Cross-Linking Chromatin Immunoprecipitation (X-ChIP) with ChIP Protocol

Introduction

ChIP is an effective technique used for specifically matching histone or protein modifications to the genomic regions. After isolating chromatin, antibodies to the antigen concerned are used to establish whether this target attaches to a specific DNA sequence or not, or to alternatively map the distribution across the genome (DNA or microarray sequencing). This can be carried out either temporally or spatially. This procedure gives explicit details on how to perform ChIP on cells.

 

Save time with validated kits and reagents

To obtain the best results from the initial ChIP experiment, Abcam’s reagents and ChIP kits are recommended:

  • Abcam’s flexible Chromatin Extraction Kit enables extracting sheared, unsheared, native, or cross-linked chromatin in less than 60 minutes
  • Abcam’s easy-to-use ChIP Kit allows users to go directly from cells to ChIP
  • Abcam’s Magnetic One-Step ChIP Kit or One-Step ChIP Kit enables to perform ChIP directly from chromatin
  • Abcam’s High Sensitivity ChIP Kit detects low-abundance events

 

Alternatively, the following protocol is recommended.

 

1. Cross-linking and cell harvesting

 

Proteins are cross-linked to the DNA using formaldehyde. Cross-linking is a time dependent protocol and hence needs to be optimized. It is recommended to cross-link the samples for 2–30 minutes, but too much cross-linking can reduce sonication efficiency and antigen accessibility. Also, epitopes might be concealed. The formaldehyde is quenched and the cross-linking reaction is terminated by adding glycine.

1.1. First, two confluent 150 cm2 dishes (1x107–5x107 cells per dish) are taken. To cross-link the proteins to DNA, formaldehyde is directly added to the media in a drop-wise fashion, bringing it to a final concentration of 0.75% and rotating mildly for 10 minutes at room temperature (RT).

1.2. Glycine is added at a final concentration of 125 mm to the media and incubated by shaking at RT for 5 minutes.

1.3. 10 ml cold PBS is used to rinse cells.

1.4. 5 ml of cold PBS is added; dishes are scraped meticulously with a cell scraper; and the cells are transferred into a 50 ml tube.

1.5. 3 ml of PBS is added to the dishes, which are again scraped and the remainder of the cells is transferred to the 50 ml tube.

1.6. The tube is centrifuged at 4 °C for 5 minutes, 1,000 x g.

1.7. Supernatant is carefully aspirated off, and the pellet is resuspended in ChIP lysis buffer (750 μl per 1x107 cells) and incubated on ice for 10 minutes.

 

If suspension cells are used, it is recommended to begin with 1x1075x107 cells and treat them with both glycine and 0.75% formaldehyde as mentioned in Step 1. Pellet the cells by centrifuging for 5 minutes, 1,000 x g. Rinse thrice with cold PBS and then resuspend the pellet in ChIP lysis buffer (750 μl per 1x107 cells). Advance to Step 2.

2. Sonication

2.1. Lysate is sonicated to shear DNA to an average fragment size of 200 to 1000 base pairs. This needs to be optimized because different sonication times are required for different cell lines.

Optimal conditions are identified by time course sonication of the cross-linked lysate. Samples need to be removed over the course of time, followed by isolating the DNA as illustrated in Step 3. The size of the fragment is analyzed on a 1.5% agarose gel as described in Figure 1.

2.2. Following sonication, cell debris is pelleted by centrifuging for 10 minutes, 4 °C, 8,000 x g and the supernatant is transferred to a new tube. This chromatin preparation will be later utilized for the immunoprecipitation (IP) in Step 4.

2.3. 50 μl of each sonicated sample is removed to establish the size of the fragments and DNA concentration.

It is recommended to snap freeze the sonicated chromatin in liquid nitrogen and store for up to 3 months at -80 °C. Multiple freeze-thaws should be avoided.

 

3. Determination of DNA concentration and fragment size

3.1. The samples of sonicated chromatin can be used to measure the concentration of DNA for subsequent IPs and determine the size of DNA fragments. 70 µl of elution buffer is added to 50 µL of chromatin.

3.2. 2 µl of RNase A (10 mg/ml) and 4.8 µl of 5m NaCl are added and incubated while shaking at 65 °C overnight.

3.3. 2 µl of proteinase K (20 mg/ml) is added and incubated while shaking for 1 hour at 60 °C.

RNase A is used to treat the samples because high RNA levels will impede with DNA purification if PCR purification kit is used. Yields can be considerably reduced with saturation of the columns.

Proteinase K is used to treat the samples because this enzyme cleaves the peptide bonds next to the carboxylic group of aromatic and aliphatic amino acids. This disrupts the cross-links between DNA and proteins which helps in DNA purification.

3.4. DNA is purified using phenol:chloroform extraction or a PCR purification kit.

3.5. To measure the concentration of DNA, 5 μl of the purified DNA is transferred into a tube that contains 995 μl TE to provide a 200-fold dilution and subsequently read the OD260. OD260 x 10,000 is the DNA concentration in μg/ml and this is applied to measure the DNA concentration of the chromatin preparation. Purified DNA is run in a 1.5% agarose gel with a 100 base pair DNA marker to determine the size of the fragments.

 

4. Immunoprecipitation

4.1. The chromatin prepared from Step 2.2 is used. It is recommended to use about 25 μg of DNA per IP. Each sample is diluted to 1:10 ratio with RIPA Buffer. One sample will be required for the specific antibody and one sample will be needed for the control (beads only). 50 µl of chromatin is removed to serve as the input sample and this is stored at a temperature of -20 °C until further use.

4.2. Excluding the beads-only control, primary antibody is added to all the samples and rotated for 1 hour at 4 °C. The amount of antibody to be added must be empirically determined; usually 1–10 μg of antibody per 25 μg of DNA is found to work well.

4.3. Preparation of protein A/G beads: When both Protein A and Protein G beads are used, an equal amounts of Protein A and Protein G beads are mixed together and washed thrice in RIPA Buffer. The RIPA buffer is aspirated and a single stranded herring sperm DNA is added to reach a final concentration of 75 ng/μl beads and next BSA is added to a final concentration of 0.1 μg/μl beads. RIPA buffer double the bead volume is added, followed by incubating for 30 minutes and rotating at RT. RIPA buffer is used to wash once, and RIPA buffer to double the bead volume is added.

4.4. 60 µl of blocked protein A/G beads is added to all samples and IP and stored overnight, rotating at 4 °C.

Protein G beads, Protein A beads, or a combination of both need to be used. Shown in Table 1 is the affinity of Protein G beads and Protein A beads to different immunoglobulin isotypes.

4.5. The immunoprecipitated samples are centrifuged for 1 minute at 2,000 x g and the supernatant is subsequently removed.

4.6. The following washes are carried out: once in high salt wash buffer, once in low salt wash buffer, and once in LiCl wash buffer. Post each wash, the supernatant is centrifuged for 1 minute at 2,000 x g and then removed.

If there is high background, some more washes may be required. Another option is to pre-clear the sonicated chromatin by incubating with the Protein A/G beads for 1 hour before Step 4.2. This is an additional step during which non-specific binding to the beads will be removed, if any. The supernatant (sonicated chromatin) is transferred to a new tube and incubated with the antibody and beads as Illustrated in Step 4.2 onwards.

 

5. Elution and reversal of cross-links

5.1. DNA is eluted by adding 120 μl of elution buffer to the protein A/G beads and shaking gently at 30 °C for 15 minutes.

5.2. The supernatant is centrifuged for 1 minute at 2,000 x g and then transferred into a fresh tube.

5.3. 2 µl RNase A (10 mg/mL) and 4.8 µl of 5m NaCl are added and incubated while vortexing at 65 °C overnight.

5.4. 2 µl of proteinase K (20 mg/ml) is added and incubated while shaking for 1 hour at 60 °C.

5.5. Phenol:chloroform extraction or a PCR purification kit can be used to purify the DNA.

5.6. Real-time PCR is used to quantitatively measure the DNA levels. Probes and primers are usually developed by using the software available with the real-time PCR apparatus. An online design tool is also used as an alternative.

 

Abcam’s website lists a range of pre-designed probes and primers.

             

Figure 1. U2OS cells were sonicated for 5, 10, 15 and 20 min. The fragment size decreases during the time course. The optimal fragment size is observed at 15 minutes. NOTE; sonicating for too long will disrupt nucleosome-DNA interactions therefore the band size should not be smaller than 200 bp.

 

Immunoglobulin isotypes

Table 1. The affinity of Protein A and G beads to different immunoglobin isotypes

Species

Immunoglobulin isotype

Protein A

Protein G

Human

IgG1

+++

+++

 

IgG2

+++

+++

 

IgG3

-

+++

 

IgG4

+++

+++

 

IgM

Use anti Human IgM

Use anti Human IgM

 

IgE

-

+

 

IgA

-

+

Mouse

IgG1

+

+++

 

IgG2a

+++

+++

 

IgG2b

++

++

 

IgG3

+

+

 

IgM

Use anti human IgM

Use anti human IgM

Rat  

IgG1

-

+

 

IgG2a

-

+++

 

IgG2b

-

++

 

IgG2c

+

++

Chicken

all isotypes

-

++

Cow

all isotypes

++

+++

Goat

all isotypes

-

++

Guinea Pig

all isotypes

+++

++

Hamster

all isotypes

+

++

Horse

all isotypes

++

+++

Pig

all isotypes

+

++

Rabbit

all isotypes

+++

++

Sheep

all isotypes

-

++

 

Solutions

ChIP Lysis Buffer

  • 50 mm HEPES-KOH pH7.5
  • 140 mm NaCl
  • 1 mm EDTA pH8
  • 0.1% Sodium Deoxycholate
  • 0.1% SDS
  • 1% Triton X-100
  • Protease Inhibitors (add fresh each time)

RIPA Buffer

  • 50 mm Tris-HCl pH8
  • 150 mm NaCl
  • 2 mm EDTA pH8
  • 0.1% SDS
  • 0.5% Sodium Deoxycholate
  • 1% NP-40
  • Protease Inhibitors (add fresh each time)

Low Salt Wash Buffer

  • 0.1% SDS
  • 1% Triton X-100
  • 2 mm EDTA
  • 20 mm Tris-HCl pH 8.0
  • 150 mm NaCl

High Salt Wash Buffer

  • 0.1% SDS
  • 1% Triton X-100
  • 2 mm EDTA
  • 20 mm Tris-HCl pH 8.0
  • 500 mm NaCl

LiCl Wash Buffer

  • 0.25m LiCl
  • 1% NP-40
  • 1% Sodium Deoxycholate
  • 1 mm EDTA
  • 10 mm Tris-HCl pH 8.0

TE Buffer

  • 1 mm EDTA
  • 10 mm Tris pH 8.0

Elution Buffer

  • 1% SDS
  • 100 mm NaHCO3

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.

Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.

Last updated: Jun 6, 2019 at 12:49 PM

Citations

Please use one of the following formats to cite this article in your essay, paper or report:

  • APA

    Abcam. (2019, June 06). Cross-Linking Chromatin Immunoprecipitation (X-ChIP) with ChIP Protocol. News-Medical. Retrieved on November 13, 2019 from https://www.news-medical.net/whitepaper/20180111/Cross-Linking-Chromatin-Immunoprecipitation-(X-ChIP)-with-ChIP-Protocol.aspx.

  • MLA

    Abcam. "Cross-Linking Chromatin Immunoprecipitation (X-ChIP) with ChIP Protocol". News-Medical. 13 November 2019. <https://www.news-medical.net/whitepaper/20180111/Cross-Linking-Chromatin-Immunoprecipitation-(X-ChIP)-with-ChIP-Protocol.aspx>.

  • Chicago

    Abcam. "Cross-Linking Chromatin Immunoprecipitation (X-ChIP) with ChIP Protocol". News-Medical. https://www.news-medical.net/whitepaper/20180111/Cross-Linking-Chromatin-Immunoprecipitation-(X-ChIP)-with-ChIP-Protocol.aspx. (accessed November 13, 2019).

  • Harvard

    Abcam. 2019. Cross-Linking Chromatin Immunoprecipitation (X-ChIP) with ChIP Protocol. News-Medical, viewed 13 November 2019, https://www.news-medical.net/whitepaper/20180111/Cross-Linking-Chromatin-Immunoprecipitation-(X-ChIP)-with-ChIP-Protocol.aspx.

Other White Papers by this Supplier