Chromatin Immunoprecipitation (ChIP) Service
- Service Details
- Q&A
What is Chromatin Immunoprecipitation?
Chromatin Immunoprecipitation (ChIP) is a pivotal technique for exploring how DNA and proteins interact within cells, and it involves two main methods: ChIP-qPCR and ChIP-seq. ChIP-qPCR is employed to confirm known DNA fragments binding to a specific protein, while ChIP-seq is used to discover unknown DNA fragments associated with the target protein.
The process begins by cross-linking cellular "protein-DNA" complexes with formaldehyde, followed by fragmenting DNA into appropriate lengths using sonication. After cell lysis, beads coupled with antibodies are used to capture the protein of interest, along with the DNA it interacts with, forming a complex that precipitates onto the beads. After removing unbound DNA through washing, the DNA-protein complexes are eluted from the beads, resulting in the ChIP product. After decrosslinking, the obtained DNA-protein complexes can be analyzed using qPCR for known DNA fragments or Next-Generation Sequencing (NGS) for unknown DNA fragments interacting with the protein.
In cases where a suitable antibody for the target protein is unavailable, a fusion protein with a flag tag can be expressed as a bait. This involves overexpressing Flag-Bait in cells, incubating the lysate with anti-Flag beads, allowing the bait fusion protein to bind to the beads, and co-precipitating DNA fragments interacting with the bait fusion protein. Subsequent steps include washing, elution, and either qPCR or NGS sequencing.
Creative Proteomics offers ChIP services for studying protein-dna interactions.
Chromatin Immunoprecipitation Services by Creative Proteomics
| Services | Service Offerings | Result Delivery | Experiment Duration | Customer to Provide |
| ChIP qPCR | Western blot detection of the bait protein in samples (input). | Western blot detection images. | 4-5 weeks | Protein information (species, sequence), protein antibodies, cell samples, information on the target gene (species, sequence). |
| ChIP retrieval of the bait protein and its bound DNA fragments (2 groups: experimental and control). | Western blot detection images for the bait protein. qPCR detection data for the target DNA sequence. ChIP experiment report. |
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| ChIP seq | Western blot detection of the bait protein in samples (input). | Western blot detection images. | 4-5 weeks | Protein information (species, sequence), protein antibodies, cell samples. |
| ChIP retrieval of the bait protein and its bound DNA fragments (2 groups: experimental and control). | Western blot detection images for the bait protein. ChIP experiment report. |
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| Sequencing (Seq) analysis of DNA fragments from ChIP products and subsequent analysis (2 groups: input and experimental). | Sequencing detection results. Detection and analysis report. |
9 weeks |
Advantages of Chromatin Immunoprecipitation
Targeted Precision: ChIP's ability to selectively enrich specific genomic regions ensures precision in molecular analysis. Researchers can focus on defined loci, minimizing noise and enhancing the accuracy of results.
Quantitative Profiling: ChIP offers quantitative insights into molecular interactions by measuring the abundance of proteins or modifications at targeted genomic sites. This quantitative profiling adds granularity to our understanding of molecular events.
Versatility for Genome-Wide Investigations: ChIP's adaptability to genome-wide studies, particularly when coupled with next-generation sequencing, facilitates comprehensive analyses. This versatility is instrumental in uncovering global regulatory networks and understanding the intricate cross-talk among genomic elements.
Translational Impact in Drug Discovery: ChIP-derived insights have direct translational implications in drug discovery. By identifying key regulatory elements associated with diseases, ChIP guides the identification of potential therapeutic targets, opening avenues for targeted drug development.
Applications of Chromatin Immunoprecipitation Analysis
Deciphering Gene Regulatory Networks: ChIP enables the precise identification of transcription factor binding sites on genomic DNA. This application is instrumental in unraveling gene regulatory networks, providing insights into cellular responses to stimuli, developmental processes, and disease mechanisms.
Mapping Epigenetic Landscapes: ChIP allows for the high-resolution mapping of histone modifications and DNA methylation patterns. This capability provides a detailed view of the epigenetic landscape, facilitating the understanding of cellular identity and behavior.
Validation of Bioinformatic Predictions: ChIP serves as a robust experimental validation tool for bioinformatic predictions of regulatory elements. This application ensures the accuracy and reliability of computational analyses by confirming the physical presence of predicted elements.
Probing Three-Dimensional Chromatin Architecture: By combining ChIP with three-dimensional chromatin conformation capture techniques, researchers can investigate the spatial organization of chromatin. This approach is crucial for understanding the impact of spatial chromatin interactions on gene expression and regulatory mechanisms.
Issue 1: High Background with Non-Specific Antibody Control
1. Possible Cause: Non-specific binding with A or G proteins.
Solution: Implement pre-clearing treatment by mixing lysed samples with beads for 1 hour before antibody addition, followed by separation and use.
2. Possible Cause: Contaminated buffers used in Chromatin Immunoprecipitation.
Solution: Freshly prepare lysis and washing buffers to prevent contamination.
3. Possible Cause: Some A or G protein beads may inherently produce high background.
Solution: Choose a supplier offering products with the lowest background in non-specific control samples.
Issue 2: Large Binding Regions Resulting in High Background and Low Resolution
- Possible Cause: Overly large DNA fragments.
Solution: Optimize DNA fragment size based on cell line, adjusting sonication and enzyme digestion times. Recommend keeping DNA fragments below 1.5 kbp.
Issue 3: Low Signal
1. Possible Cause: Small Chromatin fragments.
Solution: During sonication, ensure chromatin fragments are no smaller than 500 bp to prevent nucleosome detachment. For end-point Chromatin Immunoprecipitation, enzymatic digestion typically yields the desired fragment size of 175 bp.
2. Possible Cause: Prolonged cell cross-linking during Chromatin Immunoprecipitation.
Solution: Cross-link with formaldehyde for 10-15 minutes, wash with PBS, and add glycine to terminate the formaldehyde action. Excessive cross-linking may reduce binding site accessibility.
3. Possible Cause: Insufficient starting material.
Solution: Suggest using 25 μg of chromatin for each immunoprecipitation reaction.
4. Possible Cause: Insufficient antibody in immunoprecipitation.
Solution: Recommend starting with 3-5 μg of antibody. Increase to 10 μg if no signal is detected.
5. Possible Cause: Hindered binding of specific antibodies.
Solution: Sodium chloride concentration in wash buffer should not exceed 500 mM to avoid hindering specific antibody binding.
6. Possible Cause: Incomplete cell lysis.
Solution: Recommend using RIPA buffer for cell lysis.
7. Possible Cause: Not enough antibody concentrated in the target area.
Solution: Use a positive control antibody to confirm proper operation. For example, use H3K4me3 for activated promoters or H3K9me3 antibody for heterochromatin loci.
8. Possible Cause: Some monoclonal antibodies are not suitable for cross-linking Chromatin Immunoprecipitation.
Solution: Monoclonal antibodies may have masked epitopes during cross-linking. Suggest using polyclonal antibodies that recognize multiple epitopes to increase the chance of immunoprecipitating the target protein.
9. Possible Cause: Incorrect antibody affinity beads used.
Solution: A and G proteins have different affinities for immunoglobulins. Use specific affinity matrices. Suggest using a mixed A and G protein agarose.
Issue 4: PCR Amplification Problems During Immunoprecipitation DNA
1. Possible Cause: Contaminated reagents in real-time PCR.
Solution: Freshly prepare solutions using reserved liquid.
2. Possible Cause: No DNA amplification in samples.
Solution: Confirm primer efficiency in samples, including standard controls/sample DNA.
