ChIP-IT® Express HT

High Throughput Magnetic
Chromatin Immunoprecipitation Kit(version A4)
Catalog No. 53018

Copyright 2011 Active Motif, Inc.

Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of
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time.
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The manufacturer of this documentation is Active Motif, Inc.
© 2011 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights
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All trademarks, trade names, service marks or logos referenced herein belong to their respective
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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Flow Chart of Process & Example. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
ChIP-IT Express HT Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
ChIP-IT Express HT Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols – ChIP-IT Express HT Assay
A. Immunoprecipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
B. Wash Magnetic Beads. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
C. Elute Chromatin, Reverse Cross-links and Treat with Proteinase K . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Appendix
Section A. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section B. PCR Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section C. Analysis of PCR Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

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Overview
Chromatin Immunoprecipitation (ChIP) is a powerful tool for studying protein/DNA interactions1, 2.
In this method, intact cells are fixed using formaldehyde, which cross-links and preserves protein/
DNA interactions. The DNA is then sheared into small, uniform fragments using either sonicationor enzymatic digestion and specific protein/DNA complexes are immunoprecipitated using anantibody directed against the DNA-binding protein of interest. Following immunoprecipitation,
cross-linking is reversed, the proteins are removed by treatment with Proteinase K and the DNA
is recovered. The DNA is then analyzed to determine which DNA fragments were bound by theprotein of interest (see Figure 1 on page 2).
ChIP is extremely useful for the study of chromatin biology and transcriptional regulation because
it enables localization of chromatin proteins, modified histones and transcription factors that arebound to specific DNA regions in specific cells. Furthermore, because protein/DNA interactions
are fixed while in an endogenous, chromosomal context, ChIP results reflect the influence ofchromosomal topology and the effects of cellular regulatory proteins3, 4, 5.
ChIP, in particular ChIP performed with antibodies directed against transcription factors rather
than abundant histone proteins, can be technically demanding. The method requires high-quality
antibodies to recognize the fixed, target-bound proteins of interest, and an efficient reagent(usually protein A or G agarose beads) to precipitate the antibody/chromatin complex. In addition,
specialized buffers, inhibitor cocktails and blocking reagents are required to minimize non-specific enrichment and reduce protein degradation.
Active Motif’s ChIP-IT® Express HT Kit contains proven ChIP reagents that enable convenient,accurate monitoring of protein/DNA interactions in an high-throughput microplate-based format.
The ChIP-IT Express HT Kit utilizes protein G-coated magnetic beads, which have made it possible
to greatly simplify and streamline the ChIP protocol. A number of steps have been reduced inlength or completely eliminated (see page 3 for details), making ChIP-IT Express HT an extremely
rapid and efficient way to perform ChIP.

product

formatcatalog no.

ChIP-IT® Express HT

25 rxns

53018

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1Flow Chart of Process & Example
Cross-link protein to DNA inliving cells with formaldehyde.

Reverse cross-links, digest withProteinase K, then stop reaction.
DNA is ready for analysis.

Break open cells and shear DNA by
Sonication or Enzymatic Shearing.

Add primary
antibody of interest.

Elute chromatin, pellet beads
with magnet and transfer
supernatant to a fresh tube.

Capture antibody-boundprotein/DNA complexes andwash using Magnetic Beads.

Figure 1: Chromatin immunoprecipitation using ChIP-IT Express HT.
ChIP-IT Express HT is based on the improvements made during the development of the ChIP-IT Express, the first magnetic
bead-based ChIP kit. In ChIP-IT Express and ChIP-IT Express HT, cells are treated with formaldehyde to fix protein/DNA
interactions and then the fixed chromatin is sheared by either sonication or enzymatic digestion. The sheared chromatin is incubated with an antibody directed against a protein of interest, and antibody-bound protein/DNA complexes are precipitatedthrough use of magnetic Protein G-coated beads. The captured chromatin is then eluted and cross-links are reversed so thatthe released DNA is ready for PCR analysis to localize the protein to a specific DNA region or regions.
PCR analysis of immunoprecipitated DNA is shown. HeLa cells were fixed for 10 minutes with 1% formaldehyde and thenchromatin was prepared by sonication shearing (5 pulses). ChIP was performed in duplicate on chromatin using RNA pol II
antibody and a Negative Control IgG. The DNA isolated from these ChIP reactions was then analyzed by 36 cycles of PCR
using GAPDH positive control primers. These antibodies and primers are available as the ChIP-IT Control Kit – Human. Kits
for mouse and rat are also available (see Related Products). Ten µl of each PCR was separated on a 1% agarose gel andvisualized by UV-illumination following ethidium bromide staining. PCR using the GAPDH primers on DNA isolated with the
RNA pol II antibody (Lanes 1-4) reproducibly generated more product than similar reactions performed on DNA isolated using
the Negative Control IgG (Lanes 5-8). These results demonstrate that ChIP performed with RNA pol II antibody (compared tonegative IgG) greatly enriched for GAPDH promoter DNA. Lane 9: No DNA control. Lane 10: Input DNA control.

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2

ChIP-IT Express HT Advantages• Process up to 96 ChIP reactions simultaneously• Faster plate-based protocol• Fewer cells required per ChIP
• Compatible with ChIP-chip and ChIP-seq methodologies
The ChIP-IT Express HT Kit contains sufficient components to perform 96 ChIP reactions. The
included LSV (Low Sample Volume) Protein G-coated magnetic beads are provided ready-to-use.
These beads have a high binding capacity for IgG, enabling them to be used in smaller volumes
that reduce non-specific binding. Because of this lower non-specific binding, magnetic beads require fewer washing steps than agarose beads, and it is not necessary to pre-clear the chromatin
or to block the beads. An added advantage is that the magnetic beads pellet much more quickly
than agarose beads, which are pelleted by centrifugation. In addition, magnetic stands aredesigned to pellet the beads onto the side of the tube. This makes it easy to remove buffers without disturbing the beads, so washing can be performed using multi-channel pipettors. This dramatically reduces hands-on time and improves sample-to-sample consistency. The LSV Protein
G-coated magnetic beads are also optimized for use in the small sample size of a microplate well.
ChIP-IT Express HT is based on the improvements made during the development of the ChIP-IT
Express, the first magnetic bead-based ChIP kit. This means that many of the steps in the ChIP
protocol have been optimized for efficiency. The specialized ChIP Elution Buffer, coupled witha reagent to inactivate Proteinase K, eliminates the need for DNA purification after the ChIP iscomplete. This saves time, minimizes manipulations and eliminates the DNA loss that can occur
during purification.
These improvements enable ChIP-IT Express HT assays to be performed rapidly. As the hands-ontime is greatly reduced, it is possible to perform 24, 48 or 96 ChIP experiments at the same time.
This is not possible with traditional ChIP methods, which are time- and manipulation-intensive.
Because appropriate controls make performing, troubleshooting and interpreting ChIP mucheasier, Active Motif sells ChIP-IT Control Kits for human, mouse and rat samples. These useful kits
contain positive and negative control antibodies, appropriate positive control PCR primers, PCR
buffer and loading dye (see Related Products in Appendix).
Another product designed to make your time more productive is Ready-to-ChIP HeLa Chromatin.
High-quality chromatin from HeLa, Hep G2, K-562 and NIH/3T3 cells has been sheared by sonication, and is ready for use in ChIP.

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3Kit Performance
The ChIP-IT Express HT Kit provides reagents and protocols to perform chromatin IP experiments
in a 96-well microplate-based format. The ChIP-IT Express HT Kit can be used in conjunction
with Active Motif’s Chromatin Shearing Kits, which are used to prepare chromatin after first
determining the optimal conditions for shearing chromatin from a particular cell line. Although
not included in this kit, a brief discussion of the reagents provided in the shearing kits is included
below for your reference.
Preparation of chromatin – Sonication Shearing
The ChIP-IT Express Shearing Kit (Catalog No. 53032) includes all buffers (excluding formaldehyde) required for cell fixation, nuclei purification and chromatin shearing by sonication. A
Glycine “Stop-Fix” Buffer is included to prevent excessive cross-linking and a Protease Inhibitor
Cocktail (PIC) and PMSF are included to ensure that the protein/DNA interactions are preserved
during the chromatin purification and immunoprecipitation steps.
Preparation of chromatin – Enzymatic Digestion
The ChIP-IT Express Enzymatic Shearing Kit (Catalog No. 53035) is similar to the ChIP-IT Express
Shearing Kit, except that it uses a proprietary Enzymatic Shearing Cocktail and Digestion Buffer
to enable enzymatic shearing, rather than sonication shearing. Enzymatic shearing is easily
controlled by time and temperature to yield fragments that are ideal for performing ChIP. The
ChIP-IT Express Enzymatic Shearing Kit replaces sonication shearing, thus eliminating problems
due to variability in sonication power as well as complications arising from the emulsification ofchromatin during sonication.
Performing ChIP in a microplate-based high-throughput format
The protocol for ChIP-IT Express HT is based on that of ChIP-IT Express, modified for efficiency
using a 96-well microplate instead of Eppendorf tubes. This does not mean that you need to perform all 96 reactions at once. You may find it convenient to perform 24 or 48 reactions at once.
In this case, simply cap the unused wells with the supplied capstrips before working in the otherwells to avoid contamination during the ChIP procedure and save the plate for later use. Also, it islikely that you will wish to incorporate the use of a multi-channel pipettor and reagent reservoir.
The Thermo Fisher MatrixTech 25 ml Reagent Reservoir (part no. 8094) is a good choice, as itallows for minimal solution waste. Be careful not to use an excess of the supplied reagents if you
are not performing all 96 ChIP reactions at one time, or you may not have sufficient reagents for
subsequent experiments.
You will also need to have a 96-well magnetic sorter, such as Active Motif’s MAG-96 (Catalog No.
90096), Ambion’s Magnetic Stand-96 or the Novagen Magnetight HT96.

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4Kit Components and Storage – ChIP-IT Express HT Kit
Please store each component at the temperature indicated in the table below. Do not re-freeze
the LSV Protein G Magnetic Beads.
Reagents

Quantity

Storage / Stability

5 M NaCl

400 µl

-20°C for 6 months

Proteinase K (0.5 µg/µl)

250 µl

-20°C for 6 months

Proteinase K Stop Solution

300 µl

-20°C for 6 months

Protease Inhibitor Cocktail (PIC)

200 µl

-20°C for 6 months

ChIP Buffer 1

75 ml-20°C for 6 months

ChIP Buffer 2

75 ml-20°C for 6 months

Elution Buffer AM2

10 ml-20°C for 6 months

Reverse Cross-linking Buffer

10 ml-20°C for 6 months

LSV Protein G Magnetic Beads*

2.8 ml

4°C for 6 months

96-well microplate

1

Room temperature

capstrips

30

Room temperature* The LSV Protein G Magnetic Beads are shipped on dry ice, but should not be re-frozen by thecustomer. Upon receipt of this kit, the beads should be stored at 4ºC.

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5Additional materials required• A ChIP-validated antibody directed against the protein of interest• 96-well magnetic sorter (e.g. Active Motif’s MAG-96 (Catalog No. 90096) Ambion’s Magnetic
Stand-96 or the Novagen Magnetight HT96).
• 37% Formaldehyde (Fixation)• Glycogen, 20 mg/ml (Purification of Input DNA)• Phenol/chloroform (Purification of Input DNA and purification of sheared DNA prior to checking concentration by spectrophotometry or gel electrophoresis)• 3 M Sodium Acetate pH 5.2 (Purification of Input DNA and purification of sheared DNA priorto checking concentration by spectrophotometry or gel electrophoresis)• 100% ethanol (Purification of Input DNA and purification of sheared DNA prior to checking
concentration by spectrophotometry or gel electrophoresis)• 70% ethanol (Purification of Input DNA and purification of sheared DNA prior to checking
concentration by spectrophotometry or gel electrophoresis)• DNase-free H2O (Purification of Input DNA)• PCR strip tubes and caps (e.g. Thermo Fisher part no. AB-0451 for Purification of DNA fromchromatin after elution)• Rocking platform for culture plates• Apparatus to rotate tubes end-to-end at 4°C (e.g. a Labquake from Barnstead/Thermolyne)• Microcentrifuge and microcentrifuge tubes• Microplate centrifuge• Spectrophotometer• Pipettors and tips (a multi-channel pipettor and filter tips are recommended)• Reagent reservoir (e.g. Thermo Fisher MatrixTech 25 ml Reagent Reservoir part no. 8094)• Agarose gel electrophoresis apparatus• Minimal cell culture media• Cell scraper (rubber policeman)• 10 ml pipette, aspirator and 100 ml graduated cylinder
Optional materials• Chromatin IP DNA Purification Kit (e.g. Active Motif Catalog No. 58002) .

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ChIP-IT Express HT Experimental Design
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Points to consider:• Cell growth and chromatin preparation. This kit contains Protein G-coated magnetic beads
and other reagents sufficient to perform a total of 96 ChIP reactions. Chromatin can beprepared for use in ChIP through sonication by using Active Motif’s ChIP-IT Express Shearing Kit (Catalog No. 53032) or by enzymatic digestion with the ChIP-IT Express Enzymatic
Shearing Kit (Catalog No. 53035). ChIP-IT Express HT is also compatible for use with Active
Motif’s Ready-to-ChIP Chromatin. Before starting an experiment, calculate the number ofchromatin preparations you require and determine the number of ChIP reactions you planto perform on each chromatin preparation. Be sure to include the appropriate control ChIP
reactions in your calculations. Also, note that if you wish to analyze the effect of particularcompounds or culturing conditions on transcription factor/DNA interactions, you should
prepare chromatin from control (untreated) cells as a reference sample. See Troubleshooting in the Appendix for comments regarding chromatin yield and the amount of chromatinused for each ChIP reaction.
• Shearing by Sonication. Chromatin sheared to a size of 200-1500 bp is usually used for
ChIP experiments. You may wish to optimize shearing conditions using chromatin that is
not intended for use in ChIP. In general, shearing efficiency is improved through the use ofa small shearing volume and a V-bottom tube rather than a round-bottom tube. Also, notethat shearing is inefficient if the chromatin sample becomes emulsified. This can be avoided
by using lower shearing power and by turning the power on gradually. If a shearing reactionis allowed to emulsify, discontinue shearing and centrifuge the sample at 4ºC for 4 minutes
at 8000 rpm in a 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 15 seconds,
then place on ice for 30 seconds, sonicate again for 15 seconds, etc.).
• Shearing by Enzymatic Digestion. While sonication is the most common method used toshear chromatin for ChIP, in some cases (e.g. when the sample is limited, when nucleosome
ChIP is to be performed, when a sonicator is not available, or for high-throughput applications) enzymatic shearing is an excellent alternative. To provide a robust and user-friendly
enzymatic shearing method, Active Motif offers the ChIP-IT Express Enzymatic Shearing Kit,
which uses a proprietary Enzymatic Shearing Cocktail to quickly digest the chromatin DNA
to 200-1500 bp fragments. Enzymatic activity can be easily controlled by time and temperature.
• LSV Protein G-coated magnetic beads. The supplied LSV beads are optimized for use in
the small volumes of a 96-well plate and are ready to use following complete resuspension
to a homogeneous slurry. There is no need to pre-block the beads or pre-clear the sample.
For best results, gently shake and roll the tube. The beads settle quickly, thus resuspend
just before pipetting. The LSV Protein G Magnetic Beads are shipped on dry ice but
should not be re-frozen by the customer. Upon receipt, the beads should be stored at
4ºC.
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7• Antibodies must be suitable for ChIP. ChIP antibodies must recognize fixed, native proteinthat is bound to DNA and/or complexed with other proteins. Many antibodies that perform
well in other applications do not perform in ChIP. Thus, ChIP performed with an unprovenantibody must include appropriate controls (such as Active Motif’s RNA pol II antibody, Catalog No. 39097) to demonstrate that the antibody and the prepared chromatin are appropriate for ChIP. For your convenience, Active Motif sells ChIP-IT Control Kits for human, mouseand rat samples; these kits contain positive and negative control antibodies, appropriate
positive PCR primers, PCR buffer and loading dye (see Related Products in Appendix).
• Perform ChIP-IT Express HT in the provided 96-well plate. You will need a 96-well magnetic sorter, such as Active Motif’s MAG-96 (Catalog No. 90096), Ambion’s Magnetic Stand-96
or Novagen’s Magnetight HT96, to perform many of the steps in the ChIP-IT Express HT
protocol. First-time users of magnetic beads in the PCR tube format should familiarize
themselves with the manipulations before performing a ChIP.
• PCR analysis of immunoprecipitated DNA. A successful ChIP results in an enrichment ofchromatin fragments that are bound by the protein of interest, not complete purification.
Thus, DNA isolated by ChIP is unavoidably contaminated with non-specifically captured
DNA. For this reason, Real-Time Quantitative PCR analysis is preferred. If this method is
not available, PCR of ChIP DNA should be performed such that cycling is stopped while thereaction is still in the linear stage of amplification. Hot-start PCR methods are recommended
to ensure consistent PCR amplification results.
If you intend to analyze the binding of a known protein to a known binding site, design
the PCR primers so that they flank the binding site and generate a 100-250 bp amplicon.
Alternatively, if you hope to identify a protein binding site within a region of DNA, it may be
best to design several primer pairs so that the DNA region in question can be systematically analyzed. In this case, design a series of primer pairs that can be used to generate
amplicons that overlap one another and span the region of interest. To facilitate this, theamplicons can be 250-400 bp in length. After these primer pairs have been used to roughly
localize the binding site, design a more focused set of primers. Use of PCR design programs
can be helpful in selecting good primer pairs. Also, as PCR analysis is extremely sensitive,
precautions against contamination should be taken throughout the entire ChIP protocol.
• PCR primers. PCR primers should efficiently and specifically amplify the desired target. Thisshould be proven on a relevant template, such as genomic or Input DNA. In addition, negative control primers can be useful to control for DNA shearing efficiency and to map putative
protein binding sites. See Troubleshooting for discussion.
• Maximum volume of chromatin. Chromatin shearing (sonication) buffers usually contain
detergents (e.g. 0.1% SDS and 0.5% sodium deoxycholate is typical). If you plan to use
more than 60 µl sonicated chromatin in a ChIP reaction, use the 200 µl reaction volume
from Table 1 (page 10). This will ensure that the detergent in the shearing buffer does notinterfere with antibody activity. This is not a concern with chromatin prepared by the Active
Motif ChIP-IT Express Enzymatic Shearing Kit because the relevant buffers contain little orno detergent.

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8• Resuspend solutions completely. Thaw the PIC and the Proteinase K Stop Solution at room
temperature until fully dissolved. Vortex gently and spin down briefly before use.
• Quantity of antibody. Best results are typically obtained by use of 1-3 µg antibody. However, this will vary according to the activity of the antibody and the quality of the chromatin,
and you may need to use more of a particular antibody.
• Stopping points in the protocol. Convenient stopping points are mentioned in the Troubleshooting section in the Appendix.
• Safety precautions. Formaldehyde is a highly toxic chemical. Appropriate safety precautions (i.e. safety glasses, gloves and lab coat) should be used. Also, formaldehyde is highly
toxic by inhalation and should be used only in a ventilated hood.
• Washing of magnetic beads. During the ChIP procedure, washing of the magnetic beads is
performed as follows:a.

Briefly spin plate to collect liquid from caps.

b.

Remove the capstrips, place the microplate in the magnetic stand and allow the beads
to pellet to the sides of the wells for 1 minute.

c.

Carefully remove the supernatant and discard.

d.

Do not remove the plate from the magnetic stand, unless necessary to resuspend
the magnetic beads. Add 200 µl of wash buffer (ChIP Buffer 1 or 2; see page 11), thenresuspend pellet completely by gently pipetting up and down several times. Take careto ensure that beads are not clinging the pipet tips after pipetting. You may need tomove the tubes away from the magnetic field before resuspending.

e.

Place the plate in the magnetic stand, if necessary, and allow beads to pellet to thesides of the wells for 1 minute.

f.

Carefully remove the supernatant and discard.

g.

Repeat steps d-g for the required number washes.

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9

Protocols – ChIP-IT Express HT Assay
A. Immunoprecipitation
1.

Thaw chromatin samples (if necessary). Transfer 10 µl of each to a microcentrifuge tube; thistube is the “Input DNA” that will be processed in Step C6. It will then be used as a control
in PCR analysis. Store this reserved chromatin at 4ºC if it will be used within 6 hours; otherwise, store at -20ºC.

2.

Set up the ChIP reactions by adding the components shown in Table 1 below to the provided
96-well plate. If you are not setting up 96 ChIPs, cap the unused wells with the supplied
capstrips before beginning to avoid contamination, then save the plate for later use. Before
pipetting the magnetic beads, they should be resuspended by inverting and/or vortexing
the bottle. The LSV beads should be the first component added to the reaction; add the antibody last. If you are using Active Motif’s Ready-to-ChIP Chromatin, use 25 µl per reaction.
Note:

You can make a master mix of all the components without the magnetic beads
and antibody, which need to be added to the wells first and last. You may find it
convenient to use a multi-channel pipette reservoir such as the Thermo Fisher
MatrixTech 25 ml Reagent Reservoir (part no. 8094). If you use this procedure
for less than all 96 ChIP reactions, be careful not to use an excess of the supplied reagents, or you may run out of some components in subsequent ChIPs.

Table 1
One reaction(if using less than
60 µl of chromatin)

Reagent

One reaction(if using more than
60 µl of chromatin)

LSV Protein G Magnetic Beads

25 µl

25 µl

ChIP Buffer 1

10 µl

20 µl

20-60 µl61-100 µl

1 µl

1 µl

Sheared Chromatin (~7 µg)*
Protease Inhibitor Cocktail (PIC)dH20

Add enough so that the final Add enough so that the final
reaction volume will be 100 µl reaction volume will be 200µl
Antibody (added last)

1-3 µg1-3 µgTotal Volume

100 µl200 µl*Note:

If you used one of our shearing kits using a 15 cm plate and did not quantify thechromatin, 25 µl will contain ~7 µg of chromatin. Depending on the application,
ChIP can be performed using anywhere from 1-50 µg of chromatin. An importantfactor is the volume of the chromatin being added, especially if the chromatin
was prepared using sonication. The detergents used during sonication will
impact ChIP. Use the 200 µl ChIP in the right column above if the volume ofchromatin will be greater than 60 µl. See Troubleshooting in the Appendix for
discussions on the amount of chromatin to use and for methods to quantify
chromatin.

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10

3.

Mix thoroughly by gentle pipetting and cap wells with the supplied capstrips.

4.

Incubate on a rolling shaker (e.g. a rotating Labquake mixer from Barnstead/Thermolyne)
or a blood tube rotator for 4 hours at 4°C. In some cases, sensitivity can be improved if thisincubation is performed overnight.

5.

Spin plate briefly in a centrifuge to collect any liquid from the insides of the caps. Remove
the capstrips carefully to avoid contamination between wells.

6.

Place tubes on the magnetic sorter for 1 minute to pellet the beads to the sides of the wells.

7.

Carefully remove the supernatant and discard.

B. Wash Magnetic Beads
The recommended procedure for bead washing methods can be found in the Experimental
Design section that begins on page 7.
1.

Wash beads three times each with 200 µl of ChIP Buffer 1.

2.

Wash beads three times each with 200 µl of ChIP Buffer 2. At this point, discard the usedcapstrips to avoid cross-contamination during the subsequent chromatin elution steps.

C. Elute Chromatin, Reverse Cross-links and Treat with Proteinase K
1.

Resuspend washed beads with 50 µl Elution Buffer AM2. Cap microplate with new capstrips.

2.

Incubate the plate for 15 minutes at room temperature on an end-to-end rotator.

3.

Briefly spin plate to collect liquid from the caps.

4.

Remove the stripcaps carefully, add 50 µl of Reverse Cross-linking Buffer to the eluted
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11chromatin, then immediately place plate on magnetic sorter and allow beads to pellet tosides of wells for 1 minute.
5.

Quickly transfer the supernatant, which contains the chromatin, to PCR strip tubes.

6.

“Input DNA” sample: take the 10 µl Input DNA aliquot (that was reserved in Step C1 above)from the ice. Add 88 µl ChIP Buffer 2 and 2 µl 5M NaCl to the Input DNA sample only, sothat its final volume is 100 µl.

7.

Incubate the ChIP and Input DNA samples at 95°C for 15 minutes in a thermocycler.
Note:

If you are using larger microcentrifuge tubes, it may be easier to perform a
2.5 hour incubation at 65°C.

8.

Return tubes to room temperature, spin tubes briefly if liquid has collected on the inside of
the caps, then add 2 µl Proteinase K.

9.

Cap tubes, mix well and incubate at 37°C for 1 hour.

10. Return the tubes to room temperature and add 2 µl Proteinase K Stop Solution. Briefly
centrifuge the tubes to collect liquid from the caps. DNA can be used immediately in PCR or
stored at -20°C.
11.

For PCR analysis, we recommend to use 5 µl of immunoprecipitated DNA in a 25 µl PCR
reaction. Input DNA should be diluted 10 fold before use in PCR. (See Appendix – Section B
for recommendations for PCR.) For ChIP-chip or ChIP-seq, additional DNA cleanup may berequired.

References
1.
2.
3.
4.
5.

Solomon, M.J. et al. (1988) Cell 53(6): 937-47.
Solomon, M.J. and Varshavsky A. (1985) PNAS USA 82(19): 6470-4.
Kuo, M.H. and Allis, C.D. (1999) Methods 19(3): 425-33.
Weinman, A.S. and Farnham, P.J. (2002) Methods 26: 37-47.
Caretti, G. et al. (2003) J Biological Chem. 278: 30435-30440.

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Appendix
Section A. Troubleshooting Guide
Problem/question

Recommendation

At what points in the
protocol can I stop?

The protocol can be paused and samples stored at -20°C at any of the following
steps:
1. After bead washing
2. After the cross-link reversal
3. After DNA clean up

How much shearedchromatin should I use
for a ChIP reaction?

Best results are obtained when the amount of chromatin used in ChIP is from
1-3 x 106 cells. If it is assumed that human diploid cells contain 6.6 picograms of
DNA and recovery after cell fixation and shearing is 70%, then this is between
4.6 and 13.9 µg DNA. The minimum amount of sheared chromatin that can beused is 1 µg while the maximum is 50 µg.

Preparing a ChIP reaction with a large volume

It is better to set up several small ChIP reactions (200 µl each) and pool thesamples at the end, rather than trying to ChIP a single large sample.

Poor enrichment with
ChIP antibody

In some cases, use of an antibody in ChIP results in lower-than-expectedenrichment of a target of interest. This is often because the antibody does notefficiently recognize fixed proteins, either because the epitope is destroyed by
fixation or because the epitope is masked by other proteins in a larger complex.
In this case, use more antibody when performing the ChIP. Alternatively, try
to find an antibody that has been proven to work in ChIP, or that is known torecognize an epitope distinct from the one recognized by the unsatisfactory
antibody.

There is no difference in band intensity
betweennegative control andpositive control.

See the recommendation below regarding increasing washing stringency.
Decrease the number of PCR cycles (30, 32, 34 PCR cycles). The exponential
phase of amplification occurs in PCR cycles where reaction components arestill in excess and PCR products are accumulating at a constant rate. During
this phase, each copy of DNA is being actively amplified, making it a bettermeasure than endpoint PCR. In endpoint PCR, reagents such as nucleotides
or primers may become exhausted. This can result in inefficient amplification,
which can cause inaccurate quantification of the gene of interest. Thus, highbackground due to endpoint PCR can be decreased if the number of PCR cycles
are reduced, so the results reflect exponential PCR. Real-time PCR can also be
used in such cases.
Shearing should produce DNA fragments that are small enough to exclude background from neighboring chromosomal sequences, but still large enough that
there is a good possibility your amplicon remains intact. We recommend 2001500 bp fragments. If the DNA fragments are too large, you the background isincreased. So, consider increasing the time of the enzymatic digestion, or thetime and/or number of pulses for sonication.
Confirm species specificity of your primers. You may need to redesign yourprimers.
See the recommendation below regarding blocking the magnetic beads.

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Problem/question

Recommendation

Strong PCR signal whenusing target PCR primers to amplify ChIP DNA
that was isolated witha negative control(non-target) antibody.

In most cases, the washing procedure in the enclosed protocol is appropriate.
However, when the background is high you can increase washing stringency inseveral ways:
1) After adding ChIP Buffer 1 and/or ChIP Buffer 2 during the wash steps,
gently agitate the samples for several minutes before removing the buffer.
2) Perform additional washes. Sufficient ChIP Buffer 1 is provided for two“extra” washes per sample. Sufficient ChIP Buffer 2 is provided for one
additional wash.
3) Add two washes using a high-salt buffer (20 mM Tris-Cl, 1 mM EDTA, 0.1%
SDS,
1% Triton X-100, 500 mM NaCl, pH 7.4), which is not provided. These additional washes should be performed after the washes with ChIP Buffer 1.
Then, proceed with the ChIP Buffer 2 washes, as outlined in the protocol.
Confirm the species specificity of your primers. You may need to redesign yourprimers.
See the next recommendation regarding blocking the magnetic beads.

Is blocking of themagnetic beads everrequired?

The beads provided are ready to use for most ChIPs. However, for applications
highly sensitive to non-specific binding (such as when cloning ChIP DNA orusing antibodies that require extra blocking), you may add blocking reagentsto the ChIP reaction. In these cases, a combination of BSA (e.g. Sigma Cat. No.
4503) and either tRNA (e.g. Sigma Cat. No. R3629) or salmon sperm DNA (e.g.
Sigma Cat. No. A-7888) can be added directly to the ChIP reaction. 2.5 µg/µl
BSA and 1.25 µg/µl tRNA or 2.5 µg/µl Salmon sperm DNA (final concentrations)can be used as a starting point and more or less can be added as desired.

The PCR products are
the correct size, but arevery light.

Load more PCR product, and/or use smaller wells for the agarose gel. It should
be noted that because the PCRs should be stopped while the reactions are in
the linear phase of amplification, the yield of PCR product will be lower than in
typical PCR amplifications, which are performed for maximum product yield. You
can also perform more PCR cycles.

No PCR products with
Real-time PCR.

The DNA should be purified before performing real-time PCR. We recommend
Active Motif’s Chromatin IP DNA Purification Kit (Catalog No. 58002) prior
to amplification. Its columns yield 50 µl; 2 µl is used for each PCR, providing
enough DNA for 25 PCR reactions.

No PCR bands for Input
DNA or ChIP’d samples.

In the presence of 0.8 mM total dNTP concentration, perform a MgCl2 titration
series in 0.5 mM increments over a range of 1-4 mM. This will identify themagnesium ion concentration that produces the highest yield of a specific
PCR product. When using Taq DNA Polymerase, too little free magnesium ion
results in little or no PCR product, while excess free magnesium ion can cause
unwanted products and promote misincorporation.
Confirm species specificity of your primers. You may need to redesign yourprimers.

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Problem/question

Recommendation

How do I design PCRprimers to analyze
shearing efficiency andto map putative
DNA-binding sites?

Negative control PCR primers can be used to demonstrate that chromatin was
sufficiently sheared. For example, negative control primers can be designed
to amplify a DNA fragment that is 2 kb away from the “Target DNA” (the region
bound by the protein of interest). Following ChIP reactions (performed with antibody against the protein of interest and with a negative IgG), PCR is performed
with the negative control primers and with primers that amplify the Target
DNA. The PCR should show that the anti-protein-of-interest ChIP enriches forTarget DNA, but not for the negative control DNA. This result would support theconclusion that the enrichment was due to protein binding to (or near) the putative Target, and not due to binding elsewhere on a very large (poorly sheared)
chromatin fragment.
Similarly, appropriately designed PCR primers can be used to roughly map the
DNA Target of the protein of interest. For example, primers can be designed
to amplify short (approximately 100 bp) DNA fragments that are progressively
closer to the putative Target DNA (e.g. within 1.5 kb, 1 kb, 500 bp, 250 bp).
This type of analysis can help confirm the exact binding site of the protein ofinterest. For such higher-resolution mapping, the chromatin must be extensively
sheared (DNA fragment size should be less than 500 bp).

Section B. PCR Analysis
The protocol below is a guideline for optimizing PCR analysis of DNA collected through ChIP.
Accurate PCR analysis of ChIP DNA requires that the PCR be stopped during linear amplification.
The appropriate number of PCR cycles must be determined empirically. In the example below,
PCR is performed on four DNA templates: DNA from ChIP with the positive control RNA pol II
antibody and the Negative Control IgG (that are included in the ChIP-IT Control Kit – Human), theInput DNA and DNA from ChIP with the test antibody. A water-only control PCR is also performed
to ensure that the PCR reagents are not contaminated.
In the example below, PCR reactions are set up using 2 different PCR cocktails, which containpositive & negative PCR primer sets. If you are using Active Motif’s ChIP-IT Control Kit – Human,
only GAPDH positive control primers would be used for the first DNA template because the positive control RNA pol II antibody can bind many regions along a chromosome, making it difficult
to design “negative control” primers that function with all cell types and shearing conditions. For
PCR analysis of ChIP performed with other antibodies, we recommend that you use both positive and negative PCR primer sets that are appropriate for your antibody. See the Experimental
Design section that begins on page 7 and Troubleshooting in the Appendix for details.
Note:

PCR is extremely sensitive and all precautions should be taken to guard against
contamination. Gloves should be worn and filter-tip pipettes should be used.

1.

Program the thermocycler. The program should start with a initial melt step at 94°C for
3 minutes, then 36 cycles of [94°C for 20 seconds, 59°C for 30 seconds and 72°C for
30 seconds], then a hold cycle at 10°C. The total volume of each PCR will be 25 µl. You may
need to optimize the number of cycles for your specific system.

2.

Dilute the Input DNA 1:10 by adding 20 µl Input DNA to 180 µl dH2O.

3.

Use the table below to label PCR tubes and add the PCR templates and water-only control,
keeping the tubes on ice. Add the PCR cocktails you will make in Step 4:
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Reaction No.
1

PCR cocktail (20 µl each)

ChIP DNA – Positive control antibody Positive PCR cocktail

2

ChIP DNA – Negative control IgG

Positive PCR cocktail

3

Input DNA (diluted 1:10)

Positive PCR cocktail

4

ChIP DNA – Test antibody

Positive PCR cocktail

5

H2O (no DNA control)

Positive PCR cocktail

6

4.

PCR Template (5 µl each)

ChIP DNA – Positive control antibody Negative PCR cocktail

7

ChIP DNA – Negative control IgG

Negative PCR cocktail

8

Input DNA (diluted 1:10)

Negative PCR cocktail

9

ChIP DNA – Test antibody

Negative PCR cocktail

10

H2O (control)

Negative PCR cocktail

Set up the Positive PCR cocktail and the Negative PCR cocktail on ice according to thetables below. Add the dH2O first and the Taq polymerase last. Mix thoroughly and keepon ice. This ensures that the reaction mixture is inactive until the cycling is started. As
discussed above, if you are using the positive control RNA pol II antibody and Negative
control IgG from the ChIP-IT Control Kit – Human, only GAPDH positive control primers areprovided (as a mixture of forward and reverse primers). However, for your own test antibody
we recommend that you design and test both positive and negative PCR primer sets.

Positive PCR cocktail:
Reagent

1 reaction

5 reactions

DEPC H2O

12.3 µl61.5 µl

Positive Forward primer (5 pmol/µl)

2.0 µl

10 µl

Positive Reverse primer (5 pmol/µl)

2.0 µl10 µl

dNTP mixture (5 mM each dNTP)

1.0 µl

5.0 µl

10X PCR Buffer

2.5 µl12.5 µlTaq (5 U/µl)

0.2 µl

1.0 µlTotal Volume (Not including DNA template)

20 µl

100 µlReagent

1 reaction

5 reactions

DEPC H2O

12.3 µl61.5 µl

Negative Forward primer (5 pmol/µl)

2.0 µl

10 µl

Negative Reverse primer (5 pmol/µl)

2.0 µl

10 µl

Negative PCR cocktail:www.activemotif.com

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dNTP mixture (5 mM each dNTP)

1.0 µl

5.0 µl

10X PCR Buffer

2.5 µl12.5 µlTaq (5 U/µl)

0.2 µl

1.0 µlTotal Volume (Not including DNA template)

20 µl

100 µlNote:

We recommend that PCR analysis be performed using the 10X PCR Buffer
included with the ChIP-IT Control Kits. The composition of the 10X PCR Buffer is
750 mM Tris-Cl (pH 8.8), 200 mM (NH4)2SO4, 0.1% Tween 20 and 25 mM MgCl2.

5.

Add the appropriate PCR cocktail to each of the PCR tubes (on ice) prepared in Step 3. Cap
PCR tubes carefully and ensure that each reaction mixture is in the bottom of the tube.

6.

Place PCR tubes in thermocycler and start the PCR program described in Step 1. After 36
cycles, remove tubes 1-10 and place on ice.

7.

These PCR reactions can be immediately analyzed as described below or stored at -20°C.

Section C. Analysis of PCR Products
1.

Run ~8 µl of each PCR product on a 3% agarose gel. Save remaining PCR product in caseadditional gels must be run. Use gel combs with 2.5 mm-wide wells.

2.

PCR products obtained with the GAPDH positive control primers are 166 bp. Use either a
50 or 100 bp ladder as the migration standard. Run the gel until PCR amplification products
are well separated from PCR primers and primer dimers. Stain gel and analyze.

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17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
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E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
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+32 (0)2 653 0050
eurotech@activemotif.com

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