ChIP 101
A BEGINNER’S GUIDE TO PERFORMING
CHROMATIN IMMUNOPRECIPITATION ASSAYS

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WHAT’S INSIDE:
Introduction															25 Critical Steps in Every ChIP Experiment						

4How Many Cells are Needed for ChIP Assays?				

5

How to Properly Fix & Prepare Your Samples for ChIP		

6

How to Sonicate & Solubilize Chromatin					

8Best Type of Antibody to Use in ChIP Assays 					

11Best Way to Analyze ChIP Results								14Conclusion															15www.activemotif.com

Introduction to ChIP Assays

The chromatin immunoprecipitation (ChIP) assay has become one of the mostpopular laboratory techniques to investigate the association of DNA-binding
proteins with chromatin and to study the localization of histone modifications
throughout the genome. While ChIP assays have become routine for many
molecular biologists, there are many relatively complicated steps in ChIP
protocols, leading to multiple challenges that must be overcome in eachexperiment to achieve success.

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How to Get the Best ChIP Results in Every Experiment
For over two decades Active Motif has been a pioneer

highest quality ChIP results as easily and quickly asin the development of kits and assays to study genepossible.

regulation and protein function. We were the first

Our team of scientists have collectively done tens ofcompany to develop a ChIP kit, well over 15 years ago

thousands of ChIP-qPCR and ChIP-Seq experiments,

now, and we have been improving and innovating ever

and we’ve learned a lot over the years. We’re happy

since. Active Motif now offers an extensive portfolio ofto share some of our favorite tips and tricks to get

ChIP kits, accessory products, ChIP-validated antibodies,

the best results in your ChIP assays!

and end-to-end ChIP services to enable generating the

A Very Brief History of ChIP Assays
The ChIP assay has been around for a while now. The experimental
approach that would lead to what we know as ChIP today was
first described in the 1980s in the laboratory of John Lis at Cornell
University. The first ChIP experiments investigated the genomic
localization of RNA polymerase II, initially in bacterial cells andlater in Drosophila cells. These early experiments were analyzed byhybridization-based assays.
Chromatin immunoprecipitation assays have come a long way sincethen. The assay has developed into a well-known technique that is performed in many labs on many organisms
and sample types, leading to many publications and great discoveries. The downstream analyses of ChIP assays
have evolved from endpoint PCR, to quantitative PCR (qPCR), and more recently to next-generation sequencing.

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35 Critical Steps in Every ChIP Experiment

ChIP assays include many different steps, each of which are critical to achieve high-quality results. In every ChIP
experiment there are five crucial aspects that need to be considered, which we’ve listed below:
1. Starting Material: Before starting any ChIP assay you need

4. Immunoprecipitation: After the chromatin has

a starting material on which to perform ChIP assays. You

been prepared, the next step is the actual

can use either cells or tissues, and varying amounts ofimmunoprecipitation (IP) step. The antibody used iseach.

critical, as is the decision on whether to use magnetic or

2. Crosslinking: Cells are generally fixed when performing
ChIP to crosslink the proteins to the DNA. Most protocols

agarose beads.
5. Analysis: The last step in the ChIP workflow is theuse formaldehyde, but there are several different

downstream analysis, which is usually either qPCR or

variations to fixation conditions and additives.

next-generation sequencing.

3. Sonication: The next step in the protocol is chromatin
fragmentation, which is usually performed via sonication.

Each of these critical steps is discussed further.

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4How Many Cells Are Needed for ChIP Assays?
The first thing to consider when starting to plan yourCan You Perform ChIP with Low Cell Numbers?

ChIP assay is of course the starting material. What

More recently the number of cells required to perform

sample type should be used, and how many cells (orChIP has come down quite a bit with the development

how much tissue) should be used?

of ChIP-Seq kits designed specifically for low cell

Historically, researchers needed to use millions of cells

numbers, such as the Low Cell ChIP-Seq kit fromfor each ChIP experiment to get good results. That

Active Motif. These kits allow researchers to use fewer

requirement of high numbers of cells was previously

cells, thousands instead of millions, and there are newone of the main hurdles to getting good ChIP-qPCR andtechnologies being developed to bring that cell number

ChIP-Seq results, especially with certain sample types

requirement down even further.

like primary cells and stem cells where it is sometimes

ChIP assays for abundant histone modifications are

difficult to obtain enough cells.

especially amenable to starting with low cell numbers
because histones are so tightly associated with thechromatin and the antibodies available are very
sensitive and specific. On the other hand, certain low
abundance transcription factors are often difficult to
detect in ChIP assays performed with low cell numbers
due to the dynamic nature of their DNA-binding activity.

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5How to Properly Fix & Prepare Your Samples for ChIP
Many different sample types can be used in ChIP reactions,and an important part of all ChIP protocols is how you
actually process the cells. In most ChIP experiments,
fixation is performed to crosslink the DNA to theassociated proteins, and different cells and tissue types
require different fixation protocols. The important aspects
to consider are the type of fixative used and the length oftime the fixation occurs before stopping that reaction.

Formaldehyde is the Gold Standard for ChIP
Fixation
The standard fixation material is usually formaldehyde.
The formaldehyde you see in most labs is 37%
formaldehyde by volume and is stabilized with methanol.
However, one of the problems with having methanol inyour formaldehyde is that it can lead to increased cellpermeability, which leads to more formaldehyde getting
into your cells than if you were to use formaldehydewith no methanol, ultimately resulting in over-fixationof the chromatin. On the other hand, without methanol,
the formaldehyde is not very stable and can polymerize,
making it less effective. There are several other fixatives
that work well in certain experiments, but formaldehyde
is the most commonly used because it can covalently
link two molecules together that are in very closein proximity with each other. However, if you don’t
want that, there are other fixatives like disuccinimidyl
glutarate (DSG) that can be used to crosslink two
molecules that are farther apart in distance. These
are all things that need to be taken into account when
designing your ChIP experiment, but formaldehyde
is a pretty good starting point for fixation in mostcases, and most publications use protocols that rely on
formaldehyde to fix the cells.

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6How Long Should Cells Be Fixed for ChIP?
The next consideration after selecting a fixation method is
how long the cells should be fixed. Most protocols specify
10 to 15 minutes of fixation using formaldehyde at a final
concentration of one percent. These fixation conditions work
well for many sample types, but optimization may be required
for some cases.
If you are working with pieces of tissue where not all of thecells are exposed to the liquid they’re suspended in, thensometimes additional fixation time is needed to get theformaldehyde into those cells. Chromatin preparation fromtissue samples also require the tissues to be homogenized
before proceeding to the chromatin fragmentation.
There are also protocols where you don’t need or don’t wantformaldehyde in your assay, which are referred to as native
ChIP. In native ChIP assays you don’t want the DNA to becovalently bound to your proteins of interest. The native
ChIP method generally only works well for proteins that arevery tightly bound to the DNA, such as histones.

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How to Sonicate & Solubilize Chromatin
After the cells are fixed, they must be lysed and sonicated to generate the soluble chromatin that will be used in the
immunoprecipitation reactions. You generally want the chromatin fragments to be between 200 and 1,000 base pairs, andthere are several considerations to achieve successful fragmentation.
Accessing the Fixed Chromatin in the Nucleus
Following fixation, you need to get into the nucleus to access the chromatin to break it up so that you have fragment sizes
that are small enough to be efficiently pulled down with an antibody. Some difficult to lyse cells, like PBMCs for example,
benefit from a swelling buffer treatment to lyse the cells.
Although it isn’t an absolutely required step, and wasn’t a part of the early ChIP protocols, ChIP results are generally
improved by isolating the nucleus before attempting to fragment the chromatin. There are nuclear isolation protocols thatcan be performed after fixation of the cells, which we recommend performing for best results. Most of the ChIP kits offeredby Active Motif have nuclear isolation as a standard part of the protocol because we have found it to be an important stepfor generating high-quality data. After nuclei are isolated, they can be resuspended in a more stringent lysis buffer, which
will open up your nuclei and give you access to the chromatin inside.

ChIP protocols are generally improved by isolating thenucleus before attempting to fragment the chromatin.

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Fragmenting the Chromatin – Which Method is
Best?

Optimize Sonication Conditions for Best ChIP
Results

Once you have accessed the chromatin, you need

Just like the other steps in the ChIP workflow, there’s

to fragment the DNA into lots of little pieces. Therenot a single sonication condition that will work best forare a couple of different ways of doing this, but the

every sample type. The optimal sonication conditions

preferred method is sonication. After you resuspend

are really cell type and tissue specific, so they need toyour nuclei in some kind of a lysis buffer, usually

be optimized with your sample type.

containing SDS, you subject your cells to ultrasonicFor example, T cells tend to be very difficult tosound. There are lots of different instruments out

sonicate because they are very small, tight cells

there that do this. Essentially the idea is that you’rethat are difficult to open up, so different lysis andbreaking up all of those DNA pieces into fragmentssonication conditions are required to achieve bestthat are 200 base pairs to 1,000 base pairs inresults.

length.
You want to make sure that the DNA fragments are
not too big, but also that they are not too small.
As you can imagine, since you are sonicating cells,
you’re subjecting the DNA and proteins in the fixedchromatin to relatively harsh ultrasonic sound. If
samples are sonicated for too long, you may start tobreak up proteins and DNA beyond recognition by
an antibody. The heat created during sonication canalso denature proteins so much that the relevant
epitopes are no longer recognized by the antibodies
being used.

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Using Enzymatic Methods to Fragment Chromatinthat preferentially cuts at AT-rich sequences, and it

The majority of researchers prefer to use sonication to

can be used over a time course to generate the size offragment DNA because it’s efficient and results in truly
random fragmentation, but there are some potential
drawbacks that must be considered. One of the limitationsof sonication is that it can potentially damage proteins or
disrupt protein-DNA interactions if it’s too harsh.
Researchers that want to be gentler with their chromatin
samples but still want to fragment the chromatin toa reasonable size use enzymatic shearing. The mostpopular enzymatic shearing method uses micrococcal

chromatin fragments that you want.
The main limitation that keeps most scientists fromusing MNase to fragment their chromatin is thatthe enzymatic cleavage notoriously introduces bias
because the enzyme is known to preferentially cut atcertain DNA sequences more than others. However,some newer chromatin analysis methods use MNase
conjugated to antibodies to fragment the chromatin tofacilitate working with low cell numbers.

nuclease (referred to as MNase). MNase is an enzyme

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10What’s the Best Type of Antibody to Use in ChIP Assays?
Once you have isolated your chromatin and it has been sonicated to the ideal size, it’s time to proceed to theimmunoprecipitation. No matter how perfect the sample prep and sonication steps are, the ChIP results won’t be
informative unless the right antibody is used. There are several types of antibodies available for use in ChIP assays,
including polyclonal, monoclonal, and recombinant antibodies. We discuss the benefits of each type below to help you
decide which ones are the best to use in your experiments.
The best antibodies to use for ChIP has always been a debate in the field. Are polyclonals better than monoclonals? Well,it all depends on the details. And a new layer to the debate is recombinant antibodies. Companies like Active Motif areproducing antibodies as recombinant proteins now, which tend to be even more uniform in their antibody population
than traditional monoclonal antibodies.

The short answer is it’s very difficult to say that one
type of antibody is better than the others in all cases.

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11When it Comes to ChIP Antibodies, Specificity

western blot, that doesn’t necessarily mean that it’s

Matters More than Antibody Typegoing to work in ChIP assays. The best way to find a

The most important things to evaluate when selecting an

good antibody to use in your ChIP experiments is to

antibody for ChIP are whether it recognizes your protein

search the literature. The first and most obvious way

of interest with high specificity under fixation conditions,to search is to try to find high-profile papers fromand whether it’s able to maintain binding through the IP

respected researchers that performed ChIP assays forand the wash steps.

the same target protein or histone modification.

In the early days of ChIP, many researchers preferredusing polyclonal antibodies because you can amplify
the signal without compromising specificity by using asecondary monoclonal antibody that’s specific for the
primary antibody isotype.
However, now that there are so many different antibodies
available for most target proteins, and so many havebeen tested and validated for ChIP, it’s really clear thatpolyclonals, monoclonals, and recombinant antibodies can
all perform well in ChIP assays.

What if There Isn’t a Good ChIP-Validated
Antibody?
If you can’t find an antibody published to work wellin ChIP, the next best thing would be to look foran antibody that has been used in a publication on
crosslinked material (similar to ChIP conditions).
Specifically, immunofluorescence (IF) andimmunohistochemistry (IHC) are performed under
fixation, so antibodies that work in those assays arelikely to work in ChIP assays.

Finding the Best ChIP-Validated Antibody for Your

It is important to emphasize that although an antibody

Target Protein

may recognize the protein epitope after fixation in IF or

As mentioned previously, one of the most important steps

IHC, other conditions in the ChIP assay might preventin doing a successful ChIP experiment is finding a great

the antibody from performing well.

antibody for your protein of interest. That can be a really

Another option if you can’t find a good antibody is

big challenge for some researchers. While many of theto add an epitope tag to your protein of interest andmost commonly-studied DNA-binding proteins and histone

use an antibody specific for that tag. Active Motif

modifications have multiple antibodies available to choosedeveloped an epitope tag specifically for ChIP that

from, that’s not always the case.

works well.

Even if an antibody has been validated to work by

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12How Do You Know if the Antibody Used in YourChIP Experiment Worked?
To ensure that you can trust the results of your ChIP
reactions, it is extremely important that proper controls
are performed. The best approach is to perform qPCR
reactions specific for genomic regions where the factor
or histone modification is known to be present (positive
control) and where it’s known to be absent (negative
control).
Some researchers like performing a ChIP reaction with anunrelated normal IgG antibody as a negative control. The
thinking is that any signal seen with this antibody would
be background because it is not specific for the factor or
histone modification being studied. While this approachis better than no control at all, it isn’t great because
the specific antibody being used in the ChIP likely has
different background reactivity than the normal IgG, so it
isn’t really a fair comparison.

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What Beads Are Best for Immunoprecipitations?
After the immunoprecipitation is performed, the proteinchromatin complexes targeted with your antibody areusually isolated by incubating with a bead conjugated toeither protein A or protein G. There are different types ofbeads available that you can use, and the “best bead for
ChIP” is a polarizing debate in the field of ChIP.
Many researchers prefer using magnetic beads because
they have several benefits: they make the washingsteps very fast and easy and they are compatible
with automation if you have a robot with a magnetic
mechanism. Another advantage with magnetic beads isthat you can see them being pulled to the side of the
tube next to the magnet so you can avoid removing theimmunoprecipitated sample when you aspirate the wash
buffers.
An alternative to magnetic beads is agarose beads,
which have their own advantages. Most notably, agarose
beads have very low background and high sensitivity.
One problem that researchers experienced with agarose
beads in the past is that it was difficult and timeconsuming to perform washes with centrifugation at eachstep. Active Motif solved this problem by using protein
G agarose beads in an easy-to-use column format. You
can do all of your washes rapidly and easily on a column.
You just add your wash buffer to the column and it flows
through, you don’t have to spin down your beads foreach wash, so you can perform many ChIP reactions,side by side, using this method.

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What’s the Best Way to Analyze ChIP Results?
The final step in any ChIP reaction is analysis of the DNA that(when two adapters attach to each other and not yourwas immunoprecipitated. After reversing the crosslinking,sample DNA) to a minimum. These two factors aredigesting the associated proteins, and purifying DNA,

integral to maximizing the amount of useful sequencing

researchers usually investigate the DNA sequences they havedata you’ll produce.

isolated by either PCR or next-generation sequencing (NGS).

NGS-based analyses provide significantly more

Analysis using qPCR is a good option if you already know

information than qPCR for each ChIP experiment, butwhich loci you’re interested in and there are not very many

the downside of ChIP-Seq assays is that they require

regions being investigated. On the other hand, NGS works

more processing to generate NGS libraries as well aswell if you want to take an unbiased approach and identify

bioinformatics to interpret the sequencing results, which

all the sites across the genome where your factor or histone

many researchers find intimidating. Next-generation

modification is present.

sequencing is also more expensive than the qPCR-based
analysis, but the cost of NGS is coming down reallyConstructing NGS Libraries for your ChIP-Seq

quickly, so the field is moving more and more to NGS-

Experiments

based analysis for ChIP due to the huge increase in the

Prior to loading on a sequencing instrument, you need toamount of data generated relative to qPCR, and many

make NGS libraries from your enriched ChIP DNA. Making

high-profile journals are starting to require ChIP-Seq in

an NGS library for sequencing simply means attaching shortpublications.

oligos (called adapters) to the ends of your sample DNA so it

While bioinformatics is becoming easier due to open

can be read by the sequencing instrument. There are manysource analysis platforms that are relatively easy to use,ways to attach these adapters, so how do you know whichmost ChIP-Seq projects involve dedicated bioinformatics

way is best? Focus on methods that retain as much of yourscientists to perform the analysis and to generate theChIP sample as possible, using optimized adapter attachment

output figures to help analyze and visualize the data.

and cleanup steps. Also, you’ll want to keep adapter dimer

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CONCLUSIONChIP assays have many different steps, all of which are critical to the overall
success of the experiment. However, with good planning, using ChIPvalidated antibodies, using the right ChIP kits for each sample type, anddoing some optimization, the multiple challenges of ChIP assays can beovercome.
For those researchers that need the highest quality ChIP-Seq data as quickly
as possible, we recommend using end-to-end ChIP-Seq services. With these
services, you simply send your cells or tissue to Active Motif, and you’ll
receive the analyzed data back in a matter of weeks.

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