Histone Extraction Kit(version A2)
Catalog No. 40028

Copyright 2016 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 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.
© 2016 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.

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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Histone Extraction Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols
Buffer Preparation and Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Assay Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Section A. Cultured Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Section B. One-Step Extraction for Cell Lines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Section C. Two-Step Extraction for Cell Lines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section D. 96-well Cell Culture Plate Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Section E. Tissue Sample Extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Appendix
Section F. Extraction from Primary cells (T-cells) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section G. Bradford Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Section H. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

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Overview
Active Motif’s Histone Extraction Kit isolates core histone proteins from tissues and cell lines
using an acid extraction protocol that preserves histone post-translational modifications (e.g.
acetylation, methylation, and phosphorylation). Histones isolated by this method are suitable substrates for downstream analysis in Western blots, or Active Motif’s Histone ELISA Kits and Histone
H3 PTM Multiplex assays.
The addition or removal of modifications such as phosphorylation, acetylation and methylation at
specific amino acid residues on the tails of core histone proteins H2A, H2B, H3 and H4 can havea profound effect on cell signaling and human diseases. Many of these specific histone modifications are conserved throughout eukaryotes. Histone modifications function either by altering
chromatin structure and accessibility to transcriptional machinery and/or they create binding sites
for ‘reader’ proteins which either deposit ‘write’ or remove ‘erase’ these marks.
The Histone Extraction Kit provides enough reagents for two 96-well cell culture plates (192wells), or 100 extraction reactions from cultured cell lines and tissues. A supplemental protocol isavailable for working with primary cells (e.g. T-cells).

product

formatcatalog no.

Histone Extraction Kit

192 rxns

40028

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1

Introduction
Histone Overview
The basic structural unit of chromatin is the nucleosome, which consists of 147 base pairs (bp) of
DNA wrapped around a histone octamer. The histone octamer consists of two copies of the core
histone H2A-H2B dimers and a tetramer of H3-H4. A linker histone, histone H1, binds chromatin
outside the nucleosome unit to regulate chromatin structure.
Histone modifications such as phosphorylation, acetylation and methylation at specific aminoacid residues on the N-terminal histone tails that extend beyond the core nucleosome have beenfound to influence and regulate transcription, chromosome packaging and DNA damage repair.
While the biological significance of some histone modifications remains to be understood, somehave been demonstrated to correlate very closely with specific cellular states like transcriptional
activity1-6.
Histone acetylation is catalyzed by histone acetyltransferases (HATs) and has been shown to beimportant in the regulation of transcription, replication, DNA damage repair, and chromosomal
condensation7-10. Acetylation of histones is believed to result in decondensation of heterochromatin into the more relaxed euchromatin structure which increases accessibility of regulatory
proteins, such as transcription factors and DNA repair elements, to the underlying DNA. Thus
histone acetylation plays an important role in the active processes associated with chromatin
remodeling.
Histone methylation is a post-translational modification which takes place on the side chains ofboth lysine (K) and arginine (R) residues. Histone methylation is a reversible process which iscatalyzed by histone methyltransferase enzymes, such as PRMT1 or Suv39H and is reversed byhistone demethylases, such as LSD1 or Jumanji domain-containing proteins. The gene regulationconsequences of histone methylation on the transcriptional state depends on the methylated
residue and the degree of methylation. Lysine residues can undergo mono-, di- or trimethylation11-14. Additionally, arginine dimethylation can occur in either a symmetric or asymmetric state.
Histone phosphorylation occurs on serine and threonine residues and influences chromosome
condensation, DNA repair and apoptosis. For example, phosphorylation of serine 28 on histone
H3 has recently been shown to associate with destabilized nucleosomes in transcribed chromatin, making this an interesting indicator of both mitotic activity and transcriptional activation15-16.
Given the importance of histone post-translational modifications in development and disease, Active Motif has developed a quick and easy method to isolate cell lysates that are highly enrichedin histone proteins, while still preserving post-translational modifications. The Histone Extraction
Kit uses acid precipitation to isolate the highly basic histone proteins from cell culture, tissue andprimary cells. The histone lysate is suitable for use in downstream applications such as Western
blot, Active Motif’s Histone ELISAs or the Histone H3 PTM Multiplex assay. The histone lysate willcontain a mixture of protein samples. For a more purified histone preparation, we suggest using
one of Active Motif’s Histone Purification Kits (Catalog Nos. 40025, 40026 and 40027).

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2

References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.

Kirmizis, A., et al. (2004) Genes & Dev, 18: 1592-1605.
Squazzo, S., et al. (2006) Genome Res, 16: 890-900.
Kouzarides T. (2007) Cell, 128 (4): 693-705.
Lee J.S., et al. (2010) Cell, 142 (5): 682-685.
Berger. S.L., et al. (2007) Nature, 447 (7143): 407-412.
Sims, R.J. and Reinberg, D. (2008) Nat Rev Mol. Cell Biol, 9 (10): 815-820.
Millar, C.B., et al. (2006) Genes & Dev, 20: 711-722.
Doyon, Y., et al. (2006) Mol Cell, 21: 51-64.
Qin, S., Parthun, M.R. (2006) Mol Cell Biol, 26: 3649-3658.
Shrogen-Knaak, M., et al. (2006) Science, 311: 844-847.
Berger, S.L (2002) Curr Opin Genet Dev, 12: 142-148.
Zhang, K., et al. (2002) Annal Biochem, 306: 259-269.
Zhang, L., et al. (2003) Chromosoma, 112: 77-86.
Marmorstein, R. and Trievel, R.C. (2009) Biochim Biophys Acta, 1789: 58-68.
Cruickshank, M.N., et al. (2010) Amino Acids, 39(5): 1087-1105.
Sun, J-M., et al. (2007) Nucleic Acids Res, 35(19): 6640-6647.

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3Kit Performance and Benefits
The Histone Extraction Kit can be used to prepare lysates for evaluation of histone post-translational modifications in the analysis of normal and diseased tissues, or to screen the effects ofdrug compounds in cultured cells.
Input amounts: The amount of input material recommended per reaction will depend on thesample type. Guidelines are provided below.
Sample Type

Input range

Cultured Cell Lines

100,000 cells – 2 million cells per reaction

96-well culture plate

5,000 – 50,000 cells per well

Tissue Samples

5 mg – 50 mg per reaction

Expected yield: The yield will vary depending on the cell or tissue sample used. Below are someguidelines of expected yields.
SampleType

Input
AmountProtein
Yield

Concentration(mg/ml)Mouse Brain

51 mg

0.75 – 1.25 mg

3.0 – 4.5 mg/ml
2.0 – 3.0 mg/ml

Mouse Lung

62 mg

0.6 – 0.75 mg

Mouse Kidney

51 mg

0.6 – 0.9 mg

2.4 – 3.7 mg/ml

Mouse Spleen

58 mg

1.4 – 1.6 mg

2.4 – 5.8 mg/ml

Mouse Liver

58 mg

1.5 – 2.0 mg

6.5 – 7.6 mg/ml

Mouse Heart52 mg

0.75 – 1.0 mg

3.5 – 4.0 mg/ml

Mouse Colon

52 mg

0.75 – 1.0 mg

2.5 – 3.5 mg/ml

6-well plate

300,000 cells/well~220 µg–12-well plate

100,000 cells/well~75 µg–24-well plate

50,000 cells/well~37.5 µg–

Assay time: As little as 2.5 hours.

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4Histone Extraction Kit

Figure 1: SDS-PAGE gel of histones extracted from mouse kidney.
The Histone Extraction tissue sample protocol was used to prepare lysates from mouse kidney tissue. Twenty microgrammouse kidney lysate (Lane 1) or 10 µg mouse kidney lysate (Lane 2) were loaded onto a 16% Tris-Glycine gel to show the
purity of histone lysate.

Figure 2: Western blot confirms histone post-translational modifications are preserved during extraction.
The Histone Extraction Kit was used to isolate histone lysates from untreated (–) and treated (+) HeLa cells that underwent
the one-step extraction protocol. Twenty micrograms of each lysate were run per well and probed with a 1:500 dilution
H3S10ph mAb (Cat. No. 39636), 1:250 dilution H3K27me3 pAb (Cat. No. 39155), 1:500 dilution H3K9ac mAb (Cat. No. 61251),
and 1 µg/ml Histone H3 mAb (Cat. No. 61475). Results show the preservation of histone phosphorylation, methylation andacetylation with lysates extracted using the Histone Extraction Kit.

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5

Histone Extraction Kit Components and Storage
Histone Extraction Kits are for research use only. Not for use in diagnostic procedures. All components are guaranteed stable for 6 months from date of receipt when stored properly.
Reagents

Quantity

Storage

5 ml

RT

2 x 100 ml

4°C

Lysis Buffer AM8

25 ml

4°C

Extraction Buffer

25 ml

4°C

Deacetylase Inhibitor

1 ml

-20°C

2 x 500 µl-20°C

1 M DTT

100 µl-20°C

Phosphatase Inhibitors

1 ml

RT to -20°C

Neutralization Buffer
10X PBS

Protease Inhibitor Cocktail (PIC)

Additional materials required• 15 ml or 50 ml conical tube
• 1.5 ml low retention microcentrifuge tubes (e.g. USA Scientific, Cat. No. 1415-2600)• Refrigerated centrifuge and microcentrifuge• Multichannel pipette and tips
• Scalpel, scissors or razor blades for tissue extraction• Mortar and pestle or 1.5 ml plastic pestle (VWR, Cat. No. 47747-370) for tissue extraction• Liquid nitrogen for tissue extraction• Ice bucket• Dry ice
• Methanol• Orbital shaker (e.g. LabNet Orbit P4)• Vortex• DNase-free sterile water• 96-well clear, flat bottom microtiter plate (e.g. Dynex, Cat. No. 9205)• BSA Stock Solution (10 mg/ml)• Bio-Rad Protein Assay Dye Solution (e.g. Bio-Rad, Cat. No. 500-0006)

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6

Protocols
Buffer Preparation and Recommendations
1X PBS (See the Quick Chart for Preparing Buffers)
The table below shows the amount of PBS needed per reaction/well based on the sample typeused for extraction. Calculate the amount of 1X PBS needed based on your experiment and thenumber of samples to be tested. Prepare a 1X PBS solution by diluting 10X PBS in sterile water.
For every 10 ml of 1X PBS needed, add 1 ml 10X PBS to 9 ml sterile water. Vortex to mix. Chill on
ice.

24-well
plate(per rxn)

12-well
plate(per rxn)

6-wellplate(per rxn)

T-25
flask
(per rxn)

96-well
plate(per well)

Adherent cells

4 ml

8 ml

12 ml

20 ml

200 µlSuspension cells

2 ml

4 ml

6 ml

10 ml

N/A

Complete Lysis Buffer AM8 (See the Quick Chart for Preparing Buffers)
Lysis Buffer AM8 is only needed for the two-step extraction protocol for cultured cell lines and fortissue extraction. Before use, add Deacetylase Inhibitor, Phosphatase Inhibitors and Protease Inhibitor Cocktail as described in the Quick Chart. Prepare fresh buffer each time and keep on ice.
Extraction Buffer
Is provided ready to use. We suggest pre-chilling buffer on ice.
WARNING: The Extraction Buffer is corrosive. Wear personal protective equipment whenhandling, i.e. safety glasses, gloves and labcoat.
Protease Inhibitor Cocktail (PIC)
Is provided as a 100X stock solution. Thaw the PIC at room temperature until fully dissolved,
which takes about 30 minutes. Vortex gently and spin down briefly before use, then add to thebuffers immediately before use.
Deacetylase Inhibitor
Is provided as a 100X stock solution. Thaw on ice.
Phosphatase Inhibitors
Is provided as a 100X stock solution. Inhibitors can be stored at a range of temperatures fromroom temperature to -20°C. For long-term use we suggest storing at -20°C and thawing on ice
prior to use.
0.1 M DTT (See the Quick Chart for Preparing Buffers)
Prepare a 10-fold dilution of 1 M DTT in sterile water and keep on ice. 0.1 M DTT should be prepared fresh for each experiment and discarded after use. We suggest making aliquots of the
1 M DTT stock to minimize repeated freeze thaws.
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7Complete Neutralization Buffer (See the Quick Chart for Preparing Buffers)
Before use, add 0.1 M DTT, Deacetylase Inhibitor, Phosphatase Inhibitors and Protease Inhibitor
Cocktail to the Neutralization Buffer as described in the Quick Chart below. This should always be
prepared fresh for each extraction and discarded after use. Keep buffer on ice.

Quick Chart for Preparing Buffers
Reagents to prepare

Components

1X PBS

Sterile water
10X PBSTOTAL REQUIRED

9 ml
1 ml
10 ml

45 ml
5 ml
50 ml

90 ml
10 ml
100 ml900 ml100 ml1L

1800 ml
200 ml2L

0.1 M DTT

Sterile water
1 M DTTTOTAL REQUIRED

4.5 µl
0.5 µl
5 µl

9 µl
1 µl
10 µl

18 µl
2 µl
20 µl

45 µl
5 µl
50 µl

180 µl20 µl200 µlComplete
Lysis Buffer

Lysis Buffer AM8
Deacetylase Inhibitor
Phosphatase Inhibitors
Protease Inhibitor Cocktail
TOTAL REQUIRED

242.5 µl
2.5 µl
2.5 µl
2.5 µl250 µl970 µl
10 µl
10 µl
10 µl
1 ml

4.85 ml
50 µl
50 µl
50 µl
5 ml

12.125 ml
125 µl125 µl125 µl12.5 ml24.25 ml
250 µl250 µl250 µl25 ml

Complete
Neutralization
BufferNeutralization Buffer
0.1 M DTT
Deacetylase Inhibitor
Phosphatase Inhibitors
Protease Inhibitor Cocktail
TOTAL REQUIRED

10 µl
1.5 µl
1 µl
1 µl
1 µl
14.5 µl

50 µl
7.5 µl
5 µl
5 µl
5 µl
72.5 µl

100 µl
15 µl
10 µl
10 µl
10 µl
145 µl250 µl37.5 µl
25 µl
25 µl
25 µl
362.5 µl

1 ml
150 µl100 µl100 µl100 µl1.45 mlwww.activemotif.com

Example volumes for buffer preparation

8

Assay Protocol
Read the entire protocol before use.
The Histone Extraction Kit provides multiple protocols based on sample type: adherent cultured
cells, suspension cell lines, 96-well cell culture and tissue samples. A supplemental protocol isprovided in the appendix for working with primary cells. Select the protocol to match your sample
and follow the instructions provided.

Section A: Cultured Cell Lines
The following section describes the preparation of histone containing extracts from a minimum of
100,000 cells. Protocols are provided for adherent and suspension cell culture. Please follow the
instructions corresponding to your sample type.
For the majority of cell lines, a one-step extraction method can be used for isolation of histone
proteins for downstream analysis in WB, ELISA or Histone H3 PTM Multiplex assay. However,some cell types are more difficult to work with and may require the optional two-step extraction method. When working with a new sample type, we suggest performing a small scale pilot
experiment comparing the two extraction methods to identify the best extraction protocol foryour sample.
Adherent Cell Culture:
24-well plate

12-well plate

6-well plate

T-25 flask

Seeding Density

6

0.05 x 10

60.1 x 106

0.3 x 10

0.7 x 106

Cells at 70-80% Confluency*

0.15 x 106

0.3 x 106

0.9 x 106

2.1 x 106

Growth Medium
volume to culture cells

1 ml

2 ml

3 ml

5 ml

Trypsin

500 µl

1 ml

2 ml

3 ml

Growth medium

500 µl

1 ml

2 ml

3 ml

1 X PBS(volume per wash)

1 ml

2 ml

3 ml

5 ml

Table 1: Cell culture recommendations.
*The number of cells on a confluent plate or dish will vary with cell type. For this table, HeLa cells were used
Please adjust as needed based on your particular cell type.

1.

Follow the recommendations for cell culture seeding as shown in Table 1.

2.

Treat cells as desired. Grow cells to 70-80% confluency.

3.

Aspirate growth medium and wash the cells twice with the recommended volume of 1X PBS.

4.

Detach cells from plate using trypsin. Transfer to a 15 ml conical tube.

5.

Add an equal volume of growth medium the 15 ml conical tube. Pipet to mix.

6.

Perform a cell count and determine cell viability. Record the number of viable cells.
# viable cells per pellet: ___________________________

7.

Pellet the cells by centrifugation in a pre-chilled (4°C) centrifuge at 200 x g for 5 minutes.
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98.

Discard supernatant and wash cells two times with ice-cold 1X PBS. Centrifuge at 200 x g
for 5 minutes at 4°C for each wash.

9.

After the second wash aspirate any remaining PBS without disturbing the pellet. Use a P200
pipette to remove any traces of PBS.

10. Cell pellet can be stored at -80°C, or proceed directly into the one-step or two-step extraction protocols in Section B or Section C.
Suspension Cell Culture:
1. Follow the recommendations for cell culture seeding as shown in Table 1.
2.

Treat cells as desired.

3.

Transfer cells into a 15 ml or 50 ml conical tube.

4.

Perform a cell count and determine cell viability. Record the number of viable cells.
# viable cells per pellet: ___________________________

5.

Pellet the cells by centrifugation in a pre-chilled (4°C) centrifuge at 200 x g for 5 minutes.

8.

Discard supernatant and wash cells two times with the recommended volume of ice-cold
1X PBS. Centrifuge at 200 x g for 5 minutes at 4°C for each wash.

9.

After the second wash aspirate any remaining PBS without disturbing the pellet. Use a P200
pipette to remove any traces of PBS.

10. Cell pellet can be stored at -80°C, or proceed directly into the one-step or two-step extraction protocols in Section B or Section C.

Section B: One-Step Extraction for Cell Lines
1.

Using the calculations for cell viability, add 100 µl per one million cells of ice-cold Extraction
Buffer to each cell pellet. Pipet up and down to resuspend the cells. Transfer cells to a lowbind 1.5 ml microcentrifuge tube.
[(# viable cells per pellet) / (1,000,000 cells)] x 100 µl = _________ µl Extraction Buffer2.

Incubate the cells on an end-to-end rotator for 2 hours at 4°C.

3.

Label new low-bind 1.5 ml microcentrifuge tubes for each sample and chill on ice.

4.

Following the incubation, place the tubes in a microcentrifuge at 20,800 x g for 10 minutes
at 4°C to pellet the acid insoluble material.

5.

During the spin, prepare a dry ice/methanol bath. Place a microcentrifuge rack in an ice
bucket. Add dry ice and methanol.

6.

Transfer supernatant containing histone proteins into the pre-chilled microcentrifuge tube.
If the sample will be used multiple times we suggest making aliquots to avoid freeze thaw
cycles. Record the volume per tube. Histone lysate volume: ____________ µl

7.

Snap-freeze the samples by placing them in the dry ice/methanol bath.
Note:

Once frozen, histone samples can be stored at -80°C for long-term or short-term

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10

storage. It is better to store the histones under acidic conditions to prevent
protein aggregation that may cause the histones to fall out of solution. Histones
will need to be neutralized prior to use in downstream assays. When ready touse the histone samples in downstream analysis, proceed with the protocol.
8.

Take the frozen histone samples and allow them to thaw on ice. While the samples arethawing, prepare the Complete Neutralization Buffer as described on page 8.

9.

Add Complete Neutralization Buffer to the histone lysate and vortex to mix. Calculate theamount needed per sample:(Histone lysate volume) x 0.161 = _________ µl Complete Neutralization Buffer

10. Determine protein concentration using Bradford Quantification (see Appendix Section G).
11.

Proceed directly with downstream analysis.

Section C: Two-Step Extraction for Cell Lines1.

Add 250 µl of ice-cold Lysis Buffer AM8 supplemented with inhibitors to each cell pellet. The
volume added is not dependent upon pellet size. The same amount of Lysis Buffer is added
to each sample. Pipet up and down until solution is homogenous.

2.

Incubate on ice for 30 minutes.

3.

Centrifuge at 2,644 x g for 2 minutes at 4°C to isolate the nuclei.

4.

Add 100 µl per one million cells of ice-cold Extraction Buffer to each nuclear pellet. Pipet up
and down to resuspend. Transfer nuclei to a low-bind 1.5 ml microcentrifuge tube.
[(# viable cells per pellet) / (1,000,000 cells)] x 100 µl = _________ µl Extraction Buffer2.

Incubate on an end-to-end rotator for 2 hours at 4°C.

3.

Label new low-bind 1.5 ml microcentrifuge tubes for each sample and chill on ice.

4.

Following the incubation, place the tubes in a microcentrifuge at 20,800 x g for 10 minutes
at 4°C to pellet the acid insoluble material.

5.

During the spin, prepare a dry ice/methanol bath. Place a microcentrifuge rack in an ice
bucket. Add dry ice and methanol.

6.

Transfer supernatant containing histone proteins into the pre-chilled microcentrifuge tube.
If the sample will be used multiple times we suggest making aliquots to avoid freeze thaw
cycles. Record the volume per tube. Histone lysate volume: ____________ µl

7.

Snap-freeze the samples by placing them in the dry ice/methanol bath.
Note:

8.

Once frozen, histone samples can be stored at -80°C for long-term or short-term
storage. It is better to store the histones under acidic conditions to prevent
protein aggregation that may cause the histones to fall out of solution. Histones
will need to be neutralized prior to use in downstream assays. When ready touse the histone samples in downstream analysis, proceed with the protocol.

Take the frozen histone samples and allow them to thaw on ice. While the samples arethawing, prepare the Complete Neutralization Buffer as described on page 8.
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11

9.

Add Complete Neutralization Buffer to the histone lysate and vortex to mix. Calculate theamount needed per sample:(Histone lysate volume) x 0.161 = _________ µl Complete Neutralization Buffer

10. Determine protein concentration using Bradford Quantification (see Appendix Section G).
11.

Proceed directly with downstream analysis.

Section D: 96-well Cell Culture Plate Extraction
The following section describes the preparation of histone containing extracts from 96-well cell
culture plates (5,000 to 50,000 cells per well). Given the limited starting material from a 96-wellplate, samples should be processed for immediate use in downstream assays. Storage of thehistone lysate is not recommended and will result in a significant loss of signal. Plan your experiments accordingly to prepare fresh samples the day of the assay.
1.

Culture cells as appropriate in a 96-well cell culture plate using 100 µl growth medium.

2.

Treat cells as desired. Grow cells to 70-80% confluency.

3.

Aspirate cell growth medium and wash the cells twice with 200 µl 1X PBS.

4.

Remove excess PBS by quickly inverting plate and blotting onto paper towels to remove
residual traces of PBS.

5.

Add 100 µl per well of Extraction Buffer. Seal plate with adhesive film and place the cell culture plate on a plate shaker at 1,000 rpm for 1 hour at 4°C. While the samples are incubating, prepare the Complete Neutralization Buffer as described on page 8.

6.

Add 16.1 µl Complete Neutralization Buffer to each well. Pipet up and down to mix.

7.

Samples are now ready for downstream analysis. The amount of histones isolated fromeach 96-well is usually too low to quantify. We recommend using a specific volume in downstream Histone ELISAs or Histone H3 PTM Multiplex kit and normalizing samples based on
total H3 levels.

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12Section E: Tissue Sample Extraction
The following section describes the preparation of histone lysates from tissue samples. We recommend using 5-50 mg tissue per extraction with 50 mg as the optimal amount. Isolated histone
proteins can be used for downstream analysis in WB, ELISA or Histone H3 PTM Multiplex assay.
1.

Weigh out 50 mg of tissue and place into a conical tube on dry ice.

2.

Remove a pre-chilled mortar and pestle from -80°C freezer.

3.

Transfer tissue to a mortar and add 1-2 ml liquid nitrogen. Homogenize by grinding the tissue with a pestle until a fine powder is achieved. Carefully transfer the tissue to a microcentrifuge tube. If using small tissue samples that fit within a 1.5 ml microcentrifuge tube, use abattery powered pestle (VWR, Catalog No. 47747-370) to crush the tissue within the tube.

4.

Resuspend the tissue homogenate in 250 µl of ice-cold Lysis Buffer AM8 supplemented with
inhibitors to each cell pellet. Pipet up and down until solution is homogenous.
Note:

2.

If working with large tissue sections that are not resuspended in 250 µl volume,
you can increase the amount of Lysis Buffer AM8 to a final volume of 1 ml tocompletely resuspend the tissue. Please note that excess lysis buffer is notprovided to perform all 100 reactions of the kit using this larger volume.

Incubate on ice for 30 minutes.
Note:

We recommended monitoring the cell lysis. Take 10 µl of the tissue homogenate
and look at it under a phase contrast microscope using a hemocytometer toverify that the nuclei have been released. 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, continue toincubate on ice for an additional 10 minutes on ice. Refer to the Troubleshooting
notes in the Appendix for additional tips.

3.

Centrifuge at 2,644 x g for 2 minutes at 4°C to isolate the nuclei.

4.

Add 250 µl of ice-cold Extraction Buffer per 50 mg tissue. Pipet up and down to resuspend.
Transfer nuclei to a low-bind 1.5 ml microcentrifuge tube.

5.

Incubate on an end-to-end rotator overnight at 4°C.

6.

Label new low-bind 1.5 ml microcentrifuge tubes for each sample and chill on ice.

7.

Pellet acid insoluble material in a microcentrifuge at 20,800 x g for 10 minutes at 4°C.

8.

During the spin, prepare a dry ice/methanol bath. Place a microcentrifuge rack in an ice
bucket. Add dry ice and methanol.

9.

Transfer supernatant containing histone proteins into the pre-chilled microcentrifuge tube.
If the sample will be used multiple times we suggest making aliquots to avoid freeze thaw
cycles. Record the volume per tube. Histone lysate volume: ____________ µl

10. Snap-freeze the samples by placing them in the dry ice/methanol bath.
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Note:

11.

Once frozen, histone samples can be stored at -80°C for long-term or short-term
storage. It is better to store the histones under acidic conditions to prevent
protein aggregation that may cause the histones to fall out of solution. Histones
will need to be neutralized prior to use in downstream assays. When ready touse the histone samples in downstream analysis, proceed with the protocol.

Take the frozen histone samples and allow them to thaw on ice. While the samples arethawing, prepare the Complete Neutralization Buffer as described on page 8.

12. Add Complete Neutralization Buffer to the histone lysate and vortex to mix. Calculate theamount needed per sample:(Histone lysate volume) x 0.161 = _________ µl Complete Neutralization Buffer
13. Determine protein concentration using Bradford Quantification (see Appendix Section G).
14. Proceed directly with downstream analysis.

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Appendix
Section F: Extraction from Primary Cells (e.g. T-cells)
The following section is a supplemental protocol that describes extraction modifications required
when working with primary cells. Please note this protocol is provided as a guideline only andoptimization may be required. This protocol is based the use of 500,000 cells per reaction.
1.

Isolate primary cells according to standard protocols. Aliquot 500,000 cells per microcentrifuge tube and snap freeze cells in a dry ice/methanol bath.

2.

Resuspend the frozen cell pellet in 200 µl ice-cold T-cell Buffer (see below) supplemented
with DTT and protease inhibitor cocktail. Pipet up and down to mix.

3.

Incubate the cells on ice for 30 minutes. The cells should swell during this incubation.

4.

Add 12.5 µl of a 10% IGEPAL Solution (see below) and vortex on high speed for 10 seconds.

5.

Centrifuge the homogenate for 1 minute at 14,000 rpm in a microcentrifuge at 4°C.

6.

Carefully remove supernatant and discard.

7.

Resuspend the nuclear pellet in 100 µl ice-cold Extraction Buffer. Pipet up and down to mix.
Transfer cells to a low bind tube and vortex on high for 30 seconds.

8.

Incubate overnight on ice.

9.

Spin the sample at maximum speed in a microcentrifuge at 4°C for 5 minutes.

10. Transfer supernatant containing histones to a new low bind tube.
11.

Determine protein concentration using Bradford Quantification (see Appendix Section G).

12. Proceed directly with downstream analysis.
Additional materials required:• T-Cell Buffer:
10 mM HEPES, pH 7.9
10 mM KCl
0.1 mM EGTA
0.1 mM EDTA
1 mM DTT
1X Protease Inhibitor Cocktail (PIC)
Add DTT and PIC just before use. Discard any unused solution.
• 10% IGEPAL Solution:
IGEPAL CA-630 (Sigma, Cat # I8896) diluted 1:10 in sterile waterwww.activemotif.com

15Section G: Bradford Quantification
The following protocol is a suggested guideline for quantification of histone lysates. Other quantification methods suitable with your downstream analysis may also be used. We suggest using
a BSA standard to determine protein concentration. Standards should be prepared in the same
buffer as the lysate samples (including addition of inhibitors) for accurate quantification.
Materials required• 96-well clear, flat bottom microtiter plate (e.g. Dynex, Cat. No. 9205)• BSA Stock (10 mg/ml)• Bio-Rad Protein Assay Dye Solution (e.g. Bio-Rad, Cat. No. 500-0006)• Extraction Buffer• Complete Neutralization Buffer (with inhibitors)• 1.5 ml microcentrifuge tubes & 15 ml conical tube
• Sterile water1.

In a 15 ml conical tube, prepare the Protein Assay Dye solution according to manufacturer’s
recommendations by adding 2 ml Dye Reagent Concentrate to 8 ml sterile water. Filter
through Whatman #1 filter (or equivalent) to remove particulates. Keep at room temperature.

2.

Prepare Neutralized Extraction Buffer as follows:
Reagent

VolumeExtraction Buffer

600 µlComplete Neutralization Buffer (See page 8)

96.6 µl

Final Volume

697 µl3.

Prepare a 1 mg/ml BSA standard in 140 µl Neutralized Extraction Buffer. (e.g. Add 14 µl BSA
stock (10 mg/ml) to 126 µl Neutralized Extraction Buffer.)4.

Set up 7 microcentrifuge tubes. Add 42 µl Neutralized Extraction Buffer to each tube. Next, pipette
140 µl of the 1 mg/ml BSA standard to the first microcentrifuge tube. Vortex to mix. Prepare a dilution series as indicated below by transferring 140 µl of each diluted standard to the next tube. Mix
tubes thoroughly before each transfer.
140 µl0.77 mg/ml

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140 µl0.60 mg/ml

140 µl

0.46 mg/ml

140 µl0.35 mg/ml

16

140 µl0.27 mg/ml

140 µl0.21 mg/ml

0.16 mg/ml

5.

We suggest testing the sample neat and at a 1:2 dilution. Prepare histone lysate dilutions in
Neutralized Extraction Buffer. If your sample falls outside the linear range of the BSA standard,
we recommend preparing alternate dilutions.
Neat: Read the sample directly in the assay without further dilution.
1:2 dilution: Add 5 µl histone lysate to 5 µl Neutralized Extraction Buffer for each quantification
reaction. Scale values as needed based on the number of quantification reactions. Vortex to
mix.

6.

In a clear, flat bottomed microtiter plate, set up your standard and samples in triplicate using the template shown below. Add 10 µl of each standard or sample per well. For the blankwells, add 10 µl Neutralized Extraction Buffer.
Note:

For some samples there may not be enough material to perform sample quantification in triplicates. Adjust plate layout as needed.

BSA Standard
1

2

Lysates
3

4

5

6

7

8

9

10

11

12

A

0.77 mg/ml

Neat Lysate 1

–

–

–

–

–

–

B

0.60 mg/ml

1:2 Lysate 1

–

–

–

–

–

–

C

0.46 mg/ml

Neat Lysate 2

–

–

–

–

–

–

D

0.35 mg/ml

1:2 Lysate 2

–

–

–

–

–

–

E

0.27 mg/ml

Neat Lysate 3

–

–

–

–

–

–

F

0.21 mg/ml

1:2 Lysate 3

–

–

–

–

–

–

G

0.16 mg/ml

Neat Lysate 4

–

–

–

–

–

–

H

Blank

1:2 Lysate 4

–

–

–

–

–

–

7.

Add 200 µl diluted dye reagent to each well. Mix the sample and reagent thoroughly by
pipetting up and down. Use clean tips with each well.

8.

Cover plate and incubate at room temperature for 5-10 minutes.

9.

Measure absorbance at 595nm using a microplate reader.

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17Section H: Troubleshooting Guide
Problem/question

Possible cause

Recommendation

When using a metal pestle
freshly ground tissue issticking to the tip

After cleaning thepestle the excess liquid
was not dry prior toplacing in the freezer.
Excess liquid will form
ice and stick to freshly
homogenized tissue

Dry all tools thoroughly prior to cold storage

Incomplete homogenization of tissue

Some tissue types (e.g.
heart, skeletal muscle
and uterus) will generate a fibrous strand
that is unable to behomogenized easily

Add more liquid nitrogen to keep the tissue brittle. When working with fibrous tissues, crush tissue samples very quickly to
avoid partial thawing. This will help to avoid the formation of the
fibrous strand.

How should I quantify myhistone lysate?

Because histones
are small positively
charged proteins,
methods for determining protein concentration often give differingresults

We recommend to use a Bradford assay with a BSA standard
to quantify the histone lysate. It is important to note that core
histone proteins react poorly with Coomassie dye in solution,
but due to the presence of other proteins within the lysate,
Bradford will still provide a reliable protein concentration.
However, alternative methods such as A280nm can also be
used to quantify proteins, including histones, within the lysate.
Concentration can be compared to a BSA standard prepared in
the same buffer.

Low yield

Incomplete cleaningof fresh tissue prior to
storage will add weight
to final tissue sample
giving a higher starting
mass than actual
tissue.

Clean tissue thoroughly prior to use or storage to avoid calculating extra mass.

Sample loss due toprotein sticking to thewalls of the plastic
tube.

We recommend using low retention microcentrifuge tubes forall steps of the extraction process. If working with culture celllines of less than 100,000 cells or precious tissue samples westrongly recommend low retention tubes to avoid sample loss.

Incomplete lysis

If working with cell culture samples in excess of 1 million cells
per reaction, you may need to add additional Lysis Buffer AM8
to fully resuspend the cell pellet for complete cell lysis if using
the two-step extraction protocol. We suggest adding Lysis Buffer
AM8 proportionally at the ratio of 250 µl per 1 million cells.

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