Histone Purification Mini Kit(version B4)
Catalog No. 40026

Copyright 2014 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
any kind, either explicit or implied. Information may be changed or updated in this manual at anytime.
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.
© 2019 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 respectivecompanies.
The purification method used in this kit is covered under US patent 8,163,481.

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TABLE OF CONTENTS

Page

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Kit PerformanceCore Histones Purified from Cells and Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Method Preserves Post-translational Modifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Yield of Histones: Adherent Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Yield of Histones: Suspension Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Yield of Histones: Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
ProtocolStep 1: Prepare Sample Extracts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Step 2: Prepare Crude Core Histone Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Step 3: Purifying Core Histones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Step 4: Precipitation of Histone Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Step 5: Quantify Yield of Histone Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Appendix
Section A: Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

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Introduction
Active Motif’s Histone Purification Mini Kit enables you to purify the core histone proteins andenrich for histone fractions while preserving their post-translational modifications (e.g. acetylation, methylation, and phosphorylation). This purification method is an improvement over acidprecipitation methods and utilizes a convenient spin column and a proprietary buffer system topurify the core histones from cells and tissue samples. Histones isolated by this method aresuitable substrates for downstream assays and in vitro chromatin assembly.
Unlike standard acid extraction techniques, this kit uses proprietary technology to purify the corehistones. Post-translational modifications such as phosphorylation, acetylation and methylation
are preserved, so you can extract core histone proteins from your cell culture or animal modeland determine which modifications are present.
The Histone Purification Mini Kit provides reagents for 20 histone purifications from as little as8x105 cells up to grams of tissue due to the robust histone binding capacity of the purification
column. The kit method is simple: first, an extract is made and applied to the purification column,
then histones are eluted, enabling the purification of core histones. Histones may be quantified
by an OD reading or quantitated empirically on a gel by comparison with histone standards.

productformat

catalog no.

Histone Purification Mini Kit

20 rxns

40026

The Histone Purification Mini Kit is for research use only. Not for use in diagnostic procedures.
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Kit PerformanceCore Histones Purified from Cells and Tissue

Figure 1: SDS-PAGE of histone fractions purified using the Histone Purification Mini Kit.
Ten µg per lane of core histones purified from logarithmically growing tissue culture cells (TC) and core histones isolated fromrat brain tissue (Brain) were loaded and run on a 16% Tris-glycine gel.

Histone Purification Mini Kit preserves post-translational modifications
Active Motif’s Histone Purification Mini Kit preserves phosphoryl, acetyl and methyl post-translational modifications on histones. The Western blot data shown below of the first and second
elutions of unstimulated HeLa (lanes 1-2), sodium butyrate-treated HeLa (lanes 3-4), paclitaxeltreated HeLa (lanes 5-6) and rat brain histones (lanes 7-8) demonstrates that post-translationalmodifications are intact following purification of histones.

123Acetyl-Histone H3

Phospho-Ser28 Histone H3

Methyl-Lys4 Histone H3

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245678Yield of Histones:
The following yields are approximate. Results may vary according to cell or tissue type. It ispossible to obtain a decent yield (10 µg) of histones from as few as 8 x 105 mammalian cells.
Adherent Cells: 0.1 mg total core histones from 8 x 106 cells (one 150 mm plate).
Suspension Cells: 0.1 mg total core histones from 8 x 106 cells.
Tissue: 1 mg histone per gram of tissue** The theoretical binding limit for these columns is 1 mg, but this has not been
confirmed empirically. Loading more than the theoretical limit is not recommended.
MW123456Figure 2: Analysis of histone yield using varying numbers of HeLa cells.
Histones were extracted from different numbers of HeLa cells using the Histone Purification Mini Kit and loaded onto a 16%
SDS-PAGE gel. Purified HeLa core histones were loaded as a standard in parallel. On this particular gel system, H2A and H2Bexhibit coincident migration.
Lane 1: 15 µl of a 100 µl elution from histones purified from a single 150 mm tissue culture plate of HeLa cells at
100% confluency (8 x 106 cells).
Lane 2: 15 µl of a 50 µl elution from histones purified from half of the cells from a 150 mm tissue culture plate of HeLa
cells at 100% confluency (4 x 106 cells).
Lane 3: 15 µl of a 50 µl elution from histones purified from one-fifth of the cells from a 150 mm tissue culture plate of
HeLa cells at 100% confluency (1.6 x 106 cells).
Lane 4: 15 µl of a 50 µl elution from histones purified from one-tenth of the cells from a 150 mm tissue culture plate ofHeLa cells at 100% confluency (8 x 105 cells).
Lane 5: 2.5 µg of purified HeLa core histones.
Lane 6: 5 µg of purified HeLa core histones.

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3Kit Components and Storage
Please store each component at the temperature indicated below. All components are guaranteed stable for 6 months from date of purchase when stored at the appropriate temperatures.
Reagents

Quantity

Storage / Stability

5X Neutralization Buffer

50 ml

4°C

Extraction Buffer

100 ml4°CEquilibration Buffer

10 ml

4°C

Histone Wash Buffer

30 ml

4°C

Histone Elution Buffer

10 ml

4°C

Purification Spin Columns

20

Room Temp

Collection Tubes

40

Room Temp

Additional materials required• Dounce homogenizer with a small clearance pestle (e.g. Active Motif cat. nos. 40401 or40415; the large pestles supplied with these Dounces can be used for initial sample reduction, while the small pestles should be used to process the final homogenate).
• 1.7 ml microcentrifuge tubes• Sterile water or Tris-EDTA, pH 8.0• SDS sample buffer• SDS-PAGE and Western blot reagents• For precipitation of histones: Perchloric acid, 70% (Acros Organics, part no. 424030010)• For washing of histones:
4% perchloric acid
0.2% HCl in acetone*
Cold 100% acetone (VWR, part no. BDH1101)* Add 0.5 ml of HCl stock (36.8%, Sigma, part no. H-7020) and adjust the volume with acetone
until 92 ml. Store in a glass bottle at 4°C.

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4Protocol
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Step 1: Prepare Sample Extracts
For adherent cells:
1. Grow cells to 80-90% confluence in the appropriate medium. Discard media and wash thecells 2 times with pre-warmed (37°C) serum-free media: use 2 ml for a 35 mm dish; 10 ml
for a 100 mm dish; or 20 ml for a 150 mm dish.
2.

After the 2nd wash, aspirate any remaining wash media and add ice-cold Extraction Buffer
to each dish: use 0.3 ml for each 35 mm dish; 0.8 ml for a 100 mm dish; or 1.5 ml for a 150mm dish. Using a plastic scraper and a pipet, collect the cell protein extracts in a 1.7 ml
microcentrifuge tube or a 15 ml tube as dictated by the amount of material. Pipet the cells
up and down to homogenize them into the solution.

For suspension cells:
1. Grow cells to about 80-90% confluence in appropriate medium. If necessary, gently scrape
the cells from the sides of the dish while keeping them in their media.
2.

Pipet the cells with the media and transfer to a 15 ml tube.

3.

Centrifuge at 1,000 x g for 5 minutes at room temperature.

4.

Wash the cells twice with 20 ml pre-warmed (37°C) serum-free media. After each wash,
centrifuge as in No. 3 above, then discard the serum-free media.

5.

Resuspend the cells in ice-cold Extraction Buffer (0.5 ml per 150 cm2 flask yields goodconcentrated extracts). Pipet up and down to homogenize the cells well, then transfer theresuspended cells to a microcentrifuge tube.

For tissues:
1. Homogenize the tissue completely in an ice-cold Dounce homogenizer using as little icecold Extraction Buffer as possible, as this will help ensure a highly concentrated extract.
Keep the homogenate on ice.
2.

Transfer the homogenate into a fresh microcentrifuge tube. Continue with Step 2: Prepare
Crude Core Histone Extract on page 6.

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5Step 2: Prepare Crude Core Histone Extract
1.

Leave the cells in Extraction Buffer for 30 minutes to 2 hours (or overnight) on a rotating
platform at 4°C. (Time course experiments performed on some cell lines have shown thatmost histones are extracted in as little as 10 minutes).

2.

Transfer the cell extracts to fresh tubes and centrifuge at maximum speed in a microfuge for5 minutes at 4°C.

3.

Transfer the supernatant, which contains the crude histones, to a new tube. The pellet canbe discarded or stored at -20°C for future analysis. If the pellet is to be stored, neutralize itby adding 100 µl of 1 M Tris HCl pH 8.0.

4.

Store the crude histones at -80°C, or continue with the next step.

5.

Neutralize and equilibrate the crude histones with 1/4 volume of 5X Neutralization Buffer.
For example, add 0.25 ml of 5X Neutralization Buffer to 1 ml crude histones, 0.5 ml (5X) to2 ml, 1 ml (5X) to 4 ml of crude histones, etc.

6.

Check the pH. If still acidic, add additional 5X Neutralization Buffer until the pH reaches 8.

7.

It is recommended to remove an aliquot of the crude histones for comparison to thepurified histones (column input control).

Step 3: Purify Core Histones
1.

Equilibrate the spin columns by adding 0.5 ml Equilibration Buffer then centrifuging at
2,000 x g (approximately 4,500 rpm) for 5 minutes at 4°C.

2.

Add 0.5 ml of the sample from Step 2 to the column and centrifuge at 2,000 x g for 5minutes at 4°C. Remove the flow-through and save to a new 1.7 ml microcentrifuge tube.
Repeat this step as many times as necessary to load the entire sample on the column. (Theflow-throughs can be combined and analyzed to determine column binding efficiency.)

3.

Wash the column with 0.5 ml Histone Wash Buffer and centrifuge at 2,000 x g for 5 minutes
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at 4°C. Remove the wash buffer to a microfuge tube and save for analysis later. Repeat thisstep twice for a total of three washes.
4.

Transfer the column to a new 2 ml round-bottom microcentrifuge tube. Add 100 µl of Histone Elution Buffer and centrifuge at 2,000 x g for 5 minutes at 4°C. Save the flow-throughcontaining the eluted histone proteins.

5.

Optional second elution: repeat step 4, if desired, keeping the 2nd eluate separate fromthe first eluate.

At this point the proteins are ready for use, but contain high levels of salts in the buffer that may
interfere with some downstream binding assays or other applications. If desired, the proteinscan be used without precipitation after being desalted using columns designed for this purpose(e.g. Zeba spin columns, Thermo Fisher part no. 89889). To further concentrate the proteins andremove the salt, proceed to Step 4: Precipitate Histone Proteins.

Step 4: Precipitate Histone Proteins
1.

Precipitate the histone proteins overnight at 4°C by adding perchloric acid to a final concentration of 4%. For example add 6 µl of 70% perchloric acid to 100 µl fraction, then vortex.

2.

On the following day, spin the samples at maximum speed in a microfuge for 1 hour at 4°C.

3.

Gently wash the histone pellet with 1 ml cold 4% perchloric acid. Do not vortex the pellet.
Centrifuge for 5 minutes at maximum speed in a microfuge. Repeat this step for a total oftwo washes.

4.

Wash the histone pellet two times as in step 3 using ice cold acetone containing 0.2% HCl.

5.

Wash the histone pellet two times as in step 3 using ice cold acetone.

6.

Air dry the pellet until it is completely dry, around 10 to 20 minutes.

7.

Resuspend in sterile water or TE. Flick the bottom of the tube gently with a finger. Let thepellet resuspend 10 to 20 minutes at room temperature, then gently vortex and centrifuge
briefly. Check to make sure that pellet is completely resuspended. Store at -20°C or -80°C.

Step 5: Quantify Yield of Histone Proteins1.

Total core histone proteins can be quantified by measuring 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 / mlOD 0.42
1 mg/ml=www.activemotif.com

Your ODx mg / ml

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

7Appendix
Section A: Troubleshooting Guide
Problem/question

Recommendation

Clogging problems appear
when loading the sample
onto the column.

Perform an additional 5 minute centrifugation step at 2,000 x g.

In the precipitation step,
there is no visible pellet
after spinning the histone
samples.

In general, a white pellet is visible. Leave some of washing solutions to avoid disturbing thepellet; do not vortex it. Also, the histones will not precipitate well if the preparation is toodilute. Elute the sample in less volume to obtain more concentrated preparations.

Is it possible to eliminate
H1 from the final preparation of histones?

Yes. Precipitate the histone proteins in perchloric acid 4% (H1 protein remains soluble).
H1 protein can be recovered by re-precipitation of the soluble fraction using
20-30% trichloroacetic acid.

Do the histone proteinsreact with Coomassie dyewhile in solution?

No. Core histones react poorly with Coomassie dye while in solution, and H1 does not reactat all. However, H1 and core histones are stained by Coomassie effectively in gel.

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8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

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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
E-mail: techchina@activemotif.com

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