Active Motif
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351
sales@activemotif.com

Tools to Analyze
Nuclear Function

Magnetic Beads Make Chromatin Immunoprecipitation Faster and Easier
Active Motif’s ChIP-IT™ Express Kits use protein G-coated magnetic beads instead of the traditionally used agarose beads
to make chromatin immunoprecipitation (ChIP) simpler and faster than ever before. The use of magnetic beads has
made it possible to simplify and streamline the ChIP protocol, eliminate a number of time- and labor-intensive steps anddramatically reduce background. The result is that ChIP-IT Express makes your ChIP experiments more successful andenables you to use less starting material.

ChIP method
ChIP is a powerful tool for studying protein/DNA interactions
because it combines the speciﬁcity of immunoprecipitation,
the sensitivity of PCR and the power of array proﬁling. In ChIP,
intact cells are ﬁxed using formaldehyde, which cross-links andtherefore preserves protein/DNA interactions at that precise
time point. The DNA is then sheared into small fragments using
either sonication or enzymatic digestion and speciﬁc protein/
DNA complexes are immunoprecipitated using an antibody
directed against the DNA-binding protein of interest. Following immunoprecipitation, the chromatin is eluted, cross-linking�����������������������������
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is reversed and the proteins are removed by treatment withProteinase K. The DNA is then analyzed, most commonly byPCR, to determine which DNA fragments were bound by theprotein of interest (Figure 1).
While it is a powerful technique, performing traditional ChIP isa complicated, multi-day procedure that is not well suited forthe analysis of numerous samples. Moreover, troubleshooting
the procedure and interpreting ChIP results can be difﬁcult
without validated reagents and controls that have been provento work in ChIP.

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ChIP-IT Express improves traditional ChIP
All standard ChIP methods utilize agarose-based
afﬁnity beads to capture the antibody/chromatincomplexes. However, spin-washing these beads
is slow and tedious. In addition, DNA isolated
through these methods must be further puriﬁed(by spin columns or phenol/chloroform extraction& precipitation) prior to PCR analysis.
We have developed a greatly simpliﬁed ChIP
protocol that utilizes magnetic protein G-coatedbeads for antibody capture. Magnetic beads
enable a streamlined ChIP protocol that has
fewer steps, requires little hands-on time and iscompatible with multi-channel pipetting, whichmakes high-throughput ChIP a reality (Table 1).

Figure 1: Flow chart of chromatin immunoprecipitation with ChIP-IT Express.

www.activemotif.com/chip1

ChIP-IT™ Express

Table 1: Comparison of Standard ChIP vs. ChIP-IT Express protocols.

Standard ChIP protocolChIP-IT Express protocol

Prepare chromatin by sonication

Prepare chromatin by sonication or enzymatic digestion

Block protein G beads, then use beads to pre-clear chromatin
Combine chromatin and antibody; incubate 4 hours or overnight

Combine chromatin, antibody and magnetic beads; incubate 4 hours

Add blocked protein G beads and incubate 1.5 hours
Wash beads 8 times (each wash: pellet beads with a 2-minute centrifugation, let beads
settle, remove supernatant, resuspend)

Wash beads 3 times (each wash: pellet magnetic beads in seconds with magnet, removesupernatant, resuspend in wash buffer)

Elute chromatin from beads using two 15-minute elutions

Elute chromatin (15 minutes), pellet beads in seconds with magnet and transfer supernatant
to a fresh tube

Reverse cross links (4 hours at 65°C)

Reverse cross links (15 minutes at 94°C)

Add Proteinase K to digest proteins (1.5 hours at 42°C)

Add Proteinase K to digest proteins (1 hour at 37°C)

Purify DNA by 2 phenol/chloroform extractions, followed by precipitation and resuspension

Add Proteinase K Stop Buffer

DNA is ready for analysis (e.g. PCR)

DNA is ready for analysis (e.g. PCR)

Step 1: Cell ﬁxation
The ﬁrst step in ChIP is ﬁxing the cells that are to be studied through the use of formaldehyde; this cross-links theDNA-binding proteins to the DNA so that the protein/DNA
complexes can be immunoprecipitated. While this is a fairly
straightforward step, over-ﬁxation is a common problem.
When this occurs, the chromatin becomes resistant to shearing, which prevents efﬁcient shearing of the chromatin in thenext step of the protocol. The ChIP-IT Express Kit includes allbuffers (excluding formaldehyde) required for cell ﬁxation.
A Glycine “Stop-Fix” Buffer is included to prevent excessive
cross-linking and Protease Inhibitor Cocktail (PIC) and PMSF
are included to ensure that the protein/DNA interactions arepreserved throughout the procedure.

Step 2: Chromatin shearing by sonication or enzymatic digestion
After ﬁxation, the chromatin is sheared into small fragments.
This has traditionally been performed by subjecting the chromatin to different pulses of sonication. Although sonication
is an effective method for shearing DNA, it can be difﬁcult tooptimize due to complications arising from emulsiﬁcation andoverheating. And, because the quality of your sheared sample
depends greatly upon the quality of your sonicator, it may benecessary to purchase an expensive, “high-end” sonicator to
get reproducible shearing. Because of this, Active Motif has
developed an enzymatic method for shearing chromatin, which
we believe is more reproducible and user-friendly. Our proprietary Enzymatic Shearing Cocktail quickly shears DNA into
200-1000 bp fragments (Figure 2). Because enzymatic shearing
is solely time and temperature dependent, it is more consistentand the problems caused by sonication areFigure 2: Analysis of DNA sheared using the Enzymatic Shearing Kit.
eliminated, so ChIP results are improved.
HeLa cells were fixed for 10 minutes with 1% formaldehyde and then chromatin
was prepared using the ChIP-IT Express Enzymatic Kit protocol. Chromatin
was sheared with the Enzymatic Shearing Cocktail for 5, 10 & 15 minutes andthe reaction was stopped with the addition of cold EDTA. The sheared andunsheared chromatin samples were subjected to cross-link reversal, treated withProteinase K, phenol/chloroform extracted and precipitated as described in theprotocol. Samples were separated by electrophoresis through a 1% agarose gel.

1000bp
500bp

100bp
1223

4*

5Lane 1: 100 to 1000 bp ladder.
Lane 2: Unsheared HeLa DNA.
Lane 3: HeLa DNA treated for 5 minutes (under-digested).
Lane 4: HeLa DNA treated for 10 minutes (optimized digestion).
Lane 5: HeLa DNA treated for 15 minutes (over-digested).
*Note: From this experiment, the DNA treated for 10 minutes was optimaland used successfully in ChIP.

ChIP-IT Express Kits are offered with yourchoice of either sonication or enzymatic shearing reagents. Because chromatin from different
cells lines behaves differently, the kits provide
reagents sufﬁcient for performing 3 optimizations, then making 5 preparations of shearedchromatin. Each preparation of sheared chromatin requires three 15 cm tissue culture platesof cells and yields chromatin sufﬁcient for atleast 16 ChIP-IT Express reactions (one ChIP
reaction is considered to be the incubation ofToll Free — 1 877 222 9543

ChIP-IT™ Express

After immunoprecipitation and removal of the supernatant,the chromatin and beads are washed. ChIP-IT Express saves a
lot of time and effort because magnetic beads require fewer
washing steps than agarose beads (3 washes vs. 8). Moreover,while agarose beads must be pelleted by a 2-minute centrifugation, the magnetic beads pellet in seconds. This dramaticallyreduces hands-on time and ensures sample-to-sample consistency. The provided siliconized microcentrifuge tubes (1.7 ml)
ensure a minimal loss of beads during the procedure.
Figure 3: Use of a standard tip box for magnetic ChIP.
ChIP-IT Express Kits include a strong bar magnet that can be used to convert tip boxes
into a magnetic stand for washing the protein G-coated magnetic beads.

one sample of chromatin with one antibody). If you wish toprepare chromatin from a smaller number of plates, simply
scale the protocols down accordingly.

The included magnet can be used to turn pipette tip boxes
into magnetic stands; P1000 tip boxes are used with Eppendorf
tubes, while P200 boxes can be used with 8-well PCR strips toprocess multiple samples using a multi-channel pipettor(Figures 4 & 5). If desired, commercially available magnetic
stands from Ambion (Figure 6) and others can also be used.

Step 3: Chromatin capture on magnetic beads
In traditional ChIP, the next steps would be to block the protein G agarose beads and then to incubate the blocked beads
with the sheared sample. This pre-clearing step is designed toreduce the non-speciﬁc background binding of proteins to theprotein G and beads.
In contrast, the protein G-coated magnetic beads in ChIP-IT
Express Kits are supplied ready-to-use, so there is no needfor blocking. As the magnetic beads have a very high binding
capacity for IgG, they are used in smaller volumes that reduce non-speciﬁc binding. Because of this lower non-speciﬁcbinding, it is not necessary to pre-clear the chromatin. WithChIP-IT Express, you can skip these two steps and proceed
immediately to antibody capture. The beads are simply added
to the sheared chromatin along with the antibody against theDNA-binding protein of interest. And, incubation time is only
4 hours, as opposed to overnight for traditional ChIP assays.
Steps 4 & 5: Chromatin immunoprecipitation and washing
After incubation of the antibody, beads and chromatin, theprotein/DNA complexes are immunoprecipitated and washed.
This is extremely simple with ChIP-IT Express because themagnetic beads pellet in seconds simply by placing the tube
next to the magnet that is provided in the kit (Figure 3). An
added advantage is that the magnetic beads pellet onto theside of the tube. This makes it easy to remove buffers without
disturbing the beads.

www.activemotif.com/chip

Figures 4 & 5: Multiple-sample ChIP using ChIP-IT Express.
Immunoprecipitation and wash steps are fast and easy with magnetic beads because thepellet forms on the side of the tube, even pulling completely out of the buffer. Because
it is simple to remove the buffer without disturbing the pellet, it is now possible toperform multiple ChIPs in PCR strips using a multi-channel pipettor.

Figure 6: Use of an Ambion 6 Tube Magnetic Stand with ChIP-IT Express.
Commercially available magnetic tube racks, such as those from Ambion and Promega,can be used with ChIP-IT Express.

3

ChIP-IT Express

Step 7: PCR analysis using appropriate controls
The ﬁnal step is to perform PCR using appropriate primers to determine if the expected DNA sequences wereimmunoprecipitated by your antibody. This conﬁrms that theprotein of interest was bound to the DNA when the cells wereﬁxed (Figure 7).
Because ChIP is an enrichment of DNA bound by a particular
protein, not a complete puriﬁcation of the protein-bound
DNA, ChIPs are unavoidably contaminated with non-speciﬁcally
captured chromatin, which can make data interpretation difﬁcult due to the false positive PCR products. To overcome thisproblem, Active Motif offers species-speciﬁc ChIP-IT Control
Kits for human, mouse and rat, which provide one positive andone negative antibody, positive control primers, PCR buffer
and a DNA loading dye that makes your PCR reactions readyfor loading. In addition, we offer Ready-to-ChIP Chromatin thathas been optimally sheared by sonication and validated in ChIP.
For additional information on the Control Kits and DNA, please
visit our website.

4

25,000 cells

Neg IgG

RNA pol II

Neg IgG

RNA pol II

Neg IgG

RNA pol IINeg IgG

GAPDH Primers
RNA pol II

Step 6: Reverse cross-links, chromatin elution & Proteinase K
treatment
Following the wash steps, the chromatin is eluted from theantibody that is bound to the protein G beads. The specialized Elution and Reverse Cross-linking Buffers are optimized
to reduce incubation times and also decrease background.
The sample is then incubated with Proteinase K to digest theprotein, followed by addition of Proteinase K Stop Buffer.
The ChIP-IT Express protocol is unique because the buffers
used for elution and digestion eliminate the need to purify
the DNA before it is analyzed. With traditional ChIP, the DNA
would have to be subjected to phenol/chloroform extractions,precipitation and resuspension. These steps are not necessary
with ChIP-IT Express as the DNA is now ready for analysis. This
saves time, minimizes manipulations and eliminates DNA loss
that can occur during puriﬁcation.

12,500 cells

Figure 7: ChIP-IT Express works with 12,500 cells.
ChIP-IT Express was performed in duplicate on decreasing amounts of sonicated HeLa
cell chromatin. Two µg of RNA pol II and Neg IgG antibody was used for IP. GAPDH PCR
primers were used to analyze the immunoprecipitated DNA. Using the improved ChIP-IT
Express reagents and protocol, positive ChIP data was obtained from as few as 12,500
cells. In addition, the simplified ChIP-IT Express protocol enabled ChIP to be performed
in high-throughput using 8-tube PCR strips and a multi-channel pipettor together withthe bar magnet included in the kit.

Conclusion
ChIP-IT Express Kits transform ChIP from a labor-intensive
protocol that is appropriate for only a small number of samples
into a rapid method that is suitable for experiments requiring
multiple samples (e.g. studies of pathway induction, timecourse experiments, etc.). The kits incorporate all of the improvements described above and include the reagents required
to perform your choice of either enzymatic- or sonication-type
chromatin shearing, and perform 25 ChIP reactions, including a
powerful bar magnetic. For complete details, and to download
the comprehensive protocol, please visit our website atwww.activemotif.com/chip.

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