cross-links and preserves protein/DNA interactions. DNA is then sheared into small fragments using
sonication and incubated with an antibody directed against the AM-tag. The antibody-bound protein/DNA
complexes are immunoprecipitated through the use of Protein G agarose beads and washed via gravity filtration. Following immunoprecipitation, cross-links are reversed, the proteins are removed by Proteinase K, andthe DNA is recovered and purified. ChIP enriched DNA can be used for either gene-specific or whole-genome
analysis..

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Kit Performance and Benefits
Tag-ChIP-IT Advantages:• Ideal for proteins lacking ChIP-validated antibodies or antibodies with low binding affinities• Study the effects of sequence variants, mutations or truncations on gene regulation• High exposure of AM-tag allows for efficient enrichment of low abundance proteins• AM-tag has little to no cross-reactivity with mammalian systems while optimized reagents
reduce background levels caused by non-specific binding events• Filtration based washes are the easiest wash method available and result in increased
consistency in multi-sample experiments• Reproducible results with proven performance in both qPCR and ChIP-Seq analysis

Figure 1: Tag-ChIP-IT is used to identify Estrogen Receptor binding motifs.
Estrogen Receptor (ER) cDNA was cloned into pAM_1C empty vector (Catalog No. 53023) and sequence verified. Transient transfections were performed into Ishikawa cells. Cells were induced with estradiol (E2) andchromatin was harvested according to the instructions in the Tag-ChIP-IT Kit. The AM-Tag antibody was used
to immunoprecipitate the cross-linked AM-tag-ER fusion protein. Following reversal of cross-links, enriched
DNA was submitted for next generation sequencing. Data was compared to published ChIP-Seq results using
an anti-ER antibody in the same cell line and induction conditions. ChIP-Seq data shows the same ER peak
profile with the AM-tag immunoprecipitation as endogenous ER. The binding sites detected with the AM-tag
samples were further evaluated for binding motifs. Results show that the estrogen receptor motif was identified in both Tag-ChIP-IT samples.
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Figure 2: Specificity of AM-Tag Antibody.
ChIP-Seq was performed with the AM-Tag Antibody on chromatin prepared from Human samples. The
sequencing results show an absence of DNA binding peaks which indicates there is little to no cross-reactivityof the AM-tag with human chromatin, thereby eliminating background signal in the Tag-ChIP-IT assay.

Tag-ChIP-IT Protocol Overview and Time Table
Required Time
Cell Fixation and Lysis

1.5 hours

Chromatin Sonication

15 minutes per sample

Assessment of Chromatin Size

4.5 hours

Immunoprecipitation

Overnight incubation

Binding to Protein G agarose Beads

3 hours

Wash Immune Complexes

20 minutes

Reversal of Cross-links

2.5 hours

DNA Purification

15 minutes

qPCR Analysis

2 hourswww.activemotif.com

4Kit Components and Storage
Please store each component at the temperature indicated in the table below. Do not re-freeze
the Protein G Agarose Beads. Once thawed, Protein G beads should be stored at 4°C.
Reagents

Quantity

Storage

AM-Tag Antibody (rAb)

160 µl-20°C

RNase A (10 µg/µl)

40 µl

-20°C

Proteinase K (10 µg/µl)

200 µl-20°C

Blocker

100 µl-20°C

100 mM PMSF

500 µl-20°C

Protease Inhibitor Cocktail (PIC)

500 µl-20°C

Precipitation Buffer

1.5 ml-20°C

Carrier

35 µl

-20°C

10X PBS

120 ml-20°C

Fixation Buffer

2 x 1.5 ml

4°C

Protein G Agarose Beads*

500 µl4°C

TE pH 8.0

2 x 1.5 ml

RTDetergent

25 ml

RT

5 M NaCl

400 µlRT

Stop Solution

20 ml

RT

Chromatin Prep Buffer

85 ml

RT

ChIP Filtration Columns

16 ea

RT

ChIP Buffer

35 ml

RT

Wash Buffer AM1

100 mlRT

Elution Buffer AM4

2 x 1.5 ml

RT

DNA Purification Binding Buffer

50 ml

RT

3 M Sodium Acetate

500 µlRT

DNA Purification Wash Buffer**

10 ml

RT

DNA Purification Elution Buffer

5 ml

RT

DNA Purification Columns

16 ea

RT

* The Protein G Agarose Beads are shipped on dry ice and can be stored frozen until their firstuse. Once thawed, the Protein G beads should not be re-frozen by the customer. Store beads at
4ºC.
**Requires the addition of ethanol before use.
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5Additional materials required• Stable cells or transiently transfected cell line expressing the AM-tag fusion protein construct. See Appendix, Sections G–H for details on vector design and transfection.
• Dounce homogenizer with a small clearance pestle (e.g. Active Motif Catalog Nos. 40401& 40415) with the tight-fitting “A” pestle). Use of a homogenizer is necessary for shearing
chromatin.
• 37% formaldehyde solution with 10-15% methyl alcohol to prevent polymerization. Do not
use paraformaldehyde.
• 100% ethanol (absolute)• 70% ethanol• DNase-free H2O• Rocking platform for culture plates• Apparatus to rotate tubes end-to-end at 4°C (e.g. a Labquake from Barnstead/Thermolyne
with a tube holder for 1.5 ml microcentrifuge tubes)• Microcentrifuge (table top centrifuge 4°C) and microcentrifuge tubes• 250 µl PCR tubes• Thermocycler• 15 and 50 ml conical tubes• Spectrophotometer for DNA quantitation• Pipettors and tips (filter tips are recommended)• Sonicator (e.g. Active Motif’s EpiShear™ Sonicator with a 1/8” probe (Catalog No. 53051) with
the EpiShear™ Cooled Sonication Platform (Catalog No. 53080))• Agarose gel electrophoresis apparatus• Cell scraper (rubber policeman)• (Optional) ChIP-IT® qPCR Analysis Kit (Catalog No. 53029)• (Optional) Gene-specific qPCR primer pairs for enrichment analysis; see Appendix Section I• (Optional) SYBR Green qPCR master mix (Bio-Rad Catalog No. 170-8882)

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Protocols – Experimental Set Up
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Chromatin Preparation
Tag-ChIP-IT requires 20-30 µg chromatin isolated from stable cells or transiently transfected cells
expressing the AM-tag fusion protein. For details on preparing cells for use in Tag-ChIP-IT, pleaserefer to Appendix Sections G–H on page 20.
Chromatin should be prepared 24-72 hours post-transfection, or when the cells reach 70-80%
confluency. Refer to the table below to scale buffer volumes for chromatin preparation.
60 mm
dish

100 mmdish

150 mmdish

Seeding Density

0.8 x 106

2.2 x 106

5.0 x 106

Cells at 70-80%
Confluency*

2.4 x 10

6.6 x 1015.0 x 106

Growth Medium
Volume

5 ml

10 ml

20 ml

Cell Fixative Solution

500 µl

1 ml

2 ml

Stop Solution

275 µl

550 µl1.1 mlPBS Wash Buffer(used per wash)

2 ml

5 ml

10 ml

Chromatin Prep
Buffer

2 ml

5 ml

5 ml

ChIP Buffer

500 µl500 µl500 µl6

6* The number of cells on a confluent plate or dish will vary with cell type. For this table, HeLa
cells were used. Please adjust as needed based on your particular cell type.
** Please refer to the descriptions below for complete details on buffer preparations

Buffer Preparation
Complete Cell Fixation Solution
Buffer should be prepared fresh before each experiment. For every 10 ml of cell growth medium
used, prepare 1.25 ml of Complete Cell Fixation Solution by adding 90 µl Fixation Buffer to 785µl sterile water in a 15 ml conical tube. Using appropriate precautions (i.e. safety glasses, gloves
and lab coat), add 375 µl 37% formaldehyde to the tube and vortex to mix. Use 1/10 growth
medium volume per plate. Complete cell fixation solution can be added to the growth medium inthe presence or absence of serum.
Stop Solution
Is provided ready to use. Use 1/20 media volume per cell culture plate.

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PBS Wash Buffer
Prepare 12 ml PBS Wash Buffer for every 100 mm dish. To a 15 ml conical tube add 10.2 ml sterilewater, 1.2 ml 10X PBS and 0.6 ml Detergent. Mix by inverting. Place PBS Wash Buffer on ice tochill. PBS Wash Buffer can be prepared in large quantities and stored at 4ºC for 6 months.
100 mM PMSF and Protease Inhibitor Cocktail (PIC)
Thaw the PMSF and the PIC at room temperature until fully dissolved, which takes about
30 minutes. Vortex gently and spin down briefly before use, then add to the buffers immediately
before use.
Chromatin Prep Buffer
Is supplied ready to use.
ChIP Buffer
Is supplied ready to use.
DNA Purification Wash Buffer
The DNA Purification Wash Buffer requires the addition of ethanol before use. The final concentration of ethanol should be 80%. Add 40 ml of fresh 100% ethanol to the DNA Purification Wash
Buffer bottle. Invert repeatedly. The buffer can be stored at room temperature after the additionof ethanol. The ethanol only needs to be added before the first use, after that the Wash Buffer isready for use.
3M Sodium Acetate
It is important to check the sodium acetate before use to ensure that the salts have notprecipitated out of solution. Once the sodium acetate is in solution it should be stored at room
temperature.
Protein G Agarose Beads
The supplied agarose beads require washing prior to use. Follow the instructions in the manual
to wash the beads for use in the ChIP reactions. There is no need to pre-block the beads or
pre-clear the sample. For best results, gently shake and invert the tube to resuspend the agarose
beads. The beads settle quickly, and therefore should be resuspended just before pipetting. We
recommend cutting 2 mm from the end of a pipet tip prior to pipetting to prevent the tip frombecoming clogged. Protein G Agarose Beads are shipped on dry ice and can be stored frozen
until their first use. Once thawed, beads should not be re-frozen by the customer. Protein G
Agarose Beads should be stored at 4ºC.
AM-Tag Antibody (rAb)
We recommend using 10 µg AM-Tag antibody per ChIP reaction. The AM-Tag antibody is provided
at a concentration of 1 µg/µl, so you will need to use 10 µl per immunoprecipitation.

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Recommendations
Chromatin Shearing Tips
We suggest using a probe sonicator (i.e. Active Motif’s EpiShear Probe Sonicator) which employs
a direct sonication method to prepare chromatin for use in the Tag-ChIP-IT Kit. Indirect sonication systems may require longer sonication times to achieve optimal chromatin shearing. ChIP
experiments usually require chromatin that has been sheared to a size of 200-1200 bp. In general,
shearing efficiency is improved through the use of a small shearing volume and a V-bottom tube
rather than a round-bottom tube. Also, note that shearing is inefficient if the chromatin sample
becomes emulsified with air bubbles. To determine the appropriate shearing level for your sample,
set up a “practice” tube containing only ChIP Buffer. Slowly increase the sonication amplitude until
foaming starts to occur. Reduce the amplitude setting down slightly and mark this as the highest
possible intensity to use without foaming. If a chromatin preparation becomes emulsified inadvertently, discontinue shearing and centrifuge the sample at maximum speed for 4 minutes at 4ºC ina microcentrifuge to remove trapped air. Finally, to prevent overheating and denaturation of chromatin, samples should be kept on ice as much as possible during shearing, and shearing should be
performed discontinuously (i.e. sonicate for 20 seconds, then place on ice/water for 30 seconds,
sonicate again for 20 seconds, etc.). If possible, shear while on ice or use Active Motif’s EpiShear™
Cooled Sonication Platform (Catalog No. 53080) to help regulate sample temperature.
Chromatin Quantity
It is recommended to use 20-30 µg chromatin per IP reaction. If ChIP-Seq will be performed
following enrichment, please read the recommendations listed in Section F on page 18 prior tostarting the protocol.
Safety Precautions
Formaldehyde and PMSF are highly toxic chemicals. Appropriate safety precautions (i.e. safety
glasses, gloves and lab coat) should be used. Also, formaldehyde is highly toxic by inhalation andshould be used only in a ventilated hood. Finally, chromatin sonication should be performed in abiosafety hood if the chromatin is extracted from biohazardous or infectious materials.

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Protocols – Preparation of Sheared Chromatin
Section A: Cell Fixation Starting with Cultured Cells
This protocol describes cell fixation and chromatin preparation from one 100 mm dish (approximately 6.6 x 106 cells) containing stable cells or transiently transfected cells expressing the
AM-tag fusion protein. We recommend using 10 ml growth medium per 100 mm dish. Please referto page 7 for information on scaling the protocol for use with other amounts of cells.
1.

Follow the recommendations outlined in Appendix Sections G-H for the cloning andtransfection of your protein of interest expressing the AM-tag. We recommend using
untransfected cells to provide an estimate of background. Prepare chromatin 24-72 hours
post-transfection, or when the cells reach 70-80% confluency.

2.

Freshly prepare Complete Cell Fixation Solution for each 100 mm dish. The volumes listed
below are enough to process one 100 mm dish. Please refer to the chart on page 7 to scale
the solution volumes.

3.

To fix cells, add 1/10 growth medium volume of freshly prepared Complete Cell Fixative
Solution to the existing culture media for the cells (e.g. 10 ml growth medium would get
1 ml Complete Cell Fixation Solution). Shake gently at room temperature for 15 minutes.

4.

Stop the fixation reaction by adding 1/20 media volume of Stop Solution to the existing
culture media for the cells (e.g. 10 ml growth medium would get 550 µl Stop Solution). Swirlto mix and incubate at room temperature for 5 minutes.

5.

Following the incubation, hold the plate at an angle and using a rubber policeman scrape
cells down to collect them at the bottom edge of the plate. Use a pipette to transfer thecells to a 50 ml conical tube on ice.

6.

Pellet the cells from step 5 by centrifugation for 3 minutes at 1,250 x g at 4°C.

7.

Remove the supernatant and discard. Resuspend the pellet(s) in 5 ml ice-cold PBS Wash
Buffer by pipetting up and down. Keep samples ice-cold for the remainder of the procedure.

8.

Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. Wash thepellet(s) a second time in 5 ml ice-cold PBS Wash Buffer by pipetting up and down. Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. (Cell pellets
may be stored at -80°C at this stage).

9.

Resuspend each pellet(s) in 5 ml Chromatin Prep Buffer supplemented with 5 µl PIC and5 µl 100 mM PMSF. Pipet up and down to mix.

10. Incubate on ice for 10 minutes.
11.

Transfer the resuspended pellets individually to a chilled dounce homogenizer on ice.
Use the tight fitting pestle (Type A) to homogenize the sample for 30 strokes. Transfer thecontents to a new 15 ml conical tube and centrifuge for 3 minutes at 1,250 x g at 4°C.
Monitor Cell Lysis: To ensure cell lysis, take 10 µl of the cell lysate from the dounce andlook at it under a phase contrast microscope using a hemocytometer to verify that thenuclei have been released. It is often helpful to look at the cells before and after the lysis
step as this makes it easier to identify the nuclei versus whole cells. Intact cells should havewww.activemotif.com

10

a dark central region (nucleus) surrounded by a halo of less dense cytoplasm. In lysed cells,
the nuclei will appear as dots surrounded by asymmetric debris. If the cells are not lysed,
then dounce on ice with an additional 10 strokes, or until the cells are lysed.
12. Remove the supernatant and discard. Resuspend each pellet in 500 µl ChIP Buffer supplemented with 5 µl PIC and 5 µl 100 mM PMSF. Transfer the contents to a new 2 ml microcentrifuge tube.
13. Incubate on ice for 10 minutes. Proceed to Step B: Chromatin Sonication of Cultured Cells.

Section B. Chromatin Sonication of Cultured Cells
The section below describes the fragmentation of chromatin using sonication. Sonication results
may vary depending on cell type and sonication device being used. This protocol has been validated using Active Motif’s EpiShear™ Probe Sonicator in combination with an EpiShear™ Cooled
Sonication Platform to maintain probe height and temperature consistency between samples.
We do not recommend sonication of samples containing less than 100,000 cells and/or 350 µl
volume.
The ChIP Buffer has been optimized for immunoprecipitation performance, however, due to its
unique composition optimization of sonication conditions may be required. To maintain the highsensitivity of the assay, we recommend using our buffer system and altering the sonication time
and/or amplitude of your sonication system to achieve the desired fragmentation (e.g. somesystems may require as much as a three-fold increase in sonication time to improve chromatin
shearing). Please pay particular attention to our protocol regarding the processing of input chromatin for agarose gel analysis prior to the chromatin immunoprecipitation reaction as many steps
may differ from traditional ChIP protocols and failure to follow the outlined procedure may leadto artifacts in the gel images as shown in Figure 3 on page. 13
1.

Place the 2 ml microcentrifuge tube containing the chromatin into the tube cooler or packed
ice. Open cap and submerge the microtip into the liquid until the microtip is approximately
5 mm from the bottom of the tube. Sonicate according to optimized settings for the cell typebeing used (see Recommendations on page 9). A recommended starting range for cultured
cells is: 25% amplitude, pulse for 30 seconds on and 30 seconds off for a total sonication“on” time of 10 minutes (or 20 minutes elapsed time).

2.

Spin tubes at 4°C in a microcentrifuge at maximum speed for 2 minutes to pellet the cellulardebris.

3.

Transfer 25 µl of each chromatin preparation into a 250 µl PCR tube for analysis of shearing
efficiency and chromatin quantification. This sample will be used to generate the Input DNA.

4.

Aliquot the remainder of each chromatin preparation into 1.5 ml microcentrifuge tubes. We
recommend making aliquots of 150 µl volume and storing at -80°C.
Note:

The size of the chromatin sonication should be verified before proceeding to
the immunoprecipitation step.

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11Input Preparation
5. To each 25 µl chromatin preparation from Step 3 above, add 175 µl TE pH 8.0 and
1 µl RNAse A. Cap the PCR tubes and vortex to mix
6.

Incubate in a thermocycler at 37°C for 30 minutes.

7.

Add 2 µl Proteinase K to each tube and vortex. Incubate tubes in a thermocycler at 55°C for
30 minutes and then increase the temperature to 80°C for 2 hours.

8.

Transfer each chromatin input to a 1.5 ml microcentrifuge tube. Add 83 µl Precipitation Buffer, 2 µl Carrier and 750 µl absolute ethanol. Vortex to mix and chill at -80°C for 30 minutes
to overnight.

9.

Spin tubes at 4°C in a microcentrifuge at maximum speed for 15 minutes.

10. Carefully remove the supernatant taking care not to disturb the pellet. Wash the pellet with
500 µl 70% ethanol and spin at 4°C in a microcentrifuge at maximum speed for 5 minutes.
11.

Carefully remove the supernatant taking care not to disturb the pellet. Remove residual
ethanol with a pipet tip. Leave the tubes uncapped and air dry for 10-15 minutes.

12. When the pellets are dry, add 25 µl DNA Purification Elution Buffer to each tube. Incubate atroom temperature for 10 minutes. Then vortex to ensure the pellet is completely resuspended. This solution contains your Input DNA.
13. Read the absorbance of each sample on a NanoDrop or other spectrophotometer at
260 nm to determine the DNA concentration of each chromatin preparation. Set aside
500 ng of DNA for analysis as described in Step 14. Store the remaining Input DNA at -20°C.
14. Analyze each chromatin preparation on an agarose gel by following the instructions below.
a.

Prepare 500 mM NaCl by adding 2 µl 5M NaCl to 18 µl sterile water. Vortex to mix.

b.

Transfer 500 ng of Input DNA to a 250 µl PCR tube and add 1 µl 500 mM NaCl. Adjust
the final volume to 10 µl with sterile water if needed.

c.

Heat samples in a thermocycler at 100°C for 20 minutes followed by ramping thetemperature down to 50°C.

d.

Remove tubes from the thermocycler and incubate at room temperature for 5 minutes.

e.

Add gel loading buffer to each sample and run on a 1.5% agarose gel. Include 100 bp
and 1 kb DNA ladders to analyze chromatin size. DNA should appear as a smear anywhere between 200-1200 bp.

Note:

Chromatin prepared using the Tag-ChIP-IT protocol may look different on an
agarose gel compared to chromatin prepared using traditional ChIP methods.
However, this will not affect the sensitivity of the assay or increase backgroundsignal. Please follow the protocol as listed above for preparing Input DNA. Useof an alternative reverse cross-linking method or omitting the 20 minute incubation at 100°C in NaCl is not recommended as this will cause artifacts that makethe DNA appear larger. As long as the chromatin falls within the recommended
200-1200 bp range, proceed with the ChIP reaction. If fragments do not fallwithin this range sonication conditions should be further optimized.

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1215. If chromatin preparations were successful, the aliquots stored at -80°C from Section B, Step
4 can be used to perform the ChIP reactions in Section E.

Figure 3: Validation of chromatin shearing efficiency following reversal of cross-links at 80°C for 2 hours.
Chromatin preparations of MCF-7 cells were fixed and sonicated using the EpiShear™ Probe Sonicator and
EpiShear™ Cooled Sonication Platform from Active Motif. Input DNA was prepared in duplicate according to
Section B, Steps 5-13 in the manual. In Sample 1, Step 14 was not performed and 500 ng of input DNA wasloaded directly onto a 1.5% agarose gel without receiving the addition of NaCl and incubation at 100°C. The
omission of Step 14 has caused a buffer artifact that makes the DNA appear larger on a gel. The duplicate
sample, Sample 2, was processed according to the manual instructions and included the addition of NaCl andincubation at 100°C as stated in Step 14. Analysis of 500 ng of this input DNA on a 1.5% agarose gel showsthe expected fragmentation between 200-1200 bp. The difference in DNA sizing on the gel between the two
samples illustrates the importance of following the protocol recommendations regarding the processing of
input chromatin for agarose gel analysis prior to chromatin immunoprecipitation. Omission of key steps canlead to inaccurate analysis of chromatin shearing efficiency. If the protocol steps were followed and the DNA
fragments fall outside of the recommended range, sonication conditions should be further optimized.

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13

Protocols – Chromatin Immunoprecipitation
Section C. Immunoprecipitation
Successful chromatin immunoprecipitation depends on the efficiency of the transfection, theexpression levels of the AM-tag fusion protein of interest and the ability of the tagged protein tocompete against endogenous protein for chromatin binding sites. It is recommended to optimize
the transfection and expression of tagged protein prior to performing the immunoprecipitation.
1.

Thaw sonicated chromatin on ice. Spin chromatin at 4°C in a microcentrifuge at maximum
speed for 2 minutes.

2.

Set up the ChIP reactions by adding the components in the order shown in Table 1 below
to 1.5 ml microcentrifuge tubes. Be sure to use the DNA concentration that was determinedfor your sonicated chromatin sample to calculate the volume to use. We recommend using
20-30 µg chromatin per ChIP reaction.

3.

In a 1.5 ml microcentrifuge tube prepare the AM-Tag Antibody. To the tube add 5 µl Blocker
and 10 µg AM-Tag antibody. Incubate Antibody/Blocker mix for 1 minute at room temperature and then add to the ChIP reactions.
Table 1

Reagent

1 reaction

Sheared Chromatin (20 - 30 µg)

X µl

ChIP Buffer

adjust up to 200 µl

Protease Inhibitor Cocktail (PIC)

5 µl

Antibody/Blocker mix (from Step 3)

15 µl

Maximum Volume Allowed

240 µl4.

Cap tubes and incubate on an end-to-end rotator overnight at 4°C.

5.

The Protein G agarose beads require washing before use. Transfer 30 µl Protein G agarose
beads for each IP reaction to a 1.5 ml microcentrifuge tube. Add an equal volume of TE,
pH 8.0 and invert to mix. Spin at 1250 x g in a microcentrifuge for 1 minute. Remove thesupernatant equivalent to the volume of TE added to the agarose beads.
Note:

Before pipetting the Protein G agarose beads, they should be fully resuspended
by inverting the tube. When pipetting the beads, cut 2 mm from the end of apipet tip to prevent the tip from becoming clogged.

6.

Wash the beads a second time with the same volume of TE, pH 8.0. Invert to mix. Spin
at 1250 x g for 1 minute in a microcentrifuge. Remove the supernatant equivalent to thevolume of TE added to the agarose beads. The beads are now ready to use.

7.

Spin the ChIP reactions at 1250 x g for 1 minute to collect liquid from the inside of the cap.

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14

8.

Using a cut pipet tip, add 30 µl washed Protein G agarose beads to each immunoprecipitation reaction. Cap tubes and incubate on an end-to-end rotator at 4°C for 3 hours.

9.

Label a ChIP Filtration Column for each ChIP reaction. Remove the tab from the bottom of
the column and place in an empty 1 ml pipet tip box as a holder (see Figure 4 below).

10. Remove ChIP reactions from rotator and spin at 1250 x g for 1 minute to collect liquid frominside of the cap.
11.

Add 600 µl ChIP Buffer to each ChIP reaction then transfer the entire reaction (including theprotein G agarose beads) to its labeled column. Allow flow-through to occur by gravity.

12. During the gravity flow, transfer 100 µl per ChIP reaction of Elution Buffer AM4 to a 1.5 ml
microcentrifuge tube and allow to pre-warm at 37°C during the wash steps.
13. Wash each column with 900 µl Wash Buffer AM1. Let stand for 3 min.
14. Repeat Step 13 four more times for a total of five washes.
15. Transfer columns to a new 1.5 ml microcentrifuge tube and spin in a room temperature
microcentrifuge at 1250 x g for 3 minutes to remove residual Wash Buffer.
16. Following the spin, transfer the ChIP Filtration Columns to new 1.5 ml microcentrifuge tubes.
Add 50 µl 37°C Elution Buffer AM4 to each column. Incubate at room temperature for5 minutes. Spin in a room temperature microcentrifuge at 1250 x g for 3 minutes.
17.

With columns remaining in the same microcentrifuge tube, add another 50 µl 37°C Elution
Buffer AM4 to each column. Incubate at room temperature for 5 minutes and spin in a room
temperature microcentrifuge at 1250 x g for 3 minutes.

18. Discard the ChIP Filtration Columns. The flow-through (~100 µl volume) contains the ChIP
DNA. Proceed to Section F: DNA purification.

Figure 4: Using the ChIP Filtration Columns.
Remove the tab from the bottom of the ChIP Filtration Columns and place columns in an empty 1 ml pipet tip
box to perform the wash steps.

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15

Protocols – ChIP DNA Purification
Section D. Reversal of Cross-links and DNA Purification
1.

Transfer each eluted ChIP DNA to a 250 µl PCR tube and add 2 µl Proteinase K. Vortex tomix and heat in a thermocycler at 55°C for 30 minutes and then increase the temperature to
80°C for 2 hours.

2.

Transfer the DNA to a 1.5 ml microcentrifuge tube and add 5 volumes (500 µl) DNA Purification Binding Buffer to each tube and vortex to mix. Adjust the pH with 5 µl 3M Sodium
Acetate. The sample should be bright yellow in color to indicate a proper pH. If your sample
is not bright yellow, please refer to the Troubleshooting guide in the Appendix on page 24
for details to adjust pH prior to loading the sample into the purification column.

3.

For each sample, place a DNA purification column (AM #103928) in the collection tube andadd each pH adjusted sample to its own column. Close the cap on each column, place themwith the collection tubes in a microcentrifuge and spin them at 14,000 rpm for 1 minute.

4.

Remove the column from the collection tube, then remove and discard the flow throughfrom the collection tube. Return the column to the collection tube.

5.

Prepare DNA Purification Wash Buffer (AM #103497) before the first use. Follow the instruction on page 8 for the addition of ethanol prior to using the solution. Add 750 µl DNA
Purification Wash Buffer to each column and cap the column.

6.

Spin at 14,000 rpm for 1 minute in a microcentrifuge.

7.

Remove the column from the collection tube, then remove and discard the flow throughfrom the collection tube. Return the column to the collection tube.

8.

With the column cap open, spin at 14,000 rpm for 2 minutes in a microcentrifuge to remove
any residual Wash Buffer from the column.

9.

Transfer the column to a clean microcentrifuge tube. Depending on the method to be used
for downstream analysis of the ChIP enriched DNA, use the appropriate elution volume. For
ChIP-Seq or ChIP-chip applications, we recommend pre-warming the required elution buffer
volume at 37°C for 5 minutes prior to use.
a.

For qPCR analysis: add 100 µl of DNA Purification Elution Buffer (AM #103498) to thecenter of the column matrix and incubate for 5 minutes at room temperature. Spin at
14,000 rpm for 1 minute in a microcentrifuge. Add an additional 100 µl DNA Purification
Elution Buffer to the column and incubate for 5 minutes at room temperature. Spin at
14,000 rpm for 1 minute in a microcentrifuge. Total elution volume is 200 µl.

b.

For ChIP-Seq or ChIP-chip analysis: add 36 µl of 37°C DNA Purification Elution Buffer(AM #103498) to the center of the column matrix and incubate for 5 minutes at room
temperature. Spin at 14,000 rpm for 1 minute in a microcentrifuge.

10. Discard column. Purified DNA may be stored at -20°C for future use.

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16

Protocols – ChIP DNA Analysis
Section E. Quantitative PCR (qPCR)
ChIP DNA can be analyzed on a gene-specific basis using quantitative PCR (qPCR). Positive
control and negative control PCR primer pairs should be included in every analysis to determine
the fold enrichment. Negative control primers will amplify a region of the genome not bound by
the antibody target of interest. Active Motif recommends the use of its ChIP-IT® qPCR Analysis
Kit (Catalog No. 53029) for the analysis of qPCR data. The ChIP-IT qPCR Analysis Kit contains
positive and negative control primer pairs, standard curve DNA, standard curve primers and aqPCR Analysis spreadsheet to perform the analysis calculations. Active Motif’s analysis strategy
determines primer efficiencies and the ChIP sample values are normalized according to input,
primer efficiency, chromatin amount used in the ChIP reaction and resuspension volume. The
ChIP-IT qPCR Analysis Kit provides consistency in data analysis and allows direct comparison
across samples and experiments. If not using the ChIP-IT qPCR Analysis Kit, qPCR data normalization and graphing can be done using the methods described in Section I.
1.

Below is an example qPCR reaction. Please follow the specific instructions for your qPCR
instrument. We recommend using a commercially available SYBR Green qPCR master mix(e.g. Bio-Rad Cat # 170-8882) and preparing triplicate reactions.
Reagent

20 µl PCR reactions

2X SYBR Green master mix

10 µl

Primer mix (2.5 µM each primer)*

4 µl

Sterile water

1 µl

DNA sample (ChIP or Input)

5 µl

Total volume

20 µl* We recommend designing primers to perform at an annealing temperature of 58°C so thatall qPCR reactions can be performed under identical conditions. An amplicon length of
75-150 bp is recommended. Active Motif offers validated species-specific qPCR primers
designed according to these recommendations at www.activemotif.com/chipprimers.
2.

Place tubes in a real time PCR instrument and program as below:
95°C for 2 minutes(95°C for 15 seconds, 58°C for 20 seconds, 72°C for 20 seconds) for 40 cycles

3.

If using the ChIP-IT qPCR Analysis Kit, please refer to the product manual for analysis
instructions. Otherwise, follow the recommendations in Section I.

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17Section F. ChIP-Seq
Genome-wide analysis with Next Generation sequencing involves the preparation of libraries
from ChIP DNA by the addition of adapter sequences to the ends of the DNA fragments. The
library is then PCR amplified and validated prior to sequencing.
Commercial kits are available to prepare the ChIP DNA libraries for sequencing. Please select anappropriate library preparation method compatible with the sequencing apparatus to be used.
A. General Recommendations• We recommend using 30 µg chromatin for the ChIP Reactions, although less may be used if
chromatin amounts are limiting or you are enriching for a highly abundant protein• Library generation usually requires 10 ng of ChIP enriched DNA. However, due to the low
background level with the Tag-ChIP-IT Kit lower amounts can be used when working withlow abundance transcription factors. (See B. ChIP-Seq Requirements below for moredetails)• Sequence library yields should be in the range of 1-2 micrograms• 30 million sequencing reads is sufficient for most transcription factors• 36 bp single end reads are sufficient for unique mapping and good ChIP-Seq data, although
longer reads can be used
• Input DNA should be sequenced as a control reaction in order to identify false “peaks”
and also to reveal regions of the genome that have been duplicated. Subtracting the input
peaks from the experimental peaks will help to eliminate false data. Use 50 ng Input DNA(from Section B Step 12 for cultured cell samples) for library generation.
B. ChIP-Seq Requirements
It is commonly recommended that library generation of ChIP DNA requires 10 ng of ChIP enriched
DNA. However, 10 ng is an unrealistic number for a sequence specific DNA binding transcription
factor. Using the Tag-ChIP-IT Kit, we have generated high quality libraries from sub-nanogramlevels of ChIP DNA due to the high quality of the DNA obtained. For good ChIP-Seq data, quality
enrichments with low background is more important than the total quantity of DNA recovered.
Therefore, we recommend performing qPCR on known binding sites to verify enrichment levels
against a negative control primer set using Active Motif’s ChIP-IT qPCR Analysis Kit (Catalog No.
53029) to confirm DNA quality rather than quantifying the DNA.
C. qPCR prior to ChIP-Seq
To perform qPCR for verification of the quality of the enriched ChIP DNA, follow the instructions
for qPCR in Section E on page 17 with the following modification. The ChIP DNA will need to bediluted prior to use in qPCR due to the reduced elution volume. Dilute 6 µl of ChIP DNA in 94 µl
DNA Purification Elution Buffer (AM #103498). Use 5 µl of the diluted ChIP DNA per qPCR reaction.
Quality DNA that is suitable for use in ChIP-Seq should show enrichment of known binding sites
over negative control primer sets of 5-fold (See Figure 5). If the qPCR enrichments are satisfactory then the remaining 30 µl of ChIP DNA can be used for library generation.
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Binding events detected / 1,000 cells

Poor Quality ChIP DNA
ess than -foldenrichment

Cell Line A

4
2

Cell Line B

ow signal
strengthigh background4
2
Negative Control primer

Positive Control primer

Binding events detected / 1,000 cells

High Quality ChIP DNA
Cell Line A

4
2

4
2

Cell Line B

ow background and morethan -fold enrichment

Negative Control primer

Positive Control primer

Figure 5: Comparison of qPCR results showing good versus poor enrichment over negative control primers.
Data shows qPCR results analyzed using the ChIP-IT qPCR Analysis Kit with the Human Negative Control Primer as a reference and a gene-specific positive control primer. In the top image the negative control primer set
for Cell Line A gives high background levels with Binding events detected per 1,000 cells above a value of 2,
while Cell Line B has positive control signal levels below 5 Binding events detected per 1,000 cells. The bottom image shows low background in the negative control primer set with Binding events detected per 1,000
cells below a value of 2. The fold enrichment of the positive control primer set exceeds 5-fold. Only the ChIP
DNA from the bottom sample is recommended for use in ChIP-Seq.

D. Library Generation
ChIP DNA from the TAG-ChIP-IT Kit has been validated for ChIP-Seq using Active Motif’s Next Gen
DNA Library and Next Gen Indexing Kits (Catalog Nos. 53216 & 53264).

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19

Appendix
Section G: Prepare the AM-tag Fusion Protein Construct
Active Motif’s Tag-ChIP-IT Kit requires stable cells or transiently transfected cells expressing the
AM-tag. The following outlines the design and preparation of the AM-tag vector.
1.

Using Active Motif’s pAM_1C Empty Vector (Catalog No. 53023), design a fusion protein
construct containing the AM-tag in-frame with the C-terminus of the protein of interest. We
recommend the use of directional cloning methods such as InFusion® (Clontech) or Gibson
Assembly® (New England BioLabs). Follow the manufacturer’s recommendations for cloning.
Alternatively, the AM-tag sequence can be cloned into the expression vector of choice foryour protein of interest. Ensure that the open reading frame of interest begins with the ATG
start codon, that the AM-tag remains in-frame with the C-terminus and that a stop codon ispresent. We do not recommend the use of the AM-tag at the amino-terminus of the protein
as this may significantly reduce expression efficiency. If you require a N-terminal tag, pleasecontact Active Motif’s technical support.
AM-tag: 5´– TGCCAAGATCCTCAACGCAAAGGCAACGTGATACTCTCTCAGGCTTACGGGTGCCAAGATCCTCAACGCAAAGGCAACGTGATACTCTCTCAGGCTTAC – 3´

2.

Perform transformations of the AM-tag fusion protein construct into competent E.coli. We
recommend the use of Active Motif’s Rapid Trans TAM1 Competent cells (Catalog No. 11096).
Use the appropriate antibiotic selection for the cloning vector chosen (e.g. pAM_1C contains
the ampicillin resistance gene).

3.

Select 2-4 colonies for each construct and isolate the plasmid DNA. Sequence validate
each plasmid to confirm the correct open reading frame sequence. We suggest preparing aglycerol stock for each candidate construct.

5.

Determine the amount of DNA needed for transfection. If necessary, prepare additional
DNA. Ensure that the DNA is free of nucleases or other contaminants prior to transfection.
Quantify the DNA.

Section H: Transfection of Cells with AM-tag Construct
Following sequence validation of the AM-tag cloning vector, cells can be transiently transfectedor selected to make stable cells expressing the AM-tagged protein of interest. Chromatin will
be prepared from these cells for analysis in the Tag-ChIP-IT Kit (Catalog No. 53022). Successful
chromatin immunoprecipitation depends on the efficiency of the transfection and the expression
levels of the AM-tag fusion protein of interest. It is recommended to optimize the transfection andexpression of tagged protein prior to performing the chromatin immunoprecipitation.
Once transfection conditions have been optimized, calculate the number of chromatin preparations you will require and determine the number of ChIP reactions you plan to perform on eachchromatin preparation. Be sure to include the appropriate control ChIP reactions in your calculations. We recommend using untransfected cells (or a non-expressing cell line) as an estimate
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20

of the background. It is important to transfect enough cells to yield the necessary 20-30 µg ofchromatin per immunoprecipitation reaction.
We recommend using FuGENE® HD Transfection Reagent (Catalog No. 32042) with the pAM_1C
vector at a ratio of 3:1 (µl FuGENE : µg DNA vector). Optimization may be required for each cellline and construct tested. Below are some general guidelines.
1.

In a 100 mm dish, seed 2.2 x 106 cells per plate in the appropriate growth medium. Incubate
in a humidified incubator for 24 hours.
(Optional) Prepare transfection reactions to verify expression of the AM-tagged fusion
protein. We suggest performing a time course experiment to evaluate tagged-protein
expression levels at 24, 48 and 72 hours post-transfection to identify the optimal harvest
time point for the protein of interest. Cell lysates can be prepared from these transfection
reactions for analysis by Western blot. Use the AbFlex™ AM-Tag antibody (Catalog No. 91111)
at a 1:500 dilution for detection of the tagged protein as compared to untransfected cells(Figure 6).

2.

Prepare a separate microcentrifuge tube for each transfection reaction. Add 10 µg AM-tag
construct to Opti-MEM media in a final volume of 550 µl.

3.

Add 30 µl FuGENE HD Transfection Reagent drop wise directly to the media/DNA mixture.
Do not allow FuGENE to come directly into contact with the plastic from the tube. Mix thesolution by pipetting up and down and incubate at room temperature for 15-30 minutes.

4.

Add 580 µl of the media/DNA/FuGENE mixture drop wise to each 100 mm dish. Gently swirl
dish or incubate on a shaker at 100 rpm for 2 minutes to evenly distribute transfection
mixture.

5.

Return dish to humidified incubator for 24-72 hours before proceeding with the chromatin
preparation. If desired, treat with compounds.

6.

Follow the protocol on page 7 of the manual to prepare chromatin for use in Tag ChIP-IT.

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Figure 6: Validation of protein expression by WB.
Active Motif’s pAM_1C_JunD vector was prepared containing
the DNA sequence for transcription factor JunD in-frame with aC-terminal AM-tag. Hela or HCT116 cells were seeded at 2.2 x
106 cells and transfected with 10 µg pAM_1C_JunD DNA, mocktransfected or transfected with 10 µg pAM_1C_Empty Vector. 48
hours post-transfection nuclear lysates were prepared using Active
Motif’s Nuclear Extract Kit (Catalog No. 40010). 20 µg lysate was
loaded per lane..
Lane M: Molecular weight marker (kDa)
Lane 1: Mock transfection in HeLa + 1:500 dilution AM-Tag
antibody
Lane 2: AM-Tag-JunD in HeLa + 1:500 dilution AM-Tag antibody
Lane 3: Mock transfection in HCT116 + 1:500 dilution AM-Tag
antibody
Lane 4: Empty Vector in HCT116 + 1:500 dilution AM-Tag
antibody
Lane 5: AM-Tag-JunD in HCT116 + 1:500 dilution AM-Tag
antibody

Section I: qPCR Primer Design and Data Analysis
A. Design of the primers• Design and analyze your potential primer pairs using an in silico PCR program (i.e. Primer3
at http://frodo.wi.mit.edu/ or the UCSC Genome Browser at http://genome.ucsc.edu/cgi-bin/hgPcr).
• Primers that dimerize should be avoided, as they will be bound by SYBR Green, which willcompromise accurate quantitation. You can test your primers for self-complementarity andsecondary structure at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi.
• Ideally, the amplicons should be 75-150 bp in length.
• For use with the ChIP-IT qPCR Analysis Kit, primers should be designed to anneal optimallyat 58°C with a recommended length of 18-22 bp.
• Active Motif offers ChIP Control qPCR primer sets validated to work in our ChIP-IT qPCR
Analysis Kit. To see a list of the available species-specific primers, please visit
www.activemotif.com/chipprimers.
B. Data Analysis
If the data analysis will not be performed using Active Motif’s ChIP-IT qPCR Analysis Kit, two othersimplified methods of analysis are provided below. Both methods listed require the generationof a standard curve, containing known amounts of DNA, for each primer pair being used in theexperiment.
Method 1: Fold enrichment of positive primers relative to negative control primers
1. Produce a standard curve by performing qPCR with your primer set on known DNA
quantities of Input DNA (from Section B Step 12) in triplicate. Run three to five samples
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22that are 10-fold dilutions, e.g. 0.005 ng, 0.05 ng, 0.5 ng, 5 ng and 50 ng.
2.

Run the ChIP samples along with the dilution series of the Input DNA standards using
both positive control primers (known binding sites) and negative control primers(regions of the genome not bound by your protein of interest).

3.

Your qPCR instrument will assign values to each qPCR reaction based on the standard
curve. If your machine does not average your triplicate reactions automatically, youwill need to calculate these averages.

4.

Divide the average value from the positive control primer set by the average value ofthe negative control primer set to obtain your fold enrichment.

Method 2: Express data as a percent of input
1. Produce a standard curve by performing qPCR with your primer set on known DNA
quantities of Input DNA (from Section B Step 12) in triplicate. Run three to five samples
that are 10-fold dilutions, e.g. 0.005 ng, 0.05 ng, 0.5 ng, 5 ng and 50 ng.
2.

Run the ChIP samples along with the dilution series of the Input DNA standards using
both positive control primers (known binding sites) and negative control primers(regions of the genome not bound by your protein of interest).

3.

Your qPCR instrument will assign values (in ng) to each qPCR reaction based on thestandard curve. If your machine does not average your triplicate reactions automatically, you will need to calculate these averages.

4.

For each qPCR reaction you will have used a percentage of your total ChIP DNA. In
order to calculate the amount in the whole reaction, divide the elution volume of theentire ChIP reaction by the volume used in the qPCR reaction (e.g. if you eluted ChIP
DNA in 200 µl and used 5 µl in the qPCR reaction the formula is 200/5 = 40). Then,
multiply the average qPCR quantity by this number (e.g. qPCR quantity in ng x 40).

5.

To express data as a percent of input, divide the adjusted values from Step 4 above by
the amount of DNA that went into the ChIP reaction and then multiply by 100%. (e.g. if
20 µg was used in the ChIP reaction this is equivalent to 20,000 ng of chromatin. The
calculation would be the adjusted value from Step 4 divided by 20,000 ng and thenmultiplied by 100). Typical percent of input recovered values are 0.05% to 1%.

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23Section J. Troubleshooting Guide
Problem/question

Recommendation

At what points in the
protocol can I stop?

The protocol may be stopped and samples stored at the times and temperatures below:
1. After formaldehyde fixation and centrifugation (intact cell pellet), -80°C.
2. After chromatin shearing, -80°C.
3. After DNA clean up, -20°C.

After sonication shearing and centrifugation,a viscous or cloudy
layer is visible in thechromatin.

Depending upon the cell type, lipid or glycogen layers may be seen after
centrifugation. Avoid such layers when you remove the supernatant. However,if the whole supernatant is cloudy, it should not interfere with the IP reaction.

Poor yield of shearedchromatin.

Insufficient cell numbers were used. Repeat chromatin preparation using alarger number of cells.
Nuclei not released. It is highly recommended to perform dounce homogenization, even when using sonication. Use a dounce homogenizer with a small
clearance pestle (Active Motif Catalog Nos. 40401 & 40415). Monitor cell lysis
under a microscope. Generally, the more cells that are lysed, the higher the
sheared chromatin yield.
Sonication samples were emulsified. Avoid emulsification by turning up thepower of the sonicator gradually. If a chromatin preparation becomes emulsified inadvertently, discontinue shearing and centrifuge the sample for 4 minutes
at 8,000 rpm in a 4°C microcentrifuge to remove trapped air.
Use fresh formaldehyde when preparing Complete Cell Fixation Solution
Buffers were not scaled proportionally to the size of the sample. Use the chartin Cell Growth Recommendations to scale up or down chromatin preparation.

Shearing efficiency
is not clear from gelanalysis.

Material is stuck in the wells, and smears or streaks are seen from the top
to bottom of the lane. The sheared chromatin needs to have the cross-links
reversed, protein removed (Proteinase K) and RNA removed (RNase), followed
by DNA purification.
High molecular weight products. Decrease the size of the fragments by resonicating the sample. If an alternative reverse cross-linking method was used,or the 20 minute incubation at 100°C in NaCl was omitted prior to running the
agarose gel for analysis, please repeat the input chromatin preparation andfollow the manual instructions.

Performing ChIP witha large volume ofchromatin.

This is not recommended. It is better to set up several small ChIP reactions(240 μl each) and pool the samples at the end, rather than trying to ChIP asingle large sample. Do not perform a single scaled-up reaction, as the capture
efficiency is lower.

ChIP DNA does not turnbright yellow following
the addition of 3 M
sodiium acetate

If the color is light orange or violet, this indicates the pH is too high. Add more
3 M sodium acetate 5 µl at a time, mixing after addition until the color is brightyellow. This step is crucial to the success of DNA binding and purification.
For a full color image please see the manual for Active Motif’s Chromatin IP
DNA Purification Kit Catalog No. 58002 available online at our website www.
activemotif.com.

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24

Problem/question

Recommendation

High background.

Chromatin not sheared enough. Shearing should produce DNA fragmentsthat are small enough to exclude background from neighboring chromosomal
sequences, but still large enough that there is a good possibility your ampliconremains intact. We recommend 200-1200 bp fragments. If the DNA fragments
are too large, the background is increased. Consider increasing the number ofpulses for sonication. Check the fragment size on a gel to assess your shearing
efficiency.
Too much antibody relative to the amount of chromatin in the ChIP reaction.
Excess antibody will result in more non-specific binding, which will be detected
as increased background.

Poor or no enrichmentwith target antibody.

Too little chromatin. Generally, we recommend using 20 - 30 µg of chromatin
per ChIP reaction. ChIP reactions should not exceed 50 µg per IP reaction. Be
sure to quantitate the concentration of the sheared chromatin sample(s) being
ChIP’d to ensure both that adequate chromatin is used per sample, and that
equal mass quantities of chromatin are used in each ChIP.
Inefficient transfection Optimize transfection conditions for your expression
construct of choice.
Low protein expression levels. Confirm expression of AM-tagged protein byWestern blot with AM-Tag antibody (Catalog No. 61677). If necessary, perform
a time course experiment to determine the optimal time post-transfection toharvest chromatin.
Perform a control transfection using Active Motif’s pAM_1C_JunD vector (Catalog No. 53044) under the conditions described on the certificate of analysis.
This control transfection and chromatin preparation can be used to validate
the Tag-ChIP-IT technology. Optimization may be needed for each expression
construct and cell line tested.
Problems with PCR. Confirm the amplified sequence for the positive control
primer set is bound by the antibody target. Identify other binding sites.

No PCR products for the Increase the amount of chromatin used in the ChIP reaction, the amount of
ChIP’d samples (but the antibody used, or both.
Input DNA yields thecorrect PCR product)
No PCR products withreal-time PCR

Confirm the species specificity and efficiency of your primers. You may need toredesign your primers. Primers that work in end point PCR do not always work
in qPCR.
No ethanol in DNA Purification Wash Buffer. Make sure that ethanol has been
added to the DNA Purification Wash Buffer prior to first use.

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25Technical Services
If you need assistance at any time, please call or send an e-mail to Active Motif Technical Service
at one of the locations listed below.

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

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

Visit Active Motif online at activemotif.com

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26