Co-Immunoprecipitation (CoIP) Workflow Guide: Step-by-Step Protocol Empowered by ANT BIO PTE. LTD. Products
1. Introduction to Co-Immunoprecipitation (CoIP) Workflow
Co-Immunoprecipitation (CoIP) is a classic technique based on the specific affinity between antibodies and proteins. It captures antibodies that specifically bind to target proteins or antigens, thereby enriching target proteins from complex samples and enabling the detection of interacting proteins or other biomacromolecules. To obtain optimal experimental results, all steps must be optimized according to actual conditions.
For example, depending on the cell line or the subsequent application of the captured antigen, more suitable cell lysis conditions can be selected. For mammalian cells without a cell wall structure, detergents can be directly used for lysis; other cell types require auxiliary mechanical shear force, such as ultrasonic disruption. In addition, before performing CoIP, the cell lysate can be detected by Western Blot (WB) to confirm the presence of the target protein in the cell lysate.
The entire CoIP workflow is divided into three core stages: Preparation of Cell Lysate, Immunoprecipitation, and Analysis by Western Blot. ANT BIO PTE. LTD. provides a complete set of high-quality reagents for each stage, ensuring the smooth progress of the experiment and the reliability of results.
2.1 Stage A: Preparation of Cell Lysate
1. Aspirate the medium. Add fresh medium containing regulatory molecules to treat the cells for a predetermined time.
2. Harvest the cells, remove the medium, and wash the cells once with ice-cold 1X PBS (Cat. No.: abs961, ANT BIO PTE. LTD.).
3. Remove the PBS and add 0.5 ml of ice-cold Lysis Buffer (Cat. No.: abs9239, ANT BIO PTE. LTD.) to each cell plate (10 cm), then incubate on ice for at least 5 minutes.
Note: Immediately add 1 mM PMSF (Cat. No.: abs9146, ANT BIO PTE. LTD.) to the Lysis Buffer before use.
4. Scrape the cells from the plate, transfer the extract to a microcentrifuge tube, and place on ice.
5. Perform ultrasonic disruption 3 times on ice, 5 seconds each time.
6. Centrifuge at 14,000 x g for 10 minutes at 4°C, transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, the lysate can be stored at -80°C.
Note: After preparing the cell lysate, Western Blot detection can be performed first to confirm the presence of the target protein in the cell lysate (recommended reagent: WB Solution Base Kit, Cat. No.: abs928, ANT BIO PTE. LTD.).
2.2 Stage B: Immunoprecipitation
Pre-clearing of Cell Lysate (Optional Step)
1. Add 5 μl Protein A and 5 μl Protein G (components of Cat. No.: abs955, ANT BIO PTE. LTD.) to 500 μl of cell lysate (containing 200-1000 μg total protein).
2. Incubate with rotation at 4°C for 30-60 minutes.
3. Centrifuge at 12,000 x g for 1 minute at 4°C, retain the supernatant for later use.
1. Add 500 μl of cell lysate (which can be the pre-cleared cell lysate) containing 200-1000 μg total protein into a new centrifuge tube.
2. Add 1-5 μg of monoclonal/polyclonal antibody against the target protein (IP primary antibody).
3. Simultaneously use normal IgG, a homologous antibody for non-specific immunity, as a control (Cat. No.: abs20038, ANT BIO PTE. LTD.).
4. Incubate with gentle mixing overnight at 4°C.
1. After overnight antibody incubation, add 5 μl Protein A and 5 μl Protein G (components of Cat. No.: abs955, ANT BIO PTE. LTD.).
2. Incubate with gentle mixing at 4°C for 1-3 hours or overnight.
3. Centrifuge at 12,000 x g for 1 minute, retain the precipitate.
4. Wash the precipitate with 0.5 ml of 1× Wash Buffer (component of Cat. No.: abs955, ANT BIO PTE. LTD.), centrifuge at 12,000 x g for 1 minute, and retain the precipitate.
5. Repeat step 4 three times in total.
Note: When washing and removing the supernatant, be careful to avoid aspirating the filler.
2.3 Stage C: Analysis by Western Blot
1. Resuspend the precipitate in 20-40 μl of 1× SDS Sample Buffer (Cat. No.: abs953, ANT BIO PTE. LTD.). Vortex and then microcentrifuge for 30 seconds.
2. Heat the sample to 95–100°C for 2-5 minutes, then centrifuge briefly at 14,000 x g for 1 minute.
3. Load 15-30 μl of the sample onto an SDS-PAGE gel (recommended reagent: Precast Gel, Cat. No.: abs9313, ANT BIO PTE. LTD.).
4. Analyze the sample by Western Blot (primary antibody and secondary antibody for WB; recommended reagent: WB Solution Base Kit, Cat. No.: abs928, ANT BIO PTE. LTD.).
Note: For proteins with a molecular weight close to 50 kDa, we recommend using a secondary antibody that specifically recognizes the light chain to minimize the masking effect caused by denatured heavy chains; for proteins with a molecular weight close to 25 kDa, it is recommended to use a secondary antibody that specifically recognizes the heavy chain.
3. Product Empowerment by ANT BIO PTE. LTD.
The smooth progress of CoIP experiments relies heavily on high-quality reagents. ANT BIO PTE. LTD. provides a complete set of supporting reagents for each link of the CoIP workflow, forming a one-stop solution to meet the full needs of researchers:
• Core CoIP Kit: abs955 Immunoprecipitation/Co-Immunoprecipitation (IP/CoIP) Kit, which contains high-quality Protein A, Protein G and Wash Buffer, ensuring efficient capture of immune complexes and low non-specific binding.
• Sample Preparation Reagents: abs961 (10× PBS Buffer) for cell washing, abs9239 (Lysis Buffer without Protease/Phosphatase Inhibitors) for cell lysis, and abs9146 (PMSF) as a protease inhibitor to prevent protein degradation.
• WB Detection Reagents: abs928 (WB Solution Base Kit) for subsequent Western Blot analysis, abs953 (1× SDS Sample Buffer) for sample processing, and abs9313 (Precast Gel) for electrophoresis, ensuring accurate detection of target proteins.
• Control Reagent: abs20038 (Mouse IgG Antibody) as a negative control in CoIP experiments, helping to exclude non-specific signals and ensure the reliability of experimental results.
All reagents of ANT BIO PTE. LTD. have undergone strict quality control and verification, with stable performance and reliable results. They are widely used in various CoIP-related research fields, providing strong technical support for researchers to explore protein-protein interactions.
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 importance of standardized experimental processes and high-quality reagents for CoIP experiments. Through continuous product innovation and optimization, we strive to simplify experimental operations, improve experimental success rates, and help researchers save time and energy.
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 aim 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.
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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.
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