Deep Dive into Co-Immunoprecipitation (Co-IP): Technology, Experiments and ANT BIO PTE. LTD. abs955 Kit
1. Introduction to Co-Immunoprecipitation (Co-IP) Experiment
Co-Immunoprecipitation (Co-IP) is a classic method for studying protein-protein interactions based on the specific binding between antibodies and antigens. It is an effective technique to determine the physiological interactions between two proteins in intact cells. If an antibody against protein X is used to immunoprecipitate X, protein Y that binds to X in vivo can also be precipitated. The difference between Co-IP and IP experiments lies only in whether the detection focus is on the primary target molecule (antigen) or the secondary target molecule (interacting protein).
1. Protein extraction.
2. Add the antibody against protein X to the cell lysate.
3. After incubation, add Protein A or G (immobilized on media such as Agarose or magnetic beads). If there is a target protein bound to the protein of interest in the cell, a complex "Y—X—anti-X antibody—Protein A or G" can be formed.
4. The complex is separated by denaturation and polyacrylamide gel electrophoresis (SDS-PAGE) (including X and Y antigens, and X antibody).
5. Analysis by Western Blot and mass spectrometry.
1. All interacting proteins are post-translationally modified and in their native state.
2. Protein interactions occur under natural conditions, avoiding artificial interference.
3. Native-state interacting protein complexes can be isolated.
1. May fail to detect low-affinity and transient protein-protein interactions.
2. The binding between two proteins may not be direct; a third molecule may act as a bridge.
3. The target protein must be predicted before the experiment to select the antibody for final detection. If the prediction is incorrect, the experiment will yield no results.
4. Key Points of the Experiment
1. The most important consideration is the nature of the antibody. In particular, the specificity of polyclonal antibodies is a critical issue.
2. To prevent protein degradation and modification, protease inhibitors must be added to the buffer for dissolving antigens, and the experiment should be performed at low temperature.
3. The ratio of antibody to buffer should be considered. Insufficient antibody will fail to detect the antigen, while excessive antibody will not sediment on the beads and remain in the supernatant. Too little buffer will not dissolve the antigen, and too much will dilute the antigen.
4. It is necessary to confirm that the protein-protein interaction occurs in the cell, not after cell lysis. This requires determining the localization of the protein.
1. A mild lysis condition should be used for cell lysis to avoid disrupting all protein-protein interactions in the cell. Non-ionic denaturants (NP40 or Triton X-100) are mostly used. The lysis condition for each cell type is different and should be determined empirically.
2. High-concentration denaturants (0.2% SDS) should not be used. Various enzyme inhibitors must be added to the cell lysis buffer.
5.2 Selection of Antibodies in Immunoprecipitation
Use control antibodies (negative and positive controls):
• Negative controls: Monoclonal antibody: IgG of the same species; Rabbit polyclonal antibody: Normal rabbit IgG.
• Positive controls: Antibody against a certain protein in the cell (e.g., when studying membrane protein A, use antibody against membrane protein B).
5.3 Undetected or Sparse Target Protein
Potential causes and solutions: 1. Insufficient antibody dosage: Increase the amount of antibody appropriately based on the experimental system. 2. Weak binding affinity between antibody and antigen: Replace with a higher-specificity antibody. 3. Protein degradation during lysis: Strengthen the addition of protease inhibitors and ensure all operations are performed on ice. 4. Inappropriate lysis conditions: Optimize the type and concentration of denaturants in the lysis buffer.
5.4 High Background of Target Protein
Potential causes and solutions: 1. Non-specific binding of antibodies: Use appropriate control antibodies to exclude non-specific signals. 2. Insufficient washing steps: Increase the number of washing times or extend the washing time. 3. Contamination of beads: Use pre-washed Protein A/G beads to reduce background interference. 4. High protein concentration in the sample: Dilute the sample appropriately to reduce non-specific binding.
6. ANT BIO PTE. LTD. abs955 Immunoprecipitation/Co-Immunoprecipitation (IP/CoIP) Kit
The best-selling abs955 Immunoprecipitation/Co-Immunoprecipitation (IP/CoIP) Kit from ANT BIO PTE. LTD. is designed to support your IP/CoIP experiments with one-stop solutions. The kit selects high-quality Protein A/G agarose beads, featuring ultra-high cost-effectiveness, well-validated protocols, and supporting auxiliary reagents, providing reliable technical support for your protein interaction research.
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Cat. No. |
Product Name |
Key Features |
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Immunoprecipitation/Co-Immunoprecipitation (IP/CoIP) Kit |
High-quality Protein A/G agarose beads, cost-effective, well-validated protocol, one-stop auxiliary reagents |
|
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Immunoprecipitation/Co-Immunoprecipitation (IP/CoIP) Kit (Magnetic Bead-Based) |
Low non-specific binding, short experimental time, high efficiency, suitable for high-throughput experiments |
|
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abs9116 |
Lysis Buffer for Western Blot and Immunoprecipitation (WB/IP) |
Mild formula, maintains protein interactions, contains protease inhibitors |
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PMSF (Phenylmethylsulfonyl fluoride) |
Effective protease inhibitor, stable performance, suitable for various lysis systems |
|
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abs20035 |
Rabbit IgG Antibody |
High purity, suitable as negative control in IP/CoIP experiments |
|
abs20038 |
Mouse IgG Antibody |
High specificity, suitable as negative control in IP/CoIP experiments |
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 challenges researchers face in protein interaction studies and strive to develop products that meet diverse experimental needs. The abs955 IP/CoIP Kit, as a star product, has been widely recognized by researchers for its stable performance and high cost-effectiveness.
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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