Co-Immunoprecipitation (Co-IP) Service

Background

What is Co-Immunoprecipitation (Co-IP)?

Co-Immunoprecipitation, or Co-IP, is a well-established technique that relies on antibody-antigen interactions to isolate and enrich target proteins and their interaction buddies. Basically, it uses specific antibodies to snag the target protein along with any partners from mixtures like cell or tissue extracts. The steps include breaking open the cells to release proteins, using antibodies to grab onto the target protein and form immune complexes, and then sticking these complexes to affinity matrices for enrichment. After that, a thorough washing is done to get rid of any proteins that aren’t specifically bound, and then the proteins are analyzed to figure out their interactions.

Fig. 1. Illustration of coimmunoprecipitation method for identification of protein-protein interactions. (Bao, et al., 2020)

The progress in Co-IP allows scientists to really get into the nitty-gritty of protein relationships, shedding light on cell signaling, disease processes, and how drugs do their thing. As this method keeps getting better, it’s making its way into more research areas. For instance, when paired with mass spectrometry-driven high-throughput techniques, it really speeds up identifying and measuring protein-protein interactions.

Precautions of Co-IP

• Specific IP-level decoy protein antibodies are required (emphasis);
• If there is no specific antibody, it is necessary to construct a recombinant labeled plasmid and conduct Co-IP experiment with labeled antibody;
• Sample type experimental risk: Overexpressed cell samples are the smallest, followed by cell samples, then animal tissue samples, and finally plant samples (usually transgenic plants, it is difficult to achieve specific enrichment of labeled antibodies);
• Co-IP-MS risk: Due to the presence of specific antibody high abundance proteins, the decoy protein may not be identified by mass spectrometry.

Applications of Co-IP

• Uncovering Protein Interaction Networks

- Identifies both direct and indirect links between proteins in intricate biological settings.
- Aids in creating maps that reveal the workings of signaling pathways and cell processes.

• Verification of Protein Interactions

- Confirms potential interactions found through other techniques like yeast two-hybrid (Y2H) or computational predictions.
- Strengthens the reliability of interaction studies.

• Pathway Analysis in Signal Transduction

- Explores dynamic protein interactions within signaling pathways.
- Supports studies on condition-specific or time-dependent interactions.

• Discovery of Interaction Partners

- Unveils new proteins interacting with a target of interest.
- Contributes to understanding the functional roles of these binding partners.

• Post-Translational Modification Studies

- Examines how modifications like phosphorylation or ubiquitination influence protein binding.
- Provides insights into regulatory roles within protein complexes.

• Disease Mechanism Investigation

- Focuses on complexes linked to specific diseases, such as cancer or neurological disorders.
- Aids in identifying potential diagnostic markers or drug targets.

• Characterization of Multi-Protein Assemblies

- Studies the composition, stoichiometry, and functional organization of large protein complexes.
- Helps in understanding their roles in biological processes.

• Chromatin Interaction Analysis

- Looks into proteins related to chromatin remodeling, histone modification, and transcription regulation.
- Pushes forward studies on gene expression and epigenetic control.

• Immune Complex Dynamics

- Analyzes protein complexes related to immune responses, including antigen-antibody interactions.
- Useful for understanding immune regulation and pathology.

• Therapeutic Development Support

- Screens for drug effects on protein-protein interactions.
- Evaluates how candidate molecules influence complex stability or formation.

Service Procedure

Workflow

Creative BioMart 's co-IP platform will assist you to identify new binding partners and possible new signaling pathways of your target protein. With proper design of the study, it can also provide information regarding the presence, abundance, up regulation or down regulation, even post-translational modifications of your target protein, thereby elucidating signaling pathways. See the procedure of co-IP service and how you should submit your sample. Our experts shall design your co-IP experiment according to your specific requirements and we will provide detail data of the result and procedure.

Service Contents

Protein-Protein Interaction Analysis Service

Our Co-IP services provide highly sensitive assings in the field of protein-protein Interaction Analysis that accurately reveal direct and indirect Protein interactions and provide quantitative and qualitative insights into the formation of Protein complexes. Our technologies include stabilizing interaction complexes with crosslinkers, detecting immunoprecipitation of multiprotein complexes by flow cytometry, and accurately mapping cross-linking sites between proteins using techniques such as mass spectrometry to provide customers with comprehensive protein interaction analyses that advance biological and medical research.

Antibody Screening and Validation Service

Our Co-IP service focuses on top-notch validation for screening antibodies, making sure they effectively capture target protein complexes during high-efficiency immunoprecipitation tests. We focus on ensuring antibodies are both affinity and specific, supported by detailed checks on several platforms. Plus, we provide knowledgeable advice on picking the right antibodies for new targets, handling tasks from prepping antigens to making antibodies. This all-around support means the antibodies fit the particular needs of your experiments, boosting the chances of success and the trustworthiness of the results.

Post-Immunoprecipitation Analysis Service

Creative BioMart provides a full suite of post-immunoprecipitation analysis services. This includes a variety of downstream options like Western blot, ELISA, and proteomics. We also offer bioinformatics assistance like GO Analysis, KEGG Pathway Analysis and STRING Network Analysis to help visualize and make sense of protein interaction networks. Using LC-MS/MS technology, we can identify co-precipitated proteins in detail, while our bioinformatics tools help map protein partners accurately within complex proteomes. Our integrated services support clients throughout the entire process, from conducting experiments to analyzing data, ensuring research results are both accurate and dependable. For more protein analysis services, please click Protein Analytical Service or contact us for details.

Complex Co-IP Workflow Customization Service

Creative BioMart tailors Co-IP workflows to meet the unique needs of various cell types, tissues, and experimental conditions. Our expert team applies their in-depth knowledge to optimize buffer compositions, enhancing protein interaction stability and ensuring effective immunoprecipitation in diverse experimental settings. By customizing protocols and precisely adjusting buffer formulations, we aim to deliver the most optimized Co-IP solutions for our clients, boosting both the success rate of experiments and the reliability of their results.

Why Choose Us

Core Technologies and Equipment

• High-affinity magnetic and agarose beads for immunoprecipitation.
• Validated antibody panels for common and custom targets.
• LC-MS/MS for protein identification and interaction validation.
• Advanced bioinformatics tools for data analysis and visualization.

Our Advantages

• High specificity and sensitivity for interaction detection.
• Custom protocols tailored to challenging protein complexes.
• State-of-the-art equipment for precise and reproducible results.

Case Study

Background

BackgroundCo-Immunoprecipitation follow-Mass Spectrometry (CO-IP-MS) is a commonly used method for studying protein-protein interactions. The project uses CO-IP-MS to pinpoint proteins that interact with specific proteins in specific cell lines. The complexes formed by the target proteins were isolated by antibodies, and then these binding proteins were further verified by Western blotting or mass spectrometry.

Method

Experimental procedures include sample preparation, preparation of target cell lysate, immunoprecipitation assay using specific antibodies, Western Blot assay, and mass spectrometry identification. A variety of bioinformatics analyses are also included.

Results

Western Blot : Confirmed the enrichment of target protein complexes using specific antibodies.

Fig. 2. Results of western blot detection of IP enrichment.

Mass Spectrometry : Identified ion peaks corresponding to the proteins interacting with the target protein.

Fig. 3. Mass spectrometry identification of IP-enriched target protein.

GO Analysis : Provided insights into the biological functions of the identified proteins.

Fig. 4. Results of GO_BP enrichment analysis.

KEGG Pathway Analysis : Mapped the proteins to key signaling and metabolic pathways.

Fig. 5. KEGG Enrichment Analysis.

STRING Network : Visualized the interaction network of the enriched proteins, offering a deeper understanding of their biological roles.

Fig. 6. Diagram of STRING protein network.

FAQs

• Q: How accurate is the Co-IP service?

  A: Our Co-IP service leverages sensitive mass spectrometry to precisely identify both direct and indirect protein interactions. We employ advanced LC-MS/MS technology, ensuring high-resolution and accurate protein identification. Plus, our lab follows strict protocols and quality control standards to ensure reliable results.

• Q: Is the service customizable for various types of cells and tissue samples?

  Yes, our Co-IP service is tailored for different cell types, tissues, and experimental setups. Our expert team specializes in tweaking buffer compositions to suit unique sample characteristics, enhancing the stability of protein interactions.

• Q: How do you analyze data after mass spectrometry?

  We deliver a detailed report that includes protein IDs, coverage levels, peptide counts, unique peptides, and any possible post-translational modifications.

References:

• Bao X. (2020). Introduction to Protein Posttranslational Modifications (PTMs). In: Study on the Cellular Regulation and Function of Lysine Malonylation, Glutarylation and Crotonylation. Springer Theses. Springer, Singapore. https://doi.org/10.1007/978-981-15-2509-4_1