ChIP Experiment Protocol & Troubleshooting Guide: Empowered by ANT BIO PTE. LTD. Products

1. Introduction to Chromatin Immunoprecipitation (ChIP)

Chromatin Immunoprecipitation (ChIP) is a powerful technique used to study the interaction between proteins and DNA in living cells. Its core principle is to fix protein-DNA complexes in their native state, fragment the chromatin into small segments of a specific length range, precipitate the target complexes using immunological methods to specifically enrich DNA fragments bound to the target protein, and then purify and detect these fragments to obtain information about protein-DNA interactions. This technique plays an indispensable role in exploring gene regulation mechanisms, epigenetic modifications, and other fields of life science research.

2. Step-by-Step ChIP Experimental Protocol

2.1 Cell Cross-Linking and Lysis

Cultivate adherent cells in a 150mm petri dish containing 20ml growth medium until reaching 80%-90% confluency, approximately 10^7 cells. Stimulation or treatment can be performed if necessary. It is recommended to use 1×10^6 cells for one ChIP experiment.

1.       Add formaldehyde to the 20ml medium to a final concentration of 1%, gently vortex the petri dish to mix well, and fix the cells in formaldehyde at room temperature for 10 minutes. For transcription factors and co-factors, the cross-linking time can be appropriately extended but should not exceed 30 minutes. The color of the medium will change after adding formaldehyde.

2.       Add glycine with a concentration of 0.125M to the petri dish, gently vortex to mix well, and react at room temperature for 5 minutes to quench unreacted formaldehyde and terminate cross-linking. The color of the medium will change again after adding glycine.

3.       Discard the medium, wash the cells with 20ml pre-cooled 1× PBS (Cat. No.: abs961, ANT BIO PTE. LTD.), and repeat the washing once.

4.       Add 2ml pre-cooled 1× PBS containing protease inhibitor cocktail to the petri dish; collect the cells using a cell scraper, centrifuge at 800g for 5 minutes at 4°C.

5.       Remove the supernatant (at this step, the cells can be quickly cooled in liquid nitrogen and stored at -80°C for several months). Resuspend the cells in 0.5ml cell lysis buffer containing protease inhibitor cocktail again, incubate on ice for 15 minutes, and vortex every 5 minutes.

6.       Centrifuge at 800g for 5 minutes at 4°C. Carefully remove the supernatant, add 0.5ml nuclear lysis buffer containing protease inhibitor cocktail, and resuspend the pellet.

2.2 Sonication for DNA Fragmentation

The effect of sonication depends on cell type, cell concentration, and the instrument. Pre-experiments are required to determine the optimal sonication conditions to shear cross-linked DNA into fragments of approximately 200-1000bp in length. Chromatin fragmentation is crucial for the success of ChIP experiments. Insufficient fragmentation will lead to sample loss, while excessive fragmentation will damage the epitope of the target protein and reduce ChIP efficiency. During sonication, keep the sample at a low temperature on ice at all times. Do not let the probe touch the bottom or wall of the sonication tube. If foam is generated during sonication, pause sonication and adjust the position of the sonication tube.

After sonication, centrifuge at 12,000g for 10 minutes at 4°C. After centrifugation, take 5μl of chromatin solution for agarose gel analysis to detect sonication efficiency. The 5μl chromatin solution before and after sonication can be taken for comparative analysis.

2.3 Immunoprecipitation of Target Protein and DNA

Store the following buffers on ice: low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, and TE wash buffer. Prepare 450μl dilution buffer (containing protease inhibitor cocktail) and store on ice. For multiple samples, preparation can be combined.

1.       Take 50μl of the supernatant from the centrifuged lysate into a new centrifuge tube as the DNA mixture sample for one immunoprecipitation experiment. Each 50μl lysate contains the lysate product of 1×10^6 cells. The ChIP reaction includes a positive control antibody tube, a negative control IgG tube, and a target protein antibody tube. It is recommended that the negative control IgG and the target antibody are from the same species.

2.       Add 450μl of the prepared dilution buffer to the centrifuge tube containing 50μl of fragmented chromatin sample and mix thoroughly. Take 5μl of sample from each tube into a new centrifuge tube as the 1% "input" for the experiment, which is used for experiment optimization and data processing. Store at 4°C until the protein-DNA complex elution and cross-linking reversal steps.

3.       Add 1-10μg of immunoprecipitation antibody to each centrifuge tube after taking the input, and incubate on a rotor at 4°C for more than 4 hours or overnight.

4.       Add 20μl of Protein A/G magnetic beads to each centrifuge tube (it is recommended to cut off a small part of the front end of the pipette tip before aspirating the magnetic beads), and incubate on a rotor at 4°C for 2 hours.

5.       Adsorb on a magnetic stand, let stand for 1-2 minutes, and remove the supernatant. Wash the Protein A/G magnetic bead-antibody-protein/DNA complex according to the following steps:
        Note: Mix equal volumes of Solution A and Solution B before use; do not mix in advance, otherwise precipitation will occur.

￮       Wash once with low-salt wash buffer by incubating on a rotor at 4°C for 3-5 minutes;

￮       Wash once with high-salt wash buffer by incubating on a rotor at 4°C for 3-5 minutes;

￮       Wash once with LiCl wash buffer by incubating on a rotor at 4°C for 3-5 minutes;

￮       Wash once with TE buffer by incubating on a rotor at 4°C for 3-5 minutes.

2.4 Elution and Cross-Linking Reversal

Preparation before the experiment: Thaw Proteinase K; allow ChIP elution buffer to return to room temperature to ensure SDS dissolution; prepare elution buffer: 100μl ChIP elution buffer + 1μl Proteinase K (preparation can be combined for multiple samples).

1.       Add ChIP elution buffer (containing Proteinase K) to the sample tubes and input tubes;

2.       Incubate at 62°C for 2 hours;

3.       Incubate at 95°C for 10 minutes;

4.       Allow the sample to cool to room temperature;

5.       Centrifuge at 10,000g for 10 seconds to collect the residual sample on the tube cap and tube wall, and separate the magnetic beads using a magnetic stand. Carefully transfer the supernatant to a new test tube.

2.5 DNA Purification

Purify DNA (immunoprecipitated and input) using purification columns.

2.6 Identification and Analysis

Detect the IP-obtained DNA by QPCR or high-throughput sequencing.

3. Common Problems and Troubleshooting

4. Product Empowerment by ANT BIO PTE. LTD.

The smooth progress of ChIP experiments relies heavily on high-quality and matched experimental reagents. ANT BIO PTE. LTD. provides a complete set of supporting products for each key link of the ChIP experiment, forming a one-stop solution to meet the full needs of researchers:

Among them, the abs50034 Chromatin Immunoprecipitation (ChIP) Kit is a star product of ANT BIO PTE. LTD. It has undergone strict quality verification and optimization, with a scientific and reasonable reagent ratio and a detailed experimental guide. It can effectively ensure the efficiency and reproducibility of ChIP experiments, and is suitable for various cell types and downstream detection methods such as QPCR and high-throughput sequencing. It provides strong technical support for researchers in gene regulation and epigenetic research.

5. Brand Mission

ANT BIO PTE. LTD. is committed to advancing life science research by providing high-quality, reliable reagents and comprehensive technical solutions. We deeply understand the difficulties and challenges researchers face in ChIP experiments and have been dedicated to developing products that simplify experimental processes and improve success rates. The complete set of ChIP-related products is a concrete manifestation of this commitment.

Guided by the principles of innovation, quality, and customer-centricity, our three specialized sub-brands (Absin, Starter, and UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. We strive to establish long-term and trusted partnerships with researchers worldwide, supporting them in achieving breakthroughs in life science research and contributing to the improvement of human health.

6. Disclaimer

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7. Brand Promotion Copy

ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs

At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.