Nucleosome Preparation Kit(version A3)
Catalog No. 53504

Copyright 2014 Active Motif, Inc.

Revision History
Revision

Date

Description of Change

A2

Dec 2018

Corrected Troubleshooting Guide
recommendations

Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of
any kind, either explicit or implied. Information may be changed or updated in this manual at any
time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation is proprietary information and protected by the copyright laws of the United States and international
treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2014 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights
reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective
companies.
www.activemotif.com

TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Nucleosome Preparation Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Protocols
Section A.
Section B.
Section C.
Section D.

Optimization of Enzymatic Digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
DNA Cleanup to Assess Shearing Efficiency and DNA Concentration. . . . . 6
Nucleosome Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Nucleosome Assessment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Appendix
Section E. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

www.activemotif.com

Overview
Active Motif’s Nucleosome Preparation Kit is designed to prepare mono- and oligonucleosomesfrom your samples. Nucleosomes comprise the smallest subunit of chromatin and consist of 147
base pairs of DNA wrapped around an octamer of core histone proteins. The histone octamer iscomposed of a central heterotetramer of histones H3 and H4, flanked by two heterodimers ofhistones H2A and H2B. Each nucleosome is separated by a linker of DNA.
In the Nucleosome Preparation Kit, an enzymatic cocktail is used to digest the linker DNA. By
adjusting the digestion time, it is possible to modify the amount of mononucleosomes or oligonucleosomes extracted. Isolated nucleosomes are ideal for use as substrates in the analysis ofhistone post-translational modifications, enzyme kinetics or inhibitor screening studies as they
represent more physiologically relevant substrates than histone proteins alone or synthetic peptides. The Nucleosome Preparation Kit includes enough reagent for 20 preparations from
10 cm plates and 5 sample optimizations.

product

formatcatalog no.

Nucleosome Preparation Kit

20 rxns

53504

www.activemotif.com

1

Kit Performance and Benefits
Nucleosome Advantages• More physiologically relevant substrates than synthetic peptides• Useful for the analysis of histone post-translational modifications, enzyme kinetics orinhibitor screening studies• Easily adjust the level of mononucleosomes or oligonucleosomes by adjusting digestion
time
Figure 1: Gel analysis of Nucleosome digestion.
Nucleosomes were prepared from HeLa cells and digested with
the Enzymatic Shearing Cocktail for 5, 10 & 15 minutes. Following digestion, the reactions were stopped with the addition ofcold EDTA. The nucleosomes were then subjected to DNA clean
up to assess the digestion efficiency. Samples were separated
by electrophoresis through a 1.5% agarose gel.
Lane 1: 100 to 1000 bp ladder.
Lane 2: Unsheared HeLa DNA.
Lane 3: HeLa DNA treated for 5 minutes
Lane 4: HeLa DNA treated for 10 minutes
Lane 5: HeLa DNA treated for 15 minutes

1000bp
500bp

100bp
1

2

3

4*

5

Figure 2: Gel shift seen with intact nucleosomes as comparedto purified nucleosome DNA.
Nucleosomes were prepared from HeLa cells according the
instructions in the Nucleosome Preparation Kit. A 50 µl aliquot of
the extracted nucleosomes was then subjected to DNA clean up
to assess the digestion efficiency. Both intact nucleosomes andpurified DNA samples were analyzed by TapeStation. The results
show an increase in molecular weight banding for the intact
nucleosomes as compared to the purified DNA samples. This ispredicted since an intact nucleosome complex contains boththe DNA and histone proteins
Lane A: 25 to 1500 bp DNA ladder.
Lane B1: Intact nucleosome sample
Lane C1: Purified nucleosome DNA

www.activemotif.com

2

Kit Components and Storage
Please store each component at the temperature indicated in the table below.
Reagents

Quantity

Storage / Stability

100 mM PMSF

500 µl

-20°C for 6 months

Protease Inhibitor Cocktail (PIC)

500 µl

-20°C for 6 months

1X Lysis Buffer

15 ml-20°C for 6 months

Enzymatic Shearing Cocktail

8 µl-20°C for 6 months

5 M NaCl

400 µl

-20°C for 6 months

RNase A (10 µg/µl)

40 µl-20°C for 6 months

Proteinase K (10 µg/µl)

500 µl

-20°C for 6 months

10X PBS

100 ml

4°C for 6 months

Digestion Buffer

10 ml

4°C for 6 months

0.5 M EDTA

300 µl

4°C for 6 months

2 x 500 µl

RT for 6 months

3 M Sodium Acetate

Additional materials required• Phase contrast/tissue culture microscope and hemocytometer• Cell scraper (rubber policeman)• Microcentrifuge (table top centrifuge 4°C) and microcentrifuge tubes• Pipettors and tips (filter tips are recommended)• 50% Glycerol in dH2O
• (Optional) Dounce homogenizer with a small clearance pestle (Active Motif Catalog Nos.
40401 & 40415)• Phenol/chloroform (1:1) TE saturated pH 8 (DNA Purification, Molecular Biology Grade)• 100% ethanol• 70% ethanol• DNase-free H2O• Spectrophotometer for DNA quantitation• Agarose gel electrophoresis apparatus or Bioanalyzer or TapeStation Instruments

www.activemotif.com

3

Nucleosome Preparation Experimental Design
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Points to consider:• Cell growth and nucleosome preparation. When planning an experiment, calculate thenumber of preparations you will require. Note that if you wish to analyze the effect ofparticular compounds or culturing conditions, you should prepare nucleosomes from control(untreated) cells as a reference sample.
1 well of a
24-well plate

10 cm plate

15 cm plate

130,000

0.66 x 107

1.5x 107

1X PBS Washes

2 x 1 ml2 x 5 ml2 x 10 ml

1X PBS + PMSF

500 µl +2.5 µl PMSF

1 ml +
5 µl PMSF

5 ml +
30 µl PMSF

Lysis Buffer +PIC + PMSF

200 µl + 1 µl PIC +1 µl PMSF

500 µl + 2.5 µl PIC +2.5 µl PMSF

1 ml + 5 µl PIC +5 µl PMSF

Digestion Buffer +PIC + PMSF

50 µl + 0.25 µl PIC +0.25 µl PMSF

175 µl + 1 µl PIC +1 µl PMSF

350 µl + 1.75 µl PIC +1.75 µl PMSF

Enzymatic Shearing
Cocktail, diluted

2.5 µl

8 µl

17 µl

0.5 M EDTA

1 µl

3.5 µl7 µl

Number of Cells• Resuspend solutions completely. Thaw the PMSF at room temperature until fully dissolved,
which takes about 30 minutes. Vortex gently and spin down briefly before use.
• Safety precautions. PMSF is a highly toxic chemical. Appropriate safety precautions (i.e.
safety glasses, gloves and lab coat) should be used. Cell culturing should be performed in abiosafety hood if the nucleosomes are extracted from biohazardous or infectious materials.
• Use of a dounce homogenizer. The most critical aspect of nucleosome preparation is efficient lysis of the cells so that the enzymatic cocktail can access the chromatin. If your cells
do not lyse efficiently in the Lysis Buffer, we strongly recommend a dounce homogenizer
with a small clearance size pestle to mechanically shear the plasma membrane without
disrupting the nuclei. Monitoring the cell lysis using a microscope can be very helpful. It is
important to not add the Enzymatic Cocktail until the cells are lysed.

www.activemotif.com

4

Protocols
Section A. Optimization of Enzymatic Digestion
Prior to performing nucleosome extraction, it is recommended to optimize the digestion conditions for your cell line and treatment conditions. Modification of the digestion time may be utilized to increase or decrease the abundance of mononucleosomes or oligonucleosomes. For the
optimization protocol, nucleosomes are prepared from one 15 cm plate (approximately 1.5 x 107
cells). Note that although ~350 µl of sample is prepared, only 200 µl is used to analyze digestion
efficiency. Enough reagents are included to perform the optimization protocol with 5 samples.
1.

Grow cells to 70-80% confluency in one 15 cm plate. Stimulate cells as desired to activate
the pathway of interest.

2.

When cells are ready to harvest, wash twice with 10 ml 1X PBS.

3.

Just before use, add 30 µl 100 mM PMSF to 5 ml of ice-cold 1X PBS to each plate and scrape
cells with a rubber policeman. Hold the plate at an angle and scrape cells down to collectthem at the bottom edge of the plate. Use a 1 ml pipette to transfer the cells to a 15 ml conical tube on ice.

4.

Pellet the cells by centrifugation for 10 minutes at 2,500 rpm (720 RCF) at 4°C.

5.

Remove the supernatant and discard. At this point the protocol can be continued or thepellet can be frozen. If freezing the pellet, add 1 µl 100 mM PMSF and 1 µl PIC and freeze at-80°C.

6.

Thaw pellet (if necessary) on ice and resuspend cells in 1 ml ice-cold Lysis Buffer supplemented with 5 µl PIC + 5 µl 100 mM PMSF. Pipette gently and vortex briefly to resuspend.
Transfer to a 1.5 ml microcentrifuge tube. Incubate on ice for 30 minutes.
Monitor Cell Lysis: To ensure cell lysis, take 10 µl of the cell lysate and look at it undera phase contrast microscope using a hemocytometer to verify that the nuclei have beenreleased. It is often helpful to look at the cells before and after the lysis step as this makes it
easier to identify the nuclei versus whole cells. Intact cells should have a dark central region(nucleus) surrounded by a halo of less dense cytoplasm. In lysed cells, the nuclei will appear
as dots surrounded by asymmetric debris. If the cells are not lysed, then we recommend
using a dounce homogenizer with a small clearance size pestle to mechanically shear theplasma membrane without disrupting the nuclei. Dounce on ice with 10 strokes, or until thecells are lysed.

7.

During this incubation, prepare a working stock of Enzymatic Shearing Cocktail (200 U/ml)
by diluting the supplied Enzymatic Shearing Cocktail (2 x 104 U/ml) 1:100 with 50% glycerol
in dH2O (not provided). The 200 U/ml working stock will be used in step 11 below and isstable at 4°C for 1-2 weeks.
Reagent

5 rxnsStock Enzymatic Shearing Cocktail (2 x 104 U/ml)

0.5 µl

50% glycerol

49.5 µl

www.activemotif.com

5

8.

Centrifuge lysed cells for 10 minutes at 5,000 rpm (2,400 RCF) in a 4°C microcentrifuge topellet the nuclei.

9.

Carefully remove the supernatant and discard. Resuspend the nuclei pellet in 350 µl Digestion Buffer supplemented with 1.75 µl PIC and 1.75 µl 100 mM PMSF and incubate for5 minutes at 37°C.

10. Transfer 50 µl of the chromatin in Digestion Buffer to each of 4 fresh microcentrifuge tubes
and incubate the tubes at 37°C for 2 minutes. The remaining ~150 µl chromatin can be
stored at -80°C.
11.

To optimize enzymatic digestion conditions, set up 4 reactions as indicated below. Vortex
the tubes on a low setting to mix components. Vortex the tube briefly approximately every 2
minutes during the incubation to increase the digestion efficiency.
a.

50 µl chromatin plus 2.5 µl dH20 (No Enzyme) – incubate for 10 minutes at 37°Cb.

50 µl chromatin plus 2.5 µl working stock Enzyme – incubate for 5 minutes at 37°Cc.

50 µl chromatin plus 2.5 µl working stock Enzyme – incubate for 10 minutes at 37°Cd.

50 µl chromatin plus 2.5 µl working stock Enzyme – incubate for 15 minutes at 37°C

12. Stop the reactions by adding 1 µl ice-cold 0.5 M EDTA to each tube. Chill on ice 10 minutes.
13. Centrifuge the digested nucleosome samples for 10 minutes at 15,000 rpm (18,000 RCF) ina
4°C microcentrifuge. Collect the supernatant. Nucleosomes can be stored at -80°C. Or, continue immediately with Section B below to purify the DNA for gel analysis and quantification.

Section B. DNA Clean Up to Assess Digestion Efficiency and
DNA Concentration
1.

If necessary, thaw the 50 µl aliquots of each digested nucleosome sample.

2.

Add 150 µl dH2O, then 10 µl 5 M NaCl to each tube.

3.

Add 1 µl RNase A to each sample and incubate at 37°C for 15 minutes.

4.

Add 10 µl Proteinase K to each sample and incubate at 42°C for 1.5 hours.
Note:

If you intend to use a spectrophotometer to determine the DNA concentration,
the DNA must first be cleaned up by phenol/chloroform extraction and precipitation using the following protocol:a.

Add 200 µl 1:1 phenol/chloroform TE saturated pH 8 to the sample, vortex to mix
completely and centrifuge for 5 minutes at maximum speed in a microcentrifuge.

b.

Transfer the aqueous phase to a fresh microcentrifuge tube, then add 20 µl 3 M
Sodium Acetate pH 5.2 and 500 µl 100% ethanol. Vortex to mix completely and placeat -80°C for at least 1 hour. Alternatively, the sample can be left at -20°C overnight.

c.

Centrifuge at maximum speed for 10 minutes in a microcentrifuge at 4°C.

d.

Carefully remove and discard supernatant. Do not disturb the pellet.

e.

Add 500 µl 70% ice cold ethanol without disturbing the pellet and spin for 5 minutes atwww.activemotif.com

6

maximum speed in a 4°C microcentrifuge.

5.

f.

Carefully remove and discard supernatant. Do not disturb pellet. Allow pellet to air-dry.

g.

Resuspend pellet in 30 µl dH2O and use a spectrophotometer to measure theabsorbance at 260 nm to determine the DNA concentration (1.0 A260 unit = 50 µg/ml).

We recommended loading two different quantities of each digested sample on the gel toensure one falls within an acceptable range. Add 4 µl of a 6X Loading Buffer to 16 µl ofsample, then load 5 µl & 10 µl of each sample on a 1.5% TAE agarose gel. Run the gel at
100V until the loading dye reaches 3/4 of the way to the end of the gel.
Note:

Alternatively an instrument such as the Bioanalyzer or TapeStation may be used
to analyze the purified DNA and assess the digestion efficiency.

The enzymes used in the Enzymatic Cocktail are random cleavers (sequence independent).
However, they prefer open regions that are easily accessible, such as between nucleosomes. Therefore, digestion produces a specific DNA fragment size banding pattern:~150 bp (1 nucleosome), ~300 (2 nucleosomes), ~450 bp (3 nucleosomes) etc., with an everdecreasing band intensity as the molecular weights increase.
6.

After you have determined the condition that provides the desired nucleosome pattern,
proceed with Section C to prepare nucleosomes.
Note:

The remaining chromatin sample stored in Step A.10 (~150 µl) can be digested
using 7.5 µl working stock Enzyme. Incubate at 37°C for the time that yielded
suitable digestion. Stop the reaction with 3 µl ice-cold 0.5 M EDTA. Chill on ice
10 minutes. Centrifuge the sample for 10 minutes at 15,000 rpm (18,000 RCF) in
a 4°C microcentrifuge. Collect the supernatant. This extracted nucleosomes canbe stored at -80°C for up to a year or used immediately.

Section C. Nucleosome Preparation
This protocol describes nucleosome preparation under optimized conditions (as determinedin Sections A-B) from one 10 cm plate (approximately 6.6 x 106 cells). To scale the protocol forother cell numbers, please use the table on page 7 as a guide. Enough reagents are provided toperform 20 nucleosome preparations from 10 cm plates.
1.

Grow cells to 70-80% confluency in one 10 cm plate. Stimulate cells as desired to activate
the pathway of interest.

2.

When cells are ready to harvest, wash twice with 5 ml 1X PBS.

3.

Just before use, add 5 µl 100 mM PMSF to 1 ml of ice-cold 1X PBS to each plate and scrape
cells with a rubber policeman. Hold the plate at an angle and scrape cells down to collectthem at the bottom edge of the plate. Use a 1 ml pipette to transfer the cells to a 1.5 ml
microcentrifuge tube on ice.

4.

Pellet the cells by centrifugation for 10 minutes at 2,500 rpm (720 RCF) at 4°C.

www.activemotif.com

7

5.

Remove the supernatant and discard. At this point the protocol can be continued or thepellet can be frozen. If freezing the pellet, add 1 µl 100 mM PMSF and 1 µl PIC and freeze at-80°C.

6.

Thaw pellet (if necessary) on ice and resuspend cells in 500 µl ice-cold Lysis Buffer supplemented with 2.5 µl PIC + 2.5 µl 100 mM PMSF. Pipette gently and vortex briefly to resuspend. Incubate on ice for 30 minutes.
Monitor Cell Lysis: To ensure cell lysis, take 10 µl of the cell lysate and look at it undera phase contrast microscope using a hemocytometer to verify that the nuclei have beenreleased. It is often helpful to look at the cells before and after the lysis step as this makes it
easier to identify the nuclei versus whole cells. Intact cells should have a dark central region(nucleus) surrounded by a halo of less dense cytoplasm. In lysed cells, the nuclei will appear
as dots surrounded by asymmetric debris. If the cells are not lysed, then we recommend
using a dounce homogenizer with a small clearance size pestle to mechanically shear theplasma membrane without disrupting the nuclei. Dounce on ice with 10 strokes, or until thecells are lysed.

7.

During this incubation, prepare a working stock of Enzymatic Shearing Cocktail (200 U/ml)
by diluting the supplied Enzymatic Shearing Cocktail (2 x 104 U/ml) 1:100 with 50% glycerol
in dH2O (not provided). The 200 U/ml working stock will be used in step 10 below and isstable at 4°C for 1-2 weeks.
Reagent

1-2 rxns

3-5 rxns

6-10 rxns

Stock Enzymatic Shearing Cocktail (2 x 104 U/ml)

0.5 µl1 µl

2 µl

50% glycerol

49.5 µl

99 µl

198 µl8.

Centrifuge for 10 minutes at 5,000 rpm (2,400 RCF) in a 4°C microcentrifuge to pellet thenuclei.

9.

Carefully remove the supernatant and discard. Resuspend the nuclei pellet in 175 µl Digestion Buffer supplemented with 1 µl PIC and 1 µl PMSF and incubate for 5 minutes at 37°C.

10. Add 8 μl of the working stock of Enzymatic Shearing Cocktail (200 U/ml) to the pre-warmed
nuclei and vortex to mix.
11.

Incubate the tube at 37°C for the amount of time that you determined to be optimal for yourcell line. Vortex the tube briefly approximately every 2 minutes during the incubation toincrease the digestion efficiency.

12. Stop the reaction by adding 3.5 μl ice-cold 0.5 M EDTA to each tube; chill on ice 10 minutes.
13. Centrifuge the digested nucleosome samples for 10 minutes at 15,000 rpm (18,000 RCF)
in a 4°C microcentrifuge. Carefully transfer supernatant to a fresh 1.7 ml microcentrifuge
tube. This contains your nucleosomes. Nucleosomes should be aliquoted before freezing tominimize freeze-thaw cycles and stored at -80°C. Before freezing, remove 50 µl for use inassessing the efficiency of your digestion and determining the DNA concentration.

www.activemotif.com

8Section D. Nucleosome Assessment
1.

To assess the efficiency of the enzymatic digestion or to quantify the nucleosome DNA,use the 50 µl aliquot and follow the steps in Section B: DNA Clean Up to Assess Digestion
Efficiency and DNA concentration.

2.

To quantify nucleosome protein content, measure the absorbance at 230 nm. An OD of 0.42
from a sample diluted 1:10 indicates a protein concentration of 1 mg/ml.
Undiluted samples may be outside the linear range of your spectrophotometer. Therefore,
we recommend preparing a 1/10 dilution of your samples before quantifying.

OD of 0.42 = 1 mg / ml
OD 0.42Your OD=
1 mg/mlx mg / mlwww.activemotif.com

Solve for x = Concentration of diluted stock
Multiply by 10 = Actual histone concentration

9

Appendix
Section E. Troubleshooting Guide
Problem/question

Recommendation

Poor yield of nucleosomes

Nuclei not released. It may be necessary to perform dounce homogenization. Use a dounce
homogenizer with a small clearance pestle (see reference in Optional materials on page 3).
Monitor cell lysis under a microscope as described in the procedures. Generally, the morecells that are lysed, the higher the yield.

Digestion efficiency is notclear from gel analysis.

Material is stuck in the wells, and smears or streaks are seen from the top to bottom of
the lane. The sheared chromatin needs to have protein removed (Proteinase K) and RNA
removed (RNase), followed by DNA purification using phenol/chloroform. Follow the DNA
Clean Up protocol in Appendix – Section C. DNA purification columns are not recommended, as the high protein content may clog the columns.
Lost DNA during the purification step. Phenol should be saturated with TE pH 8. Lower pH
solutions will degrade the DNA. Column purification is not recommended due to the highprotein content of the sample, which may clog the column.
High molecular weight products. You must repeat the chromatin preparation using longer
digestion times. Be sure to vortex the sample every 2 minutes during the digestion.

The ladder-like banding
pattern is not seen on
the gelA single ~100-200 bp band is present, and its appearance is not altered by the different
digestion times. This usually indicates that the cells were not completely lysed. Follow the
instructions for douncing the sample.
No DNA. This is normal for the undigested DNA lane. The DNA is present, but in such a wide
range of sizes it is not visible on the gel. No DNA in a digested sample lane indicates thatthe sample was too small or the DNA was lost during DNA purification.
Low molecular weight smear. The digestion time was too long or the Enzymatic Shearing
Cocktail was not properly diluted. A large band/smear at bottom of gel may indicate RNA
was not removed using the provided RNase.
No specific bands, but a smear that decreases in size in lanes with longer digestion time.
This indicates degraded DNA. Process the samples (particularly tissue) more quickly, besure to keep on ice as directed and to add the appropriate proteinase inhibitors.

www.activemotif.com

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

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

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

Visit Active Motif online at activemotif.com

www.activemotif.com

11