Chromatin Assembly(version A3)
Catalog No. 53500

Copyright 2018 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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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
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Chromatin Assembly
Component Aliquoting Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Component Preparation Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Protocols
A. Chromatin Assembly Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
B. Analysis of Assembled Chromatin by Partial Digestion . . . . . . . . . . . . . . . . . . . . . . 10
C. Deproteinization with Proteinase K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
D. Agarose Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Downstream Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Appendix
Section A. Troubleshootin Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

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Overview
Active Motif’s Chromatin Assembly Kit makes it possible to generate high-quality chromatin fromsupercoiled and linear DNA. The kit is based on an ATP-dependent assembly method that uses
purified components. The Chromatin Assembly Kit utilizes purified recombinant Drosophila chromatin assembly complex ACF and human histone chaperone NAP-1 (h-NAP-1) with purified HeLa
core histones for in vitro assembly of extended, regularly ordered, periodic arrays of nucleosomes. Active Motif’s Chromatin Assembly Kit contains proven reagents that provide an easy andcomplete solution for chromatin generation.

product
Chromatin Assembly Kit

formatcatalog no.

10 reactions

53500

Each Chromatin Assembly Kit contains reagents for 10 reactions. A reaction is defined as
chromatin generated from 1 µg of DNA.

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Introduction
In eukaryotes, genomic DNA is packed into chromatin to consist as chromosomes within the
nucleus. The assembly of genomic DNA and histones into chromatin is a fundamental processthat affects a broad range of gene regulatory processes, including DNA replication, DNA repair,gene expression and progression through the cell cycle. Research investigating DNA regulatory
mechanisms would ideally be conducted with the gene sequence of interest assembled into
chromatin to more closely represent the natural in vivo state.
Recent research has indicated that the transcriptional regulation of a promoter on naked DNA
differs from that on a promoter assembled into chromatin. Thus, using unassembled DNA cangenerate misleading results1-3. This calls into question the value of using DNA constructs that are
not in a chromatin-like environment for investigating the sequence of interest’s role in regulatingtranscription. Because Active Motif’s Chromatin Assembly kit enables you to closely mimic thechromatin environment, you can study regulation under conditions that are closer to real life.
The basic structural unit of chromatin is the nucleosome, which consists of 146 base pairs (bp)of DNA wrapped around a histone octamer. The histone octamer consists of two copies each of
the core histone H2A-H2B dimers and H3-H4 dimers. Crystallographic studies have shown thatan H3-H4 tetramer occupies the central region of the nucleosome, and the H2A-H2B dimers bindto the peripheral region. Nucleosomes are regularly spaced along the DNA in what is commonlyreferred to as “beads on a string”.
The assembly of core histones and DNA into nucleosomes is mediated by chaperone proteins
and associated assembly factors. Nearly all of these factors are core histone-binding proteins.
Some of the histone chaperones, such as nucleosome assembly protein-1 (NAP-1), exhibit apreference for binding to histones H3 and H4. It has also been observed that newly synthesizedhistones are acetylated and then subsequently deacetylated after assembly into chromatin. The
factors that mediate histone acetylation or deacetylation therefore play an important role in thechromatin assembly process.
While ATP-dependent chromatin assembly yields extended periodic arrays of nucleosome, thespacing of the nucleosomes is not driven by the DNA sequence itself. However, when the DNA
sequences are assembled into chromatin in the presence of their sequence-specific regulatory factors, the resulting chromatin closely resembles the configuration of the genes in vivo3-5.
Because chromatin assembled by the ATP-dependent ordering and specific factors is more likechromatin in a natural cell, it is an excellent substrate for investigating transcriptional regulationand the mechanisms that affect chromatin-dependent gene expression1-15. Assembled chromatin
is useful for many downstream applications such as histone acetyltransferase (HAT) assays, invitro chromatin immunoprecipitation (ChIP) assays and in vitro transcription assays3.

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Methods for Chromatin Assembly
In general, two in vitro methods have been developed for reconstituting or assembling chromatin. One method is ATP-independent, while the second is ATP-dependent. The ATP-independent
method for reconstituting chromatin involves the DNA and core histones plus either a protein
like NAP-1 or salt to act as a histone chaperone. This method results in a random arrangement ofhistones on the DNA that does not accurately mimic the native core nucleosome particle in thecell. These particles are often referred to as mononucleosomes because they are not regularly
ordered, extended nucleosome arrays and the DNA sequence used is usually not longer than
250 bp13. To generate an extended array of ordered nucleosomes on a greater length of DNA
sequence, the chromatin must be assembled through an ATP-dependent process.
The ATP-dependent assembly of periodic nucleosome arrays, which are similar to those seen innative chromatin, requires the DNA sequence, core histone particles, a chaperone protein andATP-utilizing chromatin assembly factors. ACF (ATP-utilizing chromatin assembly and remodelingfactor) or RSF (remodeling and spacing factor) are two widely researched assembly factors thatare used to generate extended ordered arrays of nucleosomes into chromatin in vitro1, 13.
ACF and RSF differ slightly in chromatin assembly mechanisms. ACF requires a histone chaperone protein like NAP-1 and does not require the histones to be acetylated in the assembly of thecore particle. In contrast, RSF does require the acetylation of histones H2A and H2B, but it does
not need a protein to function as a histone chaperone13.
It is important to verify the chromatin assembly reaction resulted in an extended and ordered
array of nucleosomes spaced along the DNA. By performing a simple partial enzymatic digestion
followed by a quick deproteinization treatment, agarose gel analysis of the digestion will show aladder of DNA fragments corresponding to mono-, di-, tri- and oligonucleosomes. This indicatesthe nucleosomes are assembled into regularly spaced arrays (Figure 1). Assembled chromatinthat yields six or more DNA fragments upon digestion is considered high-quality and suitable forfurther assays13.

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Figure 1: Partial enzymatic digestion of chromatin assembled from supercoiled circular and linear DNA.
One hundred µl reactions were performed on 1 μg of either linear or circular DNA using the Chromatin Assembly Kit protocol. Following the chromatin assembly incubation, each reaction was divided in two, and partially digested with 0.5 µl
Enzymatic Shearing Cocktail for 2 and 4 minutes, respectively. Samples were subsequently deproteinated with Proteinase K,
phenol/chloroform extracted and analyzed by agarose gel electrophoresis.
Lane 1: 100 bp DNA ladder.
Lane 2: Circular DNA digested with Enzymatic Shearing Cocktail for 2 minutes .
Lane 3: Circular DNA digested with Enzymatic Shearing Cocktail for 4 minutes.
Lane 4: Linear DNA digested with Enzymatic Shearing Cocktail for 2 minutes.
Lane 5: Linear DNA digested with Enzymatic Shearing Cocktail for 4 minutes.
Both the circular and linear DNA produced high-quality assembled chromatin, with at least 11 distinct bands of DNA corresponding to mononucleosomes, dinucleosomes, etc. visible.

Chromatin Assembly Kit Applications
The Chromatin Assembly kit may be used to study any function of DNA in its native environment.
Relevant applications include:• In Vitro Transcription Assays. In vitro studies of factors (or inhibitors) that affect the initiationof transcription by a transcription factor• Studies of nucleosome core histone modifications such as acetylation and methylation• In vitro ChIP assays. In vitro ChIP assays can be performed with in vitro-assembled chromatin to find other proteins associated with the promoter during the transcription activation
process. In vitro ChIP assays could also be used for determining if an inhibitor can affect theprocess of recruitment of proteins needed to initiate the transcription process

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4Kit Components and Storage
Store components at the temperatures indicated below. Before beginning your first assay, thefollowing components must be aliquoted to eliminate subsequent freeze-thaw cycles:
10X ATP Regeneration System: Prepare 10 aliquots of 11 µl each. Store at -80°C. Use a new
aliquot each reaction.
Recombinant h-NAP-1 Protein: Prepare 5 aliquots of 3.5 μl each. Store at -80°C. Each vial of thiscomponent should only go through 2 freeze-thaw cycles.
Recombinant ACF Complex: Prepare 5 aliquots of 6 μl each. Store at -80°C. Each vial of thiscomponent should only go through 2 freeze-thaw cycles.
Creatine Kinase: Prepare 5 aliquots of 2 μl each. Store at -80°C. Each vial of this component
should only go through 2 freeze-thaw cycles. Discard unused portion after second freeze-thaw.
HeLa Core Histones: Prepare 4 aliquots of 6 µl each in the siliconized microcentrifuge tubes
provided. Do not use uncoated tubes. Leave the remaining in the supplied tube. Store at -80°C.
Each vial of this component may go through 3 freeze-thaw cycles.

Reagent

Quantity

Storage / Stability

Recombinant h-NAP-1 Protein (5.0 mg/ml)

18 µl-80°C for 6 months

HeLa Core Histones (1.5 mg/ml)

24 µl-80°C for 6 months

Recombinant ACF Complex (0.25 mg/ml)

30 µl-80ºC for 6 months

High Salt Buffer

100 µl-20ºC for 6 months

Low Salt Buffer

700 µl-20ºC for 6 months

10X ATP Regeneration System

110 µl-80ºC for 6 months

Creatine Kinase

10 µl-80ºC for 6 months

Supercoiled DNA (0.1 mg/ml)

20 µl-20ºC for 6 months

0.1 M CaCl2

30 µl

Room temp to -20ºC
for 6 months

Enzymatic Shearing Cocktail

50 µl-20ºC for 6 months

4X Enzymatic Stop Solution

400 µlRoom temp to -20ºC
for 6 months

Proteinase K

80 µl-20ºC for 6 months

Glycogen

10 µl-20ºC for 6 months

5X Orange G Dye

500 µl-20ºC for 6 months

25

Room temperature

Siliconized microcentrifuge tubes (1.5 ml)

The Chromatin Assembly Kit is for research use only. Not for use in diagnostic procedures.

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5Additional materials required•
Water bath•

Sterile distilled water (dH2O)•

Microcentrifuge Tubes•

20% SDS•

Phenol/chloroform•

5M NH4OAc•

100% and 70% ethanol•

TE (10 mM Tris, 1 mM EDTA pH 8.0)•

Reagents for purifying high-quality supercoiled DNA (e.g. Qiagen Plasmid Maxi Kit,
Catalog No. 12163).

•

100 bp DNA ladder (e.g. New England Biolabs, Catalog No. N3231S)•Standard Tris/Glycine agarose gel supplies
5X Tris Glycine Buffer: 107 g Tris base, 576 g Glycine and dH20 to 4 L.

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Chromatin Assembly
Component Aliquoting Recommendations
Before beginning the first assay, the following components should be thawed completely, mixed
by gentle vortexing and aliquoted as described below. Because the HeLa Core Histones are very
sticky, it is very important that you aliquot them into the provided siliconized tubes.
10X ATP Regeneration System
This component should be aliquoted and used fresh each time. We recommend that you make 10
aliquots of 11 µl each. Store all aliquots at -80°C.
Recombinant h-NAP-1 Protein
Prepare 5 aliquots of 3.5 μl each. Store at -80°C. This component should only go through2 freeze-thaw cycles.
Recombinant ACF Complex
Prepare 5 aliquots of 6 μl each. Store at -80°C. This component should only go through2 freeze-thaw cycles.
Creatine Kinase
Prepare 5 aliquots of 2 μl each. Store at -80°C. This component should only go through2 freeze-thaw cycles.
HeLa Core Histones
Prepare 4 aliquots of 6 µl each in the provided siliconized microcentrifuge tubes. Because core
histones frequently stick to the walls of microcentrifuge tubes, especially in small volumes suchas these, it is imperative to aliquot into the provided siliconized microcentrifuge tubes. Store at-80°C. This component may go through 3 freeze-thaw cycles.

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7Component Preparation Recommendations
Prior to starting the assay please prepare the following:
Preparation of Sample DNA
Prepare high quality DNA at 0.1 µg/µl.
Note 1:

The quality of the DNA is important for the efficiency of the chromatin assembly reaction.
The quality of the DNA may be checked on a TAE agarose gel. In general, high-quality
DNA can be prepared by standard CsCl gradient methods or a commercial kit may be
used e.g. Qiagen Plasmid Maxi Kit, Catalog No. 12163.

Note 2:

Ensure that the DNA is not contaminated with RNA. Histones can interact with RNA, which
will cause variable and inconsistent results.

Preparation of Complete 10X ATP Regeneration System
Prepare a sufficient quantity of Complete 10X ATP Regeneration System by adding 0.3 µl of Creatine Kinase to 10 µl of 10X ATP Regeneration System. Use immediately. Discard any remaining
Complete 10X ATP Regeneration System that you have made and make up fresh each time.

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Protocols - Chromatin Assembly
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
One reaction of the protocol below will generate 100 µl of assembled chromatin from 1 µg input
DNA. However, the partial enzymatic digestion protocol used to verify chromatin assembly will
use a minimum of 50 µl per reaction in order to visualize the material on a gel. Thus, dependingon what you plan to do with the assembled chromatin, you may wish to perform two or morereactions of the same material and then check only one of them. In addition, the assembled
chromatin is known to be stable at 4°C for 2 days only, and it should not be frozen. Therefore,
you should plan your assembly reactions, and how/when you plan to use them, carefully.

A. Chromatin Assembly Reaction
1.

Set water bath to 27°C.

2.

Add the following reagents in the order shown in the table below. If desired, siliconizedtubes can be used; however, uncoated microcentrifuge tubes can be used.
Note:

Keep reaction reagents on ice during reaction set-up and return to appropriate-80°C or -20°C storage immediately after addition.
Positive
Control

Negative
Control

SampleRecombinant h-NAP-1

1.4 µl–

1.4 µlHeLa Core Histones

1.8 µl–

1.8 µlHigh Salt Buffer

10 µl

10 µl

10 µl

Low Salt Buffer–

3.2 µl–Component

3.

Gently vortex the samples. Centrifuge to collect material at the bottom of the microcentrifuge tube. Incubate on ice for 15 minutes.

4.

Add 64.3 µl of Low Salt Buffer to each reaction.

5.

Add the following reagents in the order outlined in the table below.
Positive
ControlComponent

Negative
Control

SampleRecombinant ACF Complex

2.5 µl–

2.5 µlComplete 10X ATP Regeneration System

10 µl

10 µl

10 µl

Supercoiled DNA (control)

10 µl

10 µl

–

–

–

1 µg

Low Salt Buffer–

2.5 µl

–

dH2O

–

–

to 100 µl

100 µl100 µl100 µlSample DNA

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6.

Gently vortex the samples. Briefly centrifuge to collect material at the bottom of the microcentrifuge tube. Incubate at 27°C for 4 hours. After incubation, the sample may be stored at
4°C for up to 2 days. Chromatin should not be frozen at this point.
Note:

Though reaction conditions have been optimized for chromatin assembly from circular
plasmid DNA, this protocol will also work for linear DNA (see Figure 1).

The assembled chromatin prepared above can be used directly in downstream assays. Certain
applications, such as electron microscopy studies, may require further purification, which can beaccomplished by sucrose gradient sedimentation or gel filtration1.

The following steps are optional. They are included so that you can check for successful
chromatin assembly.

B. Analysis of Assembled Chromatin by Partial Digestion
Partial digestion with the Enzymatic Shearing Cocktail will reveal periodic spacing of assembled
nucleosomes. After the chromatin is digested, it is deproteinated. The resulting DNA fragments
are resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Highquality chromatin should yield 6 or more distinct bands.
1.

Add 3.0 μl of 0.1 M CaCl2 (3 mM final concentration) to each 100 μl reaction of assembled
chromatin. Mix well.

2.

Digest each reaction with 1 μl of Enzymatic Shearing Cocktail for 2 minutes at room temperature.
Note:

3.

We recommend that two time points be tested for each enzymatic digestion e.g., split the
100 μl reaction into two tubes and digest each aliquot with 0.5 μl of Enzymatic Shearing
Cocktail for 2 and 4 minutes respectively. Splitting the reaction below 50 µl will makereaction efficiency difficult to visualize by agarose gel electrophoresis.

Add 34 μl (or 17 μl for half volume reaction) of 4X Enzymatic Stop Solution. Mix well. Incubate on ice for 10 min. The reactions may be stored at -20°C at this point.
Note:

The quantity of enzyme and time of incubation may need to be optimized for the sample
DNA. The conditions in this protocol are optimized for the included positive control DNA,
which is supercoiled circular DNA.

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C. Deproteinization with Proteinase K1.

Add 20% SDS to a final concentration of 0.5% (3.45 μl per 100 μl or 1.72 µl per half volume
reaction).

2.

Add 1 μl (or 0.5 μl per half volume reaction) of Proteinase K.

3.

Incubate at 55°C for 15-30 minutes.

4.

Add 1 μl Glycogen.

5.

Add sterile dH2O to final volume of 200 μl.

6.

Perform a phenol/chloroform extraction once, i.e. add 200 μl of phenol/chlorofom, vortex
samples and spin for 5 minutes at room temperature.

7.

Remove the top 200 μl (supernatant) and mix with 200 μl of 5 M NH4OAc and 1 ml 100%
EtOH.

8.

Spin for 15 minutes at 4°C. Discard the supernatant. (A little white pellet is visible at thispoint. Take care not to disturb the pellet, leave 10-20 μl close to the pellet).

9.

Wash DNA pellet by adding 500 μl 70% EtOH, and centrifuge at 4°C for 5 minutes. Remove
as much of the EtOH as possible.

10. Air dry for approximately 5 minutes.
11.

Resuspend pellet in 8 μl dH2O (or TE). Add 2 μl 5X Orange G dye.
Note:

The Orange G dye can be replaced by another dye. However, some dyes e.g. Bromophenol Blue migrate with bands at 100 bp.

12. The sample reactions can be stored at -20°C or analyzed immediately by agarose gel electrophoresis.

D. Agarose Gel Electrophoresis
1.

Perform agarose gel electrophoresis (1.5% agarose gel in cold 1X Tris-Glycine buffer in a 4°C
cold room). Run the samples and a 100 bp ladder at 150V for approximately 1 hour and 20
minutes. Add ethidium bromide to the gel and the buffer.
Note:

It is possible to run a 1.5% TAE agarose gel in cold 1X TAE buffer. However, the band
resolution will be less than when using Tris-Glycine gels and buffers.

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Downstream Applications
The following assays are just a few examples of how to further research chromatin-dependent
processes with your assembled chromatin.
In vitro ChIP Assay
Perform an in vitro ChIP assay to verify transcription factor binding to the assembled chromatin
and to investigate cofactor or inhibitor influence on binding3, 14.
In vitro Transcription Assay
The transcription process is suppressed, in general, by chromatin but not by free DNA. An in vitrotranscription assay can be used to determine what factor is involved in transcription activationfrom the assembled chromatin3, 14.
In vitro HAT Assay
With the assembled chromatin, a HAT assay can be performed to study the acetylation of histones in their native environment, making it is possible to analyze which factors are involved in or
are necessary for histone acetylation3, 14, 15.

References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.

Fyodorov, D.V., and Kadonaga, J.T. (2003) Method Enzymol. 371: 499-515.
Nakagawa, T. et al. (2001) J. Biol. Chem. 276: 27384-27391.
Thomas, M. C., and Chiang, Cheng-M. (2005) Mol. Cell. 17: 251-164.
Tsukiyama, T. et al. (1994) Nature 367: 525-532.
Pazin, M.J. et al. (1996) Genes Dev. 10: 37-49.
Armstrong, J.A. and Emerson, B.M. (1996) Mol. Cell. Biol. 16: 5634-5644.
Pazin, M.J. et al. (1994) Science 266: 2007-2011.
Sheridan, P.L. et al. (1994) Genes Dev. 9: 2090-2104.
Mayall, T.P. et al. (1997) Genes Dev. 11: 887-899.
Kraus, W.L. and Kadonaga, J.T. (1998) Genes Dev. 12: 331-342.
Naar, A.M. et al. (1999) Nature 398, 828-832.
Pazin, M.J. et al. (1997) Science 276, 809-812.
Lusser, A. and Kadonaga, J.T. (2004) Nat. Methods 1 (1): 19-26.
Koop, R. et al. (2003) Embo J.22(3): 588-599.
Kundu, T. K. et al. (2000) Mol. Cell 6: 551-561.

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Appendix
Section A. Troubleshooting Guide
PROBLEMPOSSIBLE CAUSE

RECOMMENDATION

Less than 6 nucleosome bands on agarose
gel without a similarsmear of DNA to what isseen with the negative
control.

The chromatin assembly
reaction worked well but thechromatin was subsequently
over-digested.

The enzymatic digestion may need to be
optimized e.g. try less enzyme or a shortertime for the enzymatic digestion.

Less than 6 nucleosome
bands on agarose gelwith visible DNA of highmolecular weight.

The chromatin assembly
reaction worked well but thechromatin was subsequently
under-digested.

The enzymatic digestion may need to be
optimized e.g. try adding more enzyme ordigesting for a longer time.

Less than 6 nucleosome bands on agarose
gel with a smear of
DNA similar to what isseen with the negative
control.

Low efficiency of the assembly
reaction due to too much or too
little DNA.

Adjust the DNA concentration. Perform atitration of DNA to histone ratios ranging
around 1:11, 13.

Low efficiency of the assembly
reaction due to poor quality
DNA.

Repeat DNA purification with CsCl gradientpurification or commercially available DNA
purification kit e.g. Qiagen Plasmid Maxi
Kit, Catalog No. 12163.

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13

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

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