Chromatin Immunoprecipitation (ChIP) Assay

ChIP Assay Overview

Chromatin Immunoprecipitation (ChIP) assays are used to evaluate transcription factor-DNA interactions and they are critical for advancing our understanding of gene expression regulation and epigenetic modifications. ChIP can detect and relatively quantify specific protein-DNA and protein-protein interactions in vivo at a single locus or multiple loci. ChIP involves chemically crosslinking proteins to DNA sequences, which is followed by immunoprecipitation of the crosslinked complexes (Fig 1), and analysis of the resultant DNA by endpoint or quantitative polymerase chain reaction (qPCR) (Figs 2 and 3), microarrays (ChIP-chip), or next- generation sequencing (ChIP-seq) (Figs 4 and 5).

 

ChIP procedure.

 
Song et al., 2015
 

Fig 1. ChIP procedure.
Protein and associated chromatin in living cells or tissues are crosslinked using formaldehyde. The crosslinked DNA–protein complexes (chromatin-protein) are then sheared into ∼500 bp DNA fragments using either enzymatic digestion or physical shearing by sonication. The DNA-protein complexes are then immunoprecipitated by an appropriate protein-specific antibody. After the cross-links are reversed, the associated DNA fragments are eluted, which is followed by immunoprecipitation of the crosslinked complexes and analysis of the resultant DNA by endpoint or quantitative polymerase chain reaction (qPCR), microarrays (ChIP-chip), or next- generation sequencing (ChIP-seq).

You can use ChIP to discover:

  • DNA sequences occupied by multiple specific protein targets
  • The binding sites and distribution of a particular protein, such as a transcription factors, transcription co-factors, DNA replication factors and DNA repair proteins throughout the entire genome, under specified cellular conditions
  • Gene transcription and polymerase activity
  • Complex DNA/protein interactions underlying disease phenotypes
  • Modifications to proteins, such as histones, that may influence chromatin structure and gene expression
  • Nucleosome architecture and regulation of chromosomal maintenance

Fig 2.
A. ChIP with cell line samples.

Upper panel: Successful ChIP enrichment of DNA sequences associated with the heterochromatin protein 1d gamma (HP1gamma), an important marker of gene repression. Data collected using ChIPAb+™ Hp1g antibody/primer set, mouse IgG (non-specific control) and Magna ChIP™ G kit (Cat. #: 17-10085).

Lower panel: High throughput (96-well plate) ChIP analysis of multiple protein targets to query multiple gene loci under various conditions using antibody panels and Magna ChIP™ HT96 Kit (Cat. #: 17-10077).  

B. ChIP with tissue samples.

Upper panel: cryosection tissue and isolate region specific tissue sample with provided 1mm microdissection punch. An example of region specific tissue isolation is shown in the image. A 300 µm of coronal mouse brain section with region specific microdissection of the hippocampus (A) and cerebral cortex (B). The isolated tissue was placed above the dissected region (a: hippocampus, b: cortex).The purified tissue is then dispersed with Tissue Stabilizing Solution followed by formaldehyde treatment. Formaldehyde cross-links proteins to DNA to ensure co-precipitation.

Lower panel: Chromatin from various cell lines was subjected to immunoprecipitation with the indicated antibodies using the Magna ChIP™ G Tissue kit (Cat. #: 17-20000). The IgG used for relative comparison was either Rabbit Purified IgG (Cat.#: PP64B) or Mouse Purified IgG (Cat.#: 12- 371B), depending upon the ChIP antibody. Quantitative PCR data is presented as fold relative enrichment to IgG from independent experiments or as % input. For a biological negative control qPCR was assessed with primers upstream of the Dhfr gene (UpSt (-)). Antibodies and primers utilized were as follows: Anti-RNA Polymerase II clone CTD4H8 (Cat.# 05-623B): 1 µg of mouse monoclonal affinity purified antibody immunoprecipitated with chromatin of various mouse tissue and qPCR assayed with primers specific for mouse GAPDH promoter andAnti-SP1 (Cat.# 07-645): 1 µg of mouse monoclonal affinity purified antibody immunoprecipitated from chromatin of various mouse tissue and qPCR assayed with primers specific for mouse Dhfr promoter.

C. Competitor comparisons: Higher fold enrichment using low amounts of chromatin or shorter procedure time

Upper panel: 10,000 cell equivalents of sonicated chromatin prepared from HeLa cells were chromatin immunoprecipitated using 1 µg of purified IgG (Rabbit IgG,

Cat. #: 12-370) or specific antibodies (H3K4Me3, Cat. #:17-614; Phospho-CREB, Cat. #: 17-10131). Each assay used either the reagents and protocol

provided with the Magna ChIP™ HiSens Kit (Cat. #: 17-10460) or those from the low input ChIP kit from Supplier A or Supplier D. Immunoprecipitation of antibody-associated DNA fragments was verified by qPCR using positive control primers (GAPDH promoter primers for H3K4me3 and c-Fos promoter primers for pCREB) and negative control primers (human ß-globin promoter primers). Results reflect analysis of 2 µL out of 50 µL total DNA per qPCR reaction.

Lower panel: Complete ChIP reaction in 6 hours in flexible strip well format for Imprint® Chromatin Immunoprecipitation Kit (Cat. #: ChIP1) Comparison of time required for protocol completion from fixation through purification using different Chromatin Immunoprecipitation kits. Each ChIP experiment was carried out using each manufacturers recommended protocol and total required times were compared. A highly sensitive ChIP assay. HeLa cells were counted, fixed, and immunoprecipitated according to the Imprint CHP1 bulletin with optional High Sensitivity method. ChIP assays were performed using Anti-H3K9ac (H9286) and kit provided antibodies against Human RNA Polymerase II and non-specific Mouse IgG. To evaluate ChIP enrichment, SYBR qPCR was conducted targeting the GAPDH promoter region (highly expressed housekeeping gene). Percent Input describes the amount of DNA with and without antibody selection. For these antibodies, apparent enrichment increased with fewer cells per reaction from diminished non-specific pull-down.


ChIP DNAs analyzed by Next Generation Sequencing (ChIP-seq)

Fig 3. ChIP-Seq Analysis.
Chromatin immunoprecipitation was performed using the Magna ChIP™ HiSens kit (Cat. #: 17-10460), 2µl, 2µg, or 5 µg of the anti-H3K4me2 antibody (Cat. #s: 04-790, 05-1338 or ABE250), or 1 or 3µl or 4µg anti-H3K4me3 antibody (Cat. #s: 05-745R, 07-473 or 05-1339), or 2µg anti-H3K36me3 antibody (Cat. #: ABE435), 20 µL Protein A/G magnetic beads , and 1e6 or 4e6 or 5e6 crosslinked HeLa cell chromatin followed by DNA purification. Libraries were prepared from Input and ChIP DNA samples using standard protocols with Illumina barcoded adapters, and analyzed on an Illumina HiSeq™ instrument. An excess of twelve million reads from FastQ files were mapped using Bowtie (http://bowtiebio.sourceforge.net/manual.shtml) following TagDust (http://genome.gsc.riken.jp/osc/english/dataresource/) tag removal. Peaks were identified using MACS (http://luelab.dfci.harvard.edu/MACS/), with peaks and reads visualized as a custom track in UCSC Genome Browser (http://genome.ucsc.edu) from BigWig and BED files. The highest 25% of peaks identified in the data showed 90% -99% overlap with peaks identified in the ENCODE H3K4me2 or H3K4me3 or H3K36me3 BROAD Histone tracks for HeLa S3. Data in the region of the transcriptionally active housekeeping gene TUBA1A (Tubulin, alpha 1A),  HOXB, ACTB or GAPDH is shown.

Fig 4. Effective ChIP and Reliable Next Gen Sequencing Library Construction from Limited Amounts of DNA

Next generation sequencing libraries were constructed from Sp1 ChIP DNA using either 10 ng (upper panel) or 1 ng of quantitated immunoprecipitated DNA (middle panel). A reference library was created using 10 ng of purified DNA from the input chromatin. Samples were prepared using the Magna ChIP-Seq Kit (Cat. #: 17-1010) and a ChIPab+ Sp1 antibody/primer set (Cat. #: 17-601). The resulting libraries were sequenced on an Illumina Genome Analyzer then aligned and mapped to the human hg18 reference genome. Shown above is the resulting peak analysis (derived using QuEST) of the Sp1 locus from confidently mapped reads browsed with DNAnexus™ software. Replicate sequencing runs from 10 ng and 1 ng libraries are compared, demonstrating overlapping peak identification at the promoter of the Sp1 gene. Triangles reflect high probability Sp1 binding events associated primarily near transcriptional start sites in this genomic interval.

ChIP Experiment Guides, FAQ, Troubleshooting Tips and Supplementary Protocols

ChIP can be challenging even for the most experienced researcher. It is a multi-step technique (Fig 1) that requires high quality chromatin, robust antibodies, optimized reagents, and protocols to produce reliable and reproducible results. These are typically achieved by either using commercially prepared products and protocols, or undertaking multiple validation experiments. Regardless of which strategy you prefer, below are a comprehensive ChIP experiment guides for you to tackle some of the problems commonly encountered in ChIP, from sample preparation to downstream DNA analysis.

Which ChIP kit is right for you?

Standard ChIP kits
 

Cat. #/Description Assay Time (day) Bead type (Protein A or G) Agarose or Magnetic Inhibitor Cocktail/ Proteinase K Spin Columns Control IgG/qPCR Primers
17-10460
Magna ChIP™ HiSens Chromatin Immunoprecipitation Kit
1 A+G Magnetic    
Highlight:
1) Robust ChIP from as few as 10,000 to as many as 1,000,000 cells; 2) Single buffer system (SCW buffer) for multiiple steps of sonication, chromatin IP, and wash; 3) The ChIP elution buffer formulated to allow analysis of enrichment by qPCR without additional clean-up steps for more rapid results; 4) proven for both qPCR and ChIP-seq analysis (Figs 5 and 6)
17-10461
Magna ChIP™ A/G Chromatin Immunoprecipitation Kit
1 A+G Magnetic  
Highlight:
1) Robust ChIP from as few as 10,000 to as many as 1,000,000 cells; 2) Single buffer system (SCW buffer) for multiiple steps of sonication, chromatin IP, and wash; 3) The ChIP elution buffer formulated to allow analysis of enrichment by qPCR without additional clean-up steps for more rapid results; 4) proven for both qPCR and ChIP-seq analysis (Figs 5 and 6)
17-10085
Magna ChIP™ A/G Chromatin Immunoprecipitation Kit
1 A+G Magnetic  
Highlight:
No. of citation: 92
17-10086
EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit
1 A+G Magnetic
Highlight: No. of citation: 86
17-610
Magna ChIP™ A Chromatin Immunoprecipitation Kit
1 A Magnetic  
             
17-408
EZ-Magna ChIP™ A Chromatin Immunoprecipitation Kit
1 A Magnetic
             
17-611
Magna ChIP™ G Chromatin Immunoprecipitation Kit
1 G Magnetic  
             
17-409
EZ-Magna ChIP™ G Chromatin Immunoprecipitation Kit
1 G Magnetic
Highlight: No. of citation: 41
CHP1
Imprint® Chromatin Immunoprecipitation Kit
1 G Agarose
Highlight: 1) No. of citation: 33; 2) Shortest procedure time
17-20000
Magna ChIP™ G Tissue Kit
2 G Magnetic
Highlight: 1) Suitable for ChIP assays from tissue samples and proven performance in heart, lung, cortex and kidney; 2) Microdissected functionally related populations of cells within a heterogeneous tissue can thus be analyzed with ease, precision and certainty; 3) No. of citation :32
17-295
Chromatin Immunoprecipitation (ChIP) Assay Kit
2 A Agarose      
Highlight: 1) No. of citation: 137; 2) Containing Protein A Agarose/Salmon Sperm DNA (Cat. #: 16-157) to block un-speicific DNA binding sites on agarose beads
17-371
EZ-ChIP™
2 G Agarose
Highlight: No. of citation: 1,757
CHP2NC
Imprint® Ultra Chromatin Immunoprecipitation Kit, Without Controls
2 A StaphA
Highlight: Suitable for low abundance, medium, and highly expressed transcription factors, as well as histone modifications. Robust ChIP for a wide range of cell numbers ranging from 2 to 10 million.

Speciality ChIP kits
 

Cat. #/Description No. of Assays Assay Time (day) Bead type (Protein A or G) Agarose or Magnetic Buffers Inhibitor Cocktail/ Proteinase K Spin Columns Control IgG/qPCR Primers
17-10077
Magna ChIP™ HT96 Chromatin Immunoprecipitation Kit
96 1 A+G Magnetic    
17-10078
EZ-Magna ChIP™ HT96 Chromatin Immunoprecipitation Kit
96 1 A+G Magnetic  
17-245
Acetyl-Histone H3 Immunoprecipitation (ChIP) Assay Kit
22 2 A Agarose      
17-229
Acetyl-Histone H4 Immunoprecipitation (ChIP) Assay Kit
22 2 A Agarose      
17-1010
Magna ChIP-Seq™ Chromatin Immunoprecipitation and Next Generation Sequencing Library Preparation Kit
10 2 A+G Magnetic
17-1000
Magna ChIP2 ™ Universal Chromatin Immunoprecipitation DNA Microarray Kit
2 2 A+G Magnetic  
17-1004
Magna ChIP2 ™ Universal Chromatin Immunoprecipitation DNA Microarray Quad Kit
12 2 A+G Magnetic  
17-1002
Magna ChIP2 ™ Chromatin Immunoprecipitation Mouse Promoter 244K Microarray Kit
3 (6 slides) 2 A+G Magnetic