High Sensitivity
Chromatin Preparation(version A1)
Catalog No. 53046

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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
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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
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TABLE OF CONTENTS

Page

Overview  . . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Kit Components and Storage
High Sensitivity Chromatin Preparation Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Protocols – Experimental Set Up
Cell Growth and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Buffer Preparation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols – Preparation of Sheared Chromatin
Section A. Cell Fixation Starting with Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Section B. Chromatin Sonication of Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Section C. Cell Fixation Starting with Fresh or Frozen Tissue . . . . . . . . . . . . . . . . . . . . . . . . 12
Section D. Chromatin Sonication of Tissue  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section E. Cell Fixation Starting with PBMCs  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Section F. Chromatin Sonication of PBMCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Appendix
Section G. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Section H. Related Products  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

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Overview
Chromatin Immunoprecipitation (ChIP) is a powerful tool for studying protein/DNA interactions,
including transcription factors, co-regulatory proteins, modified histones, chromatin-modifying
enzymes and polymerases because it enables identification of the localization of proteins boundto specific DNA loci. When used in combination with whole-genome analysis such as ChIP-Seq or
ChIP-chip, insights are possible into gene regulation, gene expression, mechanisms of chromatin
modification and pathway analysis.
Active Motif’s High Sensitivity Chromatin Preparation Kit is designed to isolate formaldehydefixed chromatin from cultured cells or tissue samples for use in ChIP. The kit contains optimized
protocols and buffers to yield high quality chromatin, including recommendations for working
with difficult to lyse peripheral blood mononuclear cells (PBMCs). The High Sensitivity Chromatin
Preparation Kit is ideal for optimization of chromatin preparation and sonication conditions whenworking with different sample types. Prepared chromatin is suitable for use with Active Motif’s
ChIP-IT® High Sensitivity Kit, ChIP-IT® ChIP-Seq Kit, ChIP-IT® PBMC Kit, ChIP-exo Kit, ChIP-BisulfiteSequencing Kit, Tag ChIP-IT® Kit and enChIP Kit.
The High Sensitivity Chromatin Preparation Kit contains reagents for 16 chromatin preparations.
A chromatin preparation is defined as one 150 mm cell culture dish, 10 million PBMCs or 100 mg
tissue sample. To learn about available ChIP Kits, ChIP-Seq validated antibodies, or Active Motif’s
EpiShear™ sonication devices, please visit our website at www.activemotif.com/chip.

product

formatcatalog no.

High Sensitivity Chromatin Preparation

16 rxns

53046

ChIP-IT® High Sensitivity Kit

16 rxns

53040

Protein G Agarose Columns

30 rxns

53039

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1

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

Quantity

Storage

RNase A (10 µg/µl)

40 µl

-20°C

Proteinase K (10 µg/µl)

80 µl

-20°C

10X PBS

120 ml-20°C

100 mM PMSF

500 µl-20°C

Protease Inhibitor Cocktail (PIC)

500 µl-20°C

Precipitation Buffer

1.5 ml-20°C

Carrier

35 µl

-20°C

Fixation Buffer

2 x 1.5 ml

4°C

Swelling Buffer

120 ml4°C

Detergent

25 ml

RT

Stop Solution

20 ml

RT

Chromatin Prep Buffer

85 ml

RT

ChIP Buffer

35 ml

RT

5 M NaCl

400 µlRT

TE pH 8.0

2 x 1.5 ml

RT

5 ml

RT

DNA Purification Elution Buffer

Additional materials required• Dounce homogenizer with a small clearance pestle (e.g. Active Motif Catalog Nos. 40401& 40415) with the tight-fitting “A” pestle). Use of a homogenizer is necessary for shearing
chromatin.
• 37% formaldehyde solution with 10-15% methyl alcohol to prevent polymerization (e.g. Sigma
Aldrich Catalog No. 252549). Do not use paraformaldehyde.
• For tissue and PBMC preparations you will need phenol, saturated (DNA Purification, Molecular Biology Grade, Amresco Catalog No. 0945)• For tissue and PBMC preparations you will need chloroform/isoamyl alcohol (24:1) (DNAPurification, Molecular Biology Grade)• 100% ethanol (absolute)• 70% ethanol• DNase-free H2O• Rocking platform for culture plates
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2• Microcentrifuge (table top centrifuge 4°C) and microcentrifuge tubes• 250 µl PCR tubes• Thermocycler• 15 and 50 ml conical tubes• Spectrophotometer for DNA quantitation• Pipettors and tips (filter tips are recommended)• Sonicator (e.g. Active Motif’s EpiShear™ Sonicator with a 1/8” probe (Catalog No. 53051) with
the EpiShear™ Cooled Sonication Platform (Catalog No. 53080))• Agarose gel electrophoresis apparatus• Razor blades (for tissue preparations)• Hand-held homogenizer for tissue preparations (e.g. Biospec Products Tissue-Tearor)• Cell scraper (rubber policeman)• Dry ice (for PBMC cells)• Metal spatula for cell scraping (for PBMC cells)

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3

Protocols – Experimental Set Up
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Cell Growth Recommendations
When planning an experiment, calculate the number of chromatin preparations you will requireand determine the number of ChIP reactions you plan to perform on each chromatin preparation.
Be sure to include the appropriate positive and negative control ChIP reactions in your calculations. Also, note that if you wish to analyze the effect of particular compounds or culturingconditions on transcription factor/DNA interactions, you should prepare chromatin from control(untreated) cells as a reference sample. PBMCs (including lymphocytes and monocytes) are difficult
to lyse cells and as a result will yield approximately 30-50% as compared to cell lines.
Suggested cell number minimums for the preparation of chromatin are provided below:
Cell Culture:
We recommend the use of 100,000 – 15 million cells
PBMC Cells:
We recommend the use of 10 – 20 million cells
Tissue Samples:
We recommend the use of 100-400 mg
Cell Culture
Guidelines

24-wellplate

12-wellplate

6-well
plate

60 mm
dish

100 mmdish

150 mmdish

Seeding Density

0.05 x 106

0.1 x 106

0.3 x 106

0.8 x 106

2.2 x 106

5.0 x 106

Cells at 70-80%
Confluency*

0.15 x 106

0.3 x 106

0.9 x 106

2.4 x 106

6.6 x 106

15.0 x 106

Growth Medium
Volume

1 ml

2 ml

3 ml

5 ml

10 ml

20 ml

Cell Fixative Solution

100 µl200 µl300 µl500 µl

1 ml

2 ml

Stop Solution

55 µl

110 µl165 µl

275 µl

550 µl1.1 mlPBS Wash Buffer(used per wash)

500 µl500 µl

1 ml

2 ml

5 ml

10 ml

Chromatin Prep Buffer

500 µl500 µl

1 ml

2 ml

5 ml

5 ml

ChIP Buffer

500 µl500 µl500 µl500 µl500 µl500 µl* 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.
**Please refer to the descriptions below for complete details on buffer preparations

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4Buffer Preparation
Follow the instructions below to prepare buffers for chromatin preparation. A Quick Chart for
Buffer Preparation is provided on page 7. Buffers should be scaled accordingly if working withdifferent amounts of cells.
Complete Cell Fixation Solution
Buffer should be prepared fresh before each experiment.
For each 150 mm cell culture dish, prepare 2.5 ml of Complete Cell Fixation Solution by adding
180 µl Fixation Buffer to 1.57 ml sterile water in a 15 ml conical tube. Using appropriate precautions(i.e. safety glasses, gloves and lab coat), add 750 µl 37% formaldehyde to the tube and vortex tomix. Use 1/10 growth medium volume per plate. Complete cell fixation solution can be added to
the growth medium in the presence or absence of serum.
For each PBMC pellet, prepare 5 ml of Complete Fixation Solution by adding 0.5 ml 10X PBS to
4.36 ml sterile water in a 15 ml conical tube. Using appropriate precautions (i.e. safety glasses,
gloves and lab coat), add 140 µl 37% formaldehyde to the tube and vortex to mix. Use 5 ml Complete Fixation Solution per PBMC pellet.
Complete Tissue Fixation Solution
Buffer should be prepared fresh before each experiment. Prepare 10 ml of Tissue Fixation
Solution for each tissue sample to be processed by adding 1 ml 10X PBS to 8.7 ml sterile water in a
15 ml conical tube. Using appropriate precautions (i.e. safety glasses, gloves and lab coat), add
280 µl 37% formaldehyde to the tube and vortex to mix.
Stop Solution
Is provided ready to use. Use 1/20 media volume per cell culture plate, use 250 µl per PBMC pellet
and 515 µl per tissue sample.
Detergent
Is supplied ready to use.
PBS Wash Buffer
For each 150 mm cell culture dish or tissue sample, prepare 25 ml of PBS Wash Buffer. To a 50 ml
conical tube add 21.25 ml sterile water, 2.5 ml 10X PBS and 1.25 ml Detergent. Mix by inverting. Place
PBS Wash Buffer on ice to chill. PBS Wash Buffer can be prepared in large quantities and stored at
4ºC for 6 months.
For each PBMC pellet, prepare 10 ml of PBS Wash Buffer. To a 15 ml conical tube add 8.5 ml sterilewater, 1 ml 10X PBS and 0.5 ml Detergent. Just before use, add 20 µl 100 mM PMSF.
100 mM PMSF and Protease Inhibitor Cocktail (PIC)
Thaw PMSF and PIC at room temperature until fully dissolved, which takes about 30 minutes.
Vortex gently and spin down briefly before use, then add to the buffers immediately before use.
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5Chromatin Prep Buffer
Is supplied ready to use.
Swelling Buffer
Is supplied ready to use.
ChIP Buffer
Is supplied ready to use.

Recommendations
Chromatin Shearing Tips
We suggest using a probe sonicator (i.e. Active Motif’s EpiShear Probe Sonicator) which employs adirect sonication method to prepare chromatin for use in the ChIP. Indirect sonication systems may
require longer sonication times to achieve optimal chromatin shearing. ChIP experiments usuallyrequire chromatin that has been sheared to a size of 200-1200 bp. In general, shearing efficiency isimproved through the use of a small shearing volume and a V-bottom tube rather than a round-bottom tube. Also, note that shearing is inefficient if the chromatin sample becomes emulsified withair bubbles. To determine the appropriate shearing level for your sample, set up a “practice” tube
containing only ChIP Buffer. Slowly increase the sonication amplitude until foaming starts to occur.
Reduce the amplitude setting down slightly and mark this as the highest possible intensity to use
without foaming. If a chromatin preparation becomes emulsified inadvertently, discontinue shearing
and centrifuge the sample at maximum speed for 4 minutes at 4ºC in a microcentrifuge to remove
trapped air. Finally, to prevent overheating and denaturation of chromatin, samples should be kept
on ice as much as possible during shearing, and shearing should be performed discontinuously (i.e.
sonicate for 20 seconds, then place on ice/water for 30 seconds, sonicate again for 20 seconds, etc.).
If possible, shear while on ice or use Active Motif’s EpiShear™ Cooled Sonication Platform (Catalog
No. 53080) to help regulate sample temperature.
Chromatin Quantity
It is recommended to use 10-30 µg chromatin per IP reaction. Follow the recommendations forthe chromatin immunoprecipitation protocol to prepare the IP reaction. Chromatin prepared with
the High Sensitivity Chromatin Preparation Kit is suitable for use in Active Motif’s ChIP-IT® HighSensitivity Kit, ChIP-IT® ChIP-Seq Kit, ChIP-IT® PBMC Kit, ChIP-exo Kit, ChIP-Bisulfite-Sequencing
Kit, Tag ChIP-IT® Kit and enChIP Kit.
Safety Precautions
Formaldehyde and PMSF are highly toxic chemicals. Appropriate safety precautions (i.e. safety
glasses, gloves and lab coat) should be used. Also, formaldehyde is highly toxic by inhalation andshould be used only in a ventilated hood. Finally, chromatin sonication should be performed in abiosafety hood if the chromatin is extracted from biohazardous or infectious materials.

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6Quick Chart for Preparing Buffers
Reagents to prepare

Components

Per 150 mm dish

Per PBMC pellet

Per Tissue sample

Fixation Solution

Sterile Water
Fixation Buffer
10X PBS
37% Formaldehyde
TOTAL REQUIRED

1.57 ml
180 µl–750 µl
2.5 ml4.36 ml–
0.5 ml140 µl5 ml

8.7 ml–
1 ml
280 µl10 ml

Stop Solution

TOTAL REQUIRED

1.1 ml250 µl515 µlPBS Wash Buffer

Sterile Water
10X PBS
Detergent
100 mM PMSFTOTAL REQUIRED

21.25 ml
2.5 ml1.25 ml–
25 ml

8.5 ml1 ml
0.5 ml
20 µl
10 ml

21.25 ml
2.5 ml1.25 ml–
25 ml

Chromatin Prep Buffer

Chromatin Prep Buffer
Protease Inhibitor Cocktail
100 mM PMSFTOTAL REQUIRED

5 ml
5 µl
5 µl
5 ml

–
–
–
N/A

5 ml
5 µl
5 µl
5 ml

Swelling Buffer

Swelling Buffer
Protease Inhibitor Cocktail
100 mM PMSFTOTAL REQUIRED

–
–
–
N/A

5 ml
5 µl
5 µl
5 ml

–
–
–
N/A

Detergent

TOTAL REQUIRED

N/A

375 µlN/A

ChIP Buffer

ChIP Buffer
Protease Inhibitor Cocktail
100 mM PMSFTOTAL REQUIRED

500 µl5 µl
5 µl
510 µl500 µl5 µl
5 µl
510 µl500 µl – 1 ml
5 µl – 10 µl
5 µl – 10 µl
510 µl – 1 ml

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7

Protocols – Chromatin Preparation from Cultured Cells
Section A: Cell Fixation Starting with Cultured Cells
This protocol describes cell fixation and chromatin preparation from one 150 mm dish (approximately 1.5 x 107 cells). We recommend using 20 ml growth medium per 150 mm dish. Please refer topage 4 for information on scaling the protocol for use with other amounts of cells. The minimum
cell number to be used for the preparation of chromatin is 100,000 cells.
1.

Prepare 150 mm dishes for each cell line to be tested. Grow the cells to 70-80% confluency.
Stimulate cells as desired to activate the pathway of interest.

2.

Freshly prepare Complete Cell Fixation Solution for each 150 mm dish.

3.

To fix cells, add 1/10 growth medium volume of freshly prepared Complete Cell Fixative Solution to the existing culture media for the cells (e.g. 20 ml growth medium would get
2 ml Complete Cell Fixation Solution). Shake gently at room temperature for 15 minutes.

4.

Stop the fixation reaction by adding 1/20 media volume of Stop Solution to the existing
culture media for the cells (e.g. 20 ml growth medium would get 1.1 ml Stop Solution). Swirlto mix and incubate at room temperature for 5 minutes.

5.

Following the incubation, hold the plate at an angle and using a rubber policeman scrape
cells down to collect them at the bottom edge of the plate. Use a pipette to transfer thecells to a 50 ml conical tube on ice.

6.

Pellet the cells from step 5 by centrifugation for 3 minutes at 1,250 x g at 4°C.

7.

Remove the supernatant and discard. Resuspend the pellet(s) in 10 ml ice-cold PBS Wash Buffer by pipetting up and down. Keep samples ice-cold for the remainder of the procedure.

8.

Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. Wash thepellet(s) a second time in 10 ml ice-cold PBS Wash Buffer by pipetting up and down. Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. (Cell pellets may
be stored at -80°C at this stage).

9.

Resuspend each pellet(s) in 5 ml Chromatin Prep Buffer supplemented with 5 µl PIC and5 µl 100 mM PMSF. Pipet up and down to mix.

10. Incubate on ice for 10 minutes.
11.

Transfer the resuspended pellets individually to a chilled dounce homogenizer on ice. Use
the tight fitting pestle (Type A) to homogenize the sample for 30 strokes. Transfer the contents to a new 15 ml conical tube and centrifuge for 3 minutes at 1,250 x g at 4°C.
Monitor Cell Lysis: To ensure cell lysis, take 10 µl of the cell lysate from the dounce and lookat it under a phase contrast microscope using a hemocytometer to verify 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, thenuclei will appear as dots surrounded by asymmetric debris. If the cells are not lysed, thendounce on ice with an additional 10 strokes, or until the cells are lysed.
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8

12.

Remove the supernatant and discard. Resuspend each pellet in 500 µl ChIP Buffer supplemented with 5 µl PIC and 5 µl 100 mM PMSF. Transfer the contents to a new 2 ml microcentrifuge tube.

13.

Incubate on ice for 10 minutes. Proceed to Step B: Chromatin Sonication of Cultured Cells.

Section B. Chromatin Sonication of Cultured Cells
The section below describes the fragmentation of chromatin using sonication. Sonication results
may vary depending on cell type and sonication device being used. This protocol has beenvalidated using Active Motif’s EpiShear™ Probe Sonicator in combination with an EpiShear™ Cooled
Sonication Platform to maintain probe height and temperature consistency between samples. We
do not recommend sonication of samples containing less than 100,000 cells and/or 350 µl volume.
The ChIP Buffer has been optimized for immunoprecipitation performance, however, due to its
unique composition optimization of sonication conditions may be required. To maintain the highsensitivity of the assay, we recommend using our buffer system and altering the sonication time
and/or amplitude of your sonication system to achieve the desired fragmentation (e.g. somesystems may require as much as a three-fold increase in sonication time to improve chromatin
shearing). Please pay particular attention to our protocol regarding the processing of input chromatin for agarose gel analysis prior to the chromatin immunoprecipitation reaction as many steps
may differ from traditional ChIP protocols and failure to follow the outlined procedure may leadto artifacts in the gel images as shown in Figure 1 on page. 11
1.

Place the 2 ml microcentrifuge tube containing the chromatin into the tube cooler or packed
ice. Open cap and submerge the microtip into the liquid until the microtip is approximately
5 mm from the bottom of the tube. Sonicate according to optimized settings for the cell
type being used (see Recommendations on page 6). A recommended starting range forcultured cells is: 25% amplitude, pulse for 30 seconds on and 30 seconds off for a total
sonication “on” time of 10 minutes (or 20 minutes elapsed time).

2.

Spin tubes at 4°C in a microcentrifuge at maximum speed for 2 minutes to pellet the cellulardebris.

3.

Transfer 25 µl of each chromatin preparation into a 250 µl PCR tube for analysis of shearing
efficiency and chromatin quantification. This sample will be used to generate the Input DNA.

4.

Aliquot the remainder of each chromatin preparation into 1.5 ml microcentrifuge tubes. We
recommend making aliquots of 150 µl volume and storing at -80°C.
Note:

The size of the chromatin sonication should be verified before proceeding to theimmunoprecipitation step.

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9Input Preparation
5. To each 25 µl chromatin preparation from Step 3 above, add 175 µl TE pH 8.0 and
1 µl RNAse A. Cap the PCR tubes and vortex to mix
6.

Incubate in a thermocycler at 37°C for 30 minutes.

7.

Add 2 µl Proteinase K to each tube and vortex. Incubate tubes in a thermocycler at 55°C for
30 minutes and then increase the temperature to 80°C for 2 hours.

8.

Transfer each chromatin input to a 1.5 ml microcentrifuge tube. Add 83 µl Precipitation Buffer, 2 µl Carrier and 750 µl absolute ethanol. Vortex to mix and chill at -80°C for 30 minutes
to overnight.

9.

Spin tubes at 4°C in a microcentrifuge at maximum speed for 15 minutes.

10. Carefully remove the supernatant taking care not to disturb the pellet. Wash the pellet with
500 µl 70% ethanol and spin at 4°C in a microcentrifuge at maximum speed for 5 minutes.
11.

Carefully remove the supernatant taking care not to disturb the pellet. Remove residual
ethanol with a pipet tip. Leave the tubes uncapped and air dry for 10-15 minutes.

12.

When the pellets are dry, add 25 µl DNA Purification Elution Buffer to each tube. Incubate atroom temperature for 10 minutes. Then vortex to ensure the pellet is completely resuspended. This solution contains your Input DNA.

13.

Read the absorbance of each sample on a NanoDrop or other spectrophotometer at
260 nm to determine the DNA concentration of each chromatin preparation. Set aside
500 ng of DNA for analysis as described in Step 14. Store the remaining Input DNA at -20°C.

14.

Analyze each chromatin preparation on an agarose gel by following the instructions below.
a.

Prepare 500 mM NaCl by adding 2 µl 5M NaCl to 18 µl sterile water. Vortex to mix.

b.

Transfer 500 ng of Input DNA to a 250 µl PCR tube and add 1 µl 500 mM NaCl. Adjust
the final volume to 10 µl with sterile water if needed.

c.

Heat samples in a thermocycler at 100°C for 20 minutes followed by ramping the temperature down to 50°C.

d.

Remove tubes from the thermocycler and incubate at room temperature for 5 minutes.

e.

Add gel loading buffer to each sample and run on a 1.5% agarose gel. Include 100 bp
and 1 kb DNA ladders to analyze chromatin size. DNA should appear as a smear anywhere between 200-1200 bp.

Note:

Chromatin prepared with the High Sensitivity Chromatin Preparation Kit may
look different on an agarose gel compared to chromatin prepared using traditional ChIP methods. However, this will not affect the sensitivity of the assay or
increase background signal. Please follow the protocol as listed above for preparing Input DNA. Use of an alternative reverse cross-linking method or omitting
the 20 minute incubation at 100°C in NaCl is not recommended as this will cause
artifacts that make the DNA appear larger. As long as the chromatin falls within
the recommended 200-1200 bp range, proceed with the ChIP reaction. If fragments do not fall within this range, continue to optimize sonication conditions.

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10

15.

If chromatin preparations were successful, the aliquots stored at -80°C from Section B, Step 4
can be used to perform the ChIP reactions.

Figure 1: Validation of chromatin shearing efficiency following reversal of cross-links at 80°C for 2 hours.
Chromatin preparations of MCF-7 cells were fixed and sonicated using the EpiShear™ Probe Sonicator and EpiShear™
Cooled Sonication Platform from Active Motif. Input DNA was prepared in duplicate according to Section B, Steps 5-13 in
the manual. In Sample 1, Step 14 was not performed and 500 ng of input DNA was loaded directly onto a 1.5% agarose gel
without receiving the addition of NaCl and incubation at 100°C. The omission of Step 14 has caused a buffer artifact thatmakes the DNA appear larger on a gel. The duplicate sample, Sample 2, was processed according to the manual instructions
and included the addition of NaCl and incubation at 100°C as stated in Step 14. Analysis of 500 ng of this input DNA on a
1.5% agarose gel shows the expected fragmentation between 200-1200 bp. The difference in DNA sizing on the gel between
the two samples illustrates the importance of following the protocol recommendations regarding the processing of input
chromatin for agarose gel analysis prior to chromatin immunoprecipitation. Omission of key steps can lead to inaccurate
analysis of chromatin shearing efficiency. If the protocol steps were followed and the DNA fragments fall outside of therecommended range, sonication conditions should be further optimized.

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11

Protocols – Chromatin Preparation from Frozen Tissue
Section C: Cell Fixation Starting with Fresh or Frozen Tissue
This protocol describes cell fixation and chromatin preparation from 100-400 mg fresh or frozen
animal tissue. If performing chromatin preparation on multiple tissue samples, we recommend
completing Steps 1-7 for each sample before processing the next sample.
1.

For tissue fixation, transfer 10 ml Complete Tissue Fixation Solution (see Buffer Preparation
on page 7) to a 60 mm petri dish. Place the dish on ice.

2.

Add 100-400 mg fresh or frozen tissue sample to the petri dish and ensure that the sample isfully immersed. Cut the tissue sample into small pieces (approximately 1 mm cubes) using arazor blade.

3.

Transfer the sample plus the Complete Tissue Fixation Solution to a 15 ml conical tube androtate at room temperature for 15 minutes.

4.

Stop the fixation reaction by adding 515 µl Stop Solution to the conical tube and rotate atroom temperature for 5 minutes.

5.

Place the conical tube on ice and homogenize the contents with a hand-held tissue homogenizer set at 30,000 rpm for 45 seconds.

6.

Pellet the cells from step 5 by centrifugation for 3 minutes at 1,250 x g at 4°C.

7.

Remove the supernatant and discard. Resuspend the pellet in 10 ml ice-cold PBS Wash Buffer
by pipetting up and down. Keep samples ice-cold for the remainder of the procedure.

8.

Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. Wash thepellet(s) a second time in 10 ml ice-cold PBS Wash Buffer by pipetting up and down. Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. (Cell pellets may
be stored at -80°C at this stage).

9.

Resuspend each pellet in 5 ml Chromatin Prep Buffer supplemented with 5 µl PIC and5 µl 100 mM PMSF.

10. Incubate on ice for 10 minutes.
11.

Transfer the resuspended pellet(s) individually to a chilled dounce homogenizer on ice. Use
the tight fitting pestle (Type A) to homogenize the sample for 30 strokes. Once finished,transfer the contents to a new 15 ml conical tube.
Monitor Cell Lysis: To ensure cell lysis, take 10 µl of the cell lysate from the dounce and lookat it under a phase contrast microscope using a hemocytometer to verify 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, thenuclei will appear as dots surrounded by asymmetric debris. If the cells are not lysed, thendounce on ice with an additional 10 strokes, or until the cells are lysed.

12.

Centrifuge for 3 minutes at 1,250 x g at 4°C.
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12

13.

Remove the supernatant and discard. Resuspend each pellet in 500 µl - 1 ml ChIP Buffer
supplemented with PIC and 100 mM PMSF. (For 500 µl add 5 µl PIC and 5 µl PMSF. For 1 ml
add 10 µl PIC and 10 µl PMSF.) Transfer the contents to a new 2 ml microcentrifuge tube.

14.

Incubate on ice for 10 minutes. Proceed to Section D: Chromatin Sonication of Tissue.

Section D. Chromatin Sonication of Tissue
The section below describes the fragmentation of chromatin using sonication. Due to theincreased concentration of protein and cellular debris present in animal tissue, we recommend
following this protocol for the preparation of chromatin and input DNA from tissue. Sonication results may vary depending on tissue type and sonication device being used. This protocol has beenvalidated using Active Motifs EpiShear™ Probe Sonicator in combination with the EpiShear™ Cooled
Sonication Platform to maintain probe height and temperature consistency between samples. We
do not recommend sonication of samples containing less than 50 mg tissue and/or 350 µl volume.
The ChIP has been optimized for immunoprecipitation performance, however, due to its uniquecomposition optimization of sonication conditions may be required. To maintain the high sensitivity of the assay, we recommend using our buffer system and altering the sonication time and/or
amplitude of your sonication system to achieve the desired fragmentation (e.g. some systems may
require as much as a three-fold increase in sonication time to improve chromatin shearing). Please
pay particular attention to our protocol regarding the processing of input chromatin for agarose
gel analysis prior to the chromatin immunoprecipitation reaction as many steps may differ fromtraditional ChIP protocols and failure to follow the outlined procedure may lead to artifacts in thegel images as shown in Figure 2 on page 15.
1.

Place the 2 ml microcentrifuge tube containing the chromatin into the tube cooler or packed
ice. Open cap and submerge the microtip into the liquid until the microtip is approximately
5 mm from the bottom of the tube. Sonicate according to optimized settings for the tissue
type being used (see Recommendations on page 6). A recommended starting range for tissue
samples is: 25% amplitude, pulse for 30 seconds on and 30 seconds off for a total sonication“on” time of 10 minutes (or 20 minutes elapsed time).

2.

Spin tubes at 4°C in a microcentrifuge at maximum speed for 2 minutes to pellet the cellulardebris.

3.

Transfer 25 µl of each chromatin preparation into a 250 µl PCR tube for analysis of shearing
efficiency and chromatin quantification. This sample will be used to generate the Input DNA.

4.

Aliquot the remainder of each chromatin preparation into 1.5 ml microcentrifuge tubes. We
recommend making aliquots of 150 µl volume and storing at -80°C.
Note:

The size of the chromatin sonication should be verified before proceeding to theimmunoprecipitation step.

www.activemotif.com

13Input Preparation
5. To each 25 µl chromatin preparation from Step 3 above, add 175 µl TE pH 8.0 and
2 µl RNAse A. Cap the PCR tubes and vortex to mix.
6.

Incubate in a thermocycler at 37°C for 1 hour.

7.

Add 5 µl Proteinase K to each tube, vortex and incubate in a thermocycler at 37°C for 3 hours.

8.

Add 10 µl 5 M NaCl, vortex and incubate at 65°C for 6-16 hours to reverse cross-links.

9.

Remove tubes from the thermocycler and add 250 µl phenol and 125 µl chloroform:isoamyl
alcohol (24:1). Vortex vigorously and spin tubes in a room temperature microcentrifuge at
maximum speed for 2 minutes.

10. Transfer each upper aqueous layer to a new 1.5 ml microcentrifuge tube and add 250 µl
chloroform:isoamyl alcohol (24:1). Vortex vigorously and spin tubes in a room temperature
microcentrifuge at maximum speed for 2 minutes.
11.

Transfer the upper aqueous layer to a new 1.5 ml microcentrifuge tube. Add 83 µl Precipitation Buffer, 2 µl Carrier and 900 µl absolute ethanol. Vortex to mix and chill at -80°C for 30
minutes to overnight.

12.

Spin at 4°C in a microcentrifuge at maximum speed for 15 minutes.

13.

Carefully remove the supernatant taking care not to disturb the pellet. Wash the pellet with
500 µl 70% ethanol and spin at 4°C in a microcentrifuge at maximum speed for 5 minutes.

14.

Carefully remove the supernatant taking care not to disturb the pellet. Remove residual
ethanol with a pipet tip. Leave the tubes uncapped and air dry for 10-15 minutes.

15.

When the pellets are dry, add 25 µl DNA Purification Elution Buffer to each tube. Incubate atroom temperature for 10 minutes. Then vortex to ensure the pellet is completely resuspended. This solution contains your Input DNA.

16.

Read the absorbance of each sample on a NanoDrop or other spectrophotometer at
260 nm to determine the DNA concentration of each chromatin preparation. Set aside
500 ng of DNA for analysis as described in Step 17. Store the remaining Input DNA at -20°C.

17.

Analyze each chromatin preparation on an agarose gel by following the instructions below.
a.

Prepare 500 mM NaCl by adding 2 µl 5M NaCl to 18 µl sterile water. Vortex to mix.

b.

Transfer 500 ng of Input DNA to a 250 µl PCR tube and add 1 µl 500 mM NaCl. Adjust
the final volume to between 10 µl with sterile water if needed.

c.

Heat samples in a thermocycler at 100°C for 20 minutes followed by ramping the temperature down to 50°C.

d.

Remove tubes from the thermocycler and incubate at room temperature for 5 minutes.

e.

Add gel loading buffer to each sample and run on a 1.5% agarose gel. Include 100 bp
and 1 kb DNA ladders to analyze chromatin size. DNA should appear as a smear anywhere between 200-1200 bp.

Note:

Chromatin prepared with the High Sensitivity Chromatin Preparation Kit may
look different on an agarose gel than chromatin prepared using traditional ChIP

www.activemotif.com

14methods. However, this will not affect the sensitivity of the assay or increase
background signal. Please follow the protocol as listed above for preparing Input
DNA. Use of an alternative reverse cross-linking method or omitting the 20
minute incubation at 100°C in NaCl is not recommended as this will cause artifacts that make the DNA appear larger. As long as the chromatin falls within therecommended 200-1200 bp range, proceed with the ChIP reaction. If fragments
do not fall within this range sonication conditions should be further optimized.
18. If chromatin preparations were successful, the aliquots stored at -80°C from Section D,
Step 4 can be used to perform the ChIP reactions.

Figure 2: Validation of chromatin shearing efficiency following reversal of cross-links overnight at 65°C.
Chromatin preparations were fixed and sonicated using the EpiShear™ Probe Sonicator and EpiShear™ Cooled Sonication Platform from Active Motif. Input DNA was prepared in duplicate according to Section D, Steps 5-16 in the manual.
In Sample 1, Step 17 was not performed and 500 ng of input DNA was loaded directly onto a 1.5% agarose gel without
receiving the addition of NaCl and incubation at 100°C. The omission of Step 17 has caused a buffer artifact that makes
the DNA appear larger on a gel. The duplicate sample, Sample 2, was processed according to the manual instructions andincluded the addition of NaCl and incubation at 100°C as stated in Step 17. Analysis of 500 ng of this input DNA on a 1.5%
agarose gel shows the expected fragmentation between 200-1200 bp. The difference in DNA sizing on the gel between
the two samples illustrates the importance of following the protocol recommendations regarding the processing of input
chromatin for agarose gel analysis prior to chromatin immunoprecipitation. Omission of key steps can lead to inaccurate
analysis of chromatin shearing efficiency. If the protocol steps were followed and the DNA fragments fall outside of therecommended range, sonication conditions should be further optimized.

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15

Protocols – Chromatin Preparation from PBMCs
Section E: Cell Fixation Starting with PBMCs
PBMCs (including lymphocytes and monocytes) are difficult to lyse cells and as a result will yieldapproximately 30-50% as compared to cell lines. We recommend using a minimum of 10 million
cells for the preparation of chromatin.
1.

Collect your PBMCs, T cells, B cells, NK cells or moncytes according to your preferred protocol. Transfer the appropriate number of cells required for each chromatin preparation into
a conical tube and centrifuge at 1,250 x g at 4°C to collect the cells. If there are cells that are
floating or are stuck to the sides of the tube following the spin, use a metal spatula to scrape
the cells down into solution and repeat the centrifugation at 1,250 x g at 4°C for 5 minutes.
Continue to repeat the scraping and spin as many times as needed to pellet all the cells.
Remove and discard supernatant.
Note:

PBMCs may stick to certain plastics. Therefore, it is recommended to use a metal
spatula for cell scraping to avoid losing any sample material.

2.

Flash freeze the cell pellet(s) by imersing conical tube into dry ice. Incubate on dry ice for 10
minutes. Cell pellets may be stored at -80°C at this stage, or proceed with the next step.

3.

Prepare fresh Fixation Solution. To fix cells, add 5 ml of freshly prepared Fixative Solution to
the cell pellet and resuspend cells by pipetting up and down. Incubate at room temperaturefor 15 minutes on a roller.

4.

Stop the fixation reaction by adding 250 µl Stop Solution to the existing fixation solution.
Swirl to mix and incubate at room temperature for 5 minutes on a roller.

5.

Following the incubation, add 250 µl Detergent to each conical tube. Invert to mix. Pellet thecells by centrifugation for 5 minutes at 1,250 x g at 4°C.

6.

If there are cells that are floating or are stuck to the sides of the tube following the spin, use
a metal spatula to scrape the cells down into solution and repeat the centrifugation at
1,250 x g at 4°C for 5 minutes. Continue to repeat the scraping and spin as many times asneeded to pellet all the cells.

7.

Remove the supernatant and discard. Resuspend the pellet(s) in 5 ml ice-cold PBS Wash Buffer by pipetting up and down. Keep samples ice-cold for the remainder of the procedure.

8.

Centrifuge for 5 minutes at 3,200 x g at 4°C. The cell pellet will tend to be loose, so carefully
remove the supernatant and discard taking care to avoid disturbing the pellet. Wash thepellet(s) a second time in 5 ml ice-cold PBS Wash Buffer by pipetting up and down. Centrifuge for 5 minutes at 3,200 x g at 4°C. Carefully remove the supernatant and discard.

9.

Flash freeze the cell pellet(s) by imersing conical tube into dry ice. The freeze/thaw will tohelp facilitate cell lysis. Incubate on dry ice for 10 minutes. Cell pellets may be stored at -80°C
at this stage, or proceed with the next step.

10. Resuspend each pellet(s) in 5 ml ice-cold Swelling Buffer supplemented with 5 µl PIC and5 µl 100 mM PMSF. Pipet up and down to mix.
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16

11.

Incubate on ice for 30 minutes.

12.

Add 125 µl Detergent to each cell pellet. Vortex for 30 sec on highest setting to lyse the cells.
Note:

We do not recommend the use of a dounce homogenizer to assist in cell lysis
as the transfer process to and from the dounce increases sample loss. If cells
continue to have difficulty lysing, we suggest additional sonication.

13.

Centrifuge for 10 minutes at 3,200 x g at 4°C. Remove and discard the supernatant.

14.

Resuspend each pellet in 500 µl ChIP Buffer supplemented with 5 µl PIC and 5 µl 100 mM
PMSF. Transfer the contents to a new 2 ml microcentrifuge tube.

15.

Incubate on ice for 10 minutes. Proceed to Step F: Chromatin Sonication of PBMCs.

Section F. Chromatin Sonication of PBMCs
The section below describes the fragmentation of chromatin using sonication. Sonication results
may vary depending on cell type and sonication device being used. This protocol has beenvalidated using Active Motif’s EpiShear™ Probe Sonicator in combination with an EpiShear™ Cooled
Sonication Platform to maintain probe height and temperature consistency between samples. We
do not recommend sonication of samples containing less than 10 million cells or 350 µl volume.
The ChIP Buffer has been optimized for immunoprecipitation performance, however, due to its
unique composition optimization of sonication conditions may be required. To maintain the highsensitivity of the assay, we recommend using our buffer system and altering the sonication time
and/or amplitude of your sonication system to achieve the desired fragmentation (e.g. somesystems may require as much as a three-fold increase in sonication time to improve chromatin
shearing). Please pay particular attention to our protocol regarding the processing of input chromatin for agarose gel analysis prior to the chromatin immunoprecipitation reaction as many steps
may differ from traditional ChIP protocols and failure to follow the outlined procedure may leadto artifacts in the gel images as shown in Figure 3 on page 19.
1.

Place the 2 ml microcentrifuge tube containing the chromatin into the tube cooler or packed
ice. Open cap and submerge the microtip into the liquid until the microtip is approximately
5 mm from the bottom of the tube. Sonicate according to optimized settings for the cell
type being used (see Recommendations on page 6). A recommended starting range for
PBMCs is: 4 rounds of sonication at 42% amplitude, pulse for 30 seconds on and 30 seconds
off for a total sonication “on” time of 5 minutes per round. (Total sonication “on” time is 20
minutes and total elapsed time is 40 minutes per sample.)2.

Spin tubes at 4°C in a microcentrifuge at maximum speed for 5 minutes to pellet the cellulardebris.

3.

Transfer 25 µl of each chromatin preparation into a 250 µl PCR tube for analysis of shearing
efficiency and chromatin quantification. This sample will be used to generate the Input DNA.

4.

Aliquot the remainder of each chromatin preparation into 1.5 ml microcentrifuge tubes. We
recommend making aliquots of 250 µl volume.
www.activemotif.com

17

5.

Flash freeze the chromatin by imersing tubes into dry ice for 10 minutes and store at -80°C.
Note:

The size of the chromatin sonication should be verified before proceeding to theimmunoprecipitation step.

Input Preparation
6. To each 25 µl chromatin preparation from Step 5 above, add 175 µl TE pH 8.0 and
2 µl RNAse A. Cap the PCR tubes and vortex to mix.
7.

Incubate in a thermocycler at 37°C for 1 hour.

8.

Add 5 µl Proteinase K to each tube, vortex and incubate in a thermocycler at 37°C for 3 hours.

9.

Add 10 µl 5 M NaCl, vortex and incubate at 65°C for 6-16 hours to reverse cross-links.

10. Remove tubes from the thermocycler and add 250 µl phenol and 125 µl chloroform:isoamyl
alcohol (24:1). Vortex vigorously and spin tubes in a room temperature microcentrifuge at
maximum speed for 2 minutes.
11.

Transfer each upper aqueous layer to a new 1.5 ml microcentrifuge tube and add 250 µl
chloroform:isoamyl alcohol (24:1). Vortex vigorously and spin tubes in a room temperature
microcentrifuge at maximum speed for 2 minutes.

12.

Transfer the upper aqueous layer to a new 1.5 ml microcentrifuge tube. Add 83 µl Precipitation Buffer, 2 µl Carrier and 900 µl absolute ethanol. Vortex to mix and chill at -80°C for
30 minutes to overnight.

13.

Spin at 4°C in a microcentrifuge at maximum speed for 15 minutes.

14.

Carefully remove the supernatant taking care not to disturb the pellet. Wash the pellet with
500 µl 70% ethanol and spin at 4°C in a microcentrifuge at maximum speed for 5 minutes.

15.

Carefully remove the supernatant taking care not to disturb the pellet. Remove residual
ethanol with a pipet tip. Leave the tubes uncapped and air dry for 10-15 minutes.

16.

When the pellets are dry, add 25 µl DNA Purification Elution Buffer to each tube. Incubate atroom temperature for 10 minutes. Then vortex to ensure the pellet is completely resuspended. This solution contains your Input DNA.

17.

Read the absorbance of each sample on a NanoDrop or other spectrophotometer at
260 nm to determine the DNA concentration of each chromatin preparation. Set aside
500 ng of DNA for analysis as described in Step 18. Store the remaining Input DNA at -20°C.

18. Analyze each chromatin preparation on an agarose gel by following the instructions below.
a.

Prepare 500 mM NaCl by adding 2 µl 5M NaCl to 18 µl sterile water. Vortex to mix.

b.

Transfer 500 ng of Input DNA to a 250 µl PCR tube and add 1 µl 500 mM NaCl. Adjust
the final volume to between 10 µl with sterile water if needed.

c.

Heat samples in a thermocycler at 100°C for 20 minutes followed by ramping the temperature down to 50°C.

d.

Remove tubes from the thermocycler and incubate at room temperature for 5 minutes.

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18

e.

19.

Add gel loading buffer to each sample and run on a 1.5% agarose gel. Include 100 bp
and 1 kb DNA ladders to analyze chromatin size. DNA should appear as a smear anywhere between 200-1200 bp.

If chromatin preparations were successful, the aliquots stored at -80°C from Section F, Step 5
can be used to perform the ChIP reactions.

Figure 3: Validation of chromatin shearing efficiency following reversal of cross-links overnight at 65°C.
Chromatin preparations of PBMC cells were fixed and sonicated using the EpiShear™ Probe Sonicator and EpiShear™ Cooled
Sonication Platform from Active Motif. Input DNA was prepared in duplicate according to Section F, Steps 6-17 in the
manual. In Sample 1, Step 18 was not performed and 500 ng of input DNA was loaded directly onto a 1.5% agarose gel
without receiving the addition of NaCl and incubation at 100°C. The omission of Step 18 has caused a buffer artifact thatmakes the DNA appear larger on a gel. The duplicate sample, Sample 2, was processed according to the manual instructions
and included the addition of NaCl and incubation at 100°C as stated in Step 18. Analysis of 500 ng of this input DNA on a
1.5% agarose gel shows the expected fragmentation between 200-1200 bp. The difference in DNA sizing on the gel between
the two samples illustrates the importance of following the protocol recommendations regarding the processing of input
chromatin for agarose gel analysis prior to chromatin immunoprecipitation. Omission of key steps can lead to inaccurate
analysis of chromatin shearing efficiency. If the protocol steps were followed and the DNA fragments fall outside of therecommended range, sonication conditions should be further optimized.

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19

Appendix
Section G. Troubleshooting Guide
Problem/question

Recommendation

At what points in the
protocol can I stop?

The protocol may be stopped and samples stored at the times and temperatures below:
1. After formaldehyde fixation and centrifugation (intact cell pellet), -80°C.
2. After chromatin shearing, -80°C.
3. After Input DNA clean up, -20°C.

After sonication shearing
and centrifugation, aviscous or cloudy layer isvisible in the chromatin.

Depending upon the cell type, lipid or glycogen layers may be seen after centrifugation.
For example, fatty tissues may have a lipid layer. Avoid such layers when you remove thesupernatant. However, if the whole supernatant is cloudy, it should not interfere with the
IP reaction.

Poor yield of shearedchromatin.

Insufficient cell numbers were used. Repeat chromatin preparation using a larger numberof cells.
Nuclei not released. It is highly recommended to perform dounce homogenization, evenwhen using sonication. Use a dounce homogenizer with a small clearance pestle (Active
Motif Catalog Nos. 40401 & 40415). Monitor cell lysis under a microscope. Generally, themore cells that are lysed, the higher the sheared chromatin yield.
Sonication samples were emulsified. Avoid emulsification by turning up the power ofthe sonicator gradually. If a chromatin preparation becomes emulsified inadvertently,
discontinue shearing and centrifuge the sample for 4 minutes at 8,000 rpm in a 4°C
microcentrifuge to remove trapped air.
Use fresh formaldehyde when preparing Complete Cell Fixation Solution and Complete
Tissue Fixation Solution.
Buffers were not scaled proportionally to the size of the sample. Use the chart in Cell
Growth Recommendations to scale up or down chromatin preparation.

Shearing efficiency is notclear from gel analysis or isinefficient.

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 the cross-links reversed, protein removed(Proteinase K) and RNA removed (RNase), followed by DNA purification.
High molecular weight products. Decrease the size of the fragments by re-sonicating thesample. If an alternative reverse cross-linking method was used, or the 20 minute incubation at 100°C in NaCl was omitted prior to running the agarose gel for analysis, please
repeat the input chromatin preparation and follow the manual instructions.
Chromatin not sheared enough. Shearing should produce DNA fragments that are smallenough to exclude background from neighboring chromosomal sequences, but still large
enough that there is a good possibility your amplicon remains intact. We recommend
200-1200 bp fragments. If necessary, optimize sonication settings. Conditions provided
are recommended starting points. Increase amplitude of sonication to 42% with cultured
cells in a 500 µl volume. Increase amplitude of sonication to 63% with tissue samples in a
1 ml volume. Check the fragment size on a gel to assess your shearing efficiency.

www.activemotif.com

20Section H. Related Products
ChIP-IT® Kits		

FormatCatalog No.

ChIP-IT® Express		ChIP-IT® Express Enzymatic		
ChIP-IT® Express Shearing Kit		
ChIP-IT® Express Enzymatic Shearing Kit		
ChIP-IT® High Sensitivity		
High Sensitivity Chromatin Preparation Kit		
ChIP-IT® qPCR Analysis Kit		
ChIP-IT® ChIP-Seq		
ChIP-Bis-Seq		
ChIP-IT® FFPE		ChIP-IT® FFPE Chromatin Preparation Kit		
ChIP-exo		
enChIP		
Tag ChIP-IT®		
ChIP-IT® Express HT		
Re-ChIP-IT®		RNA ChIP-IT®		
Chromatin IP DNA Purification Kit		
EpiShear™ Probe Sonicator		
EpiShear™ Cooled Sonication Platform, 1.5 ml		
ChIP-IT® Protein G Magnetic Beads		
Protein G Agarose Columns		
Siliconized Tubes, 1.7 ml		
ChIP-IT® Control qPCR Kit – Human		
ChIP-IT® Control qPCR Kit – Mouse		ChIP-IT® Control qPCR Kit – Rat		ChIP-IT® Control Kit – Human		
ChIP-IT® Control Kit – Mouse		ChIP-IT® Control Kit – Rat		
Ready-to-ChIP HeLa Chromatin		
Ready-to-ChIP Hep G2 Chromatin		
Ready-to-ChIP K-562 Chromatin		
Ready-to-ChIP NIH/3T3 Chromatin		Bridging Antibody for Mouse IgG		
Dounce Homogenizer		Dounce Homogenizer		

25 rxns
25 rxns
10 rxns
10 rxns
16 rxns
16 rxns
10 rxns
10 libraries
10 libraries
16 rxns
5 rxns
12 rxns
16 rxns
16 rxns
96 rxns
25 rxns
25 rxns
50 rxns
110 V
1 platform
25 rxns
30 rxns
25 tubes
5 rxns
5 rxns
5 rxns
5 rxns
5 rxns
5 rxns
10 rxns
10 rxns
10 rxns
10 rxns
500 µg
1 ml
15 ml

53008
53009
53032
53035
53040
53046
53029
53041
53048
53045
53030
53043
53125
53022
53018
53016
53024
58002
53051
53080
53014
53039
53036
53026
53027
53028
53010
53011
53012
53015
53019
53020
53021
53017
40401
40415

ChIP-validated Antibodies
For an up-to-date list of over 125 ChIP-validated antibodies, please visit www.activemotif.com/chipabs.

www.activemotif.com

21

Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008, USA
E-mail: tech_service@activemotif.com

Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351

Active Motif Europe
Avenue Reine Astrid, 92
B-1310 La Hulpe, Belgium
E-mail: eurotech@activemotif.com
Direct: +32 (0)2 653 0001

Germany Free Phone: 0800 181 99 10
France Free Phone: 0800 90 99 79
UK Free Phone: 0800 169 31 47
Fax: +32 (0)2 653 0050

Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan

Direct: +81 3 5225 3638
Fax: +81 3 5261 8733
E-mail: japantech@activemotif.com

Active Motif China
787 Kangqiao Road
Building 10, Suite 202
Pudong District
Shanghai, 201315, China

Direct: (86)-21-20926090
Hotline: 400-018-8123
E-mail: techchina@activemotif.com

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22