Co-immunoprecipitation (Co-IP) Analysis Service

What is Co-IP?

The Co-IP technique is widely recognized as one of the most robust methods employed for investigating protein-protein interactions in vivo under non-denaturing conditions. These interacting proteins may be complex partners, structural proteins, co-factors, and signaling molecules. Co-IP as an enrichment method is also essential to identify low abundance target protein in a complex biological milieu. Following Co-IP, these protein complexes can be identified in various methods, such as Western Blot (WB) and LC-MS/MS, to identify partner proteins' ID, as well as exploration of undiscovered functions associated with bait protein. Additionally, quantitative proteomics such as SILAC, TMT/iTRAQ technologies can be equally applied in conjunction with Co-IP for precise quantification.

However, there poses a challenge due to the presence of highly abundant proteins and the vast array of proteins in cell lysates. Especially, this challenge arises from the inherent instability bindings, non-specific bindings, and potential antibodies contamination, which can potentially hinder the accurate detection of protein-protein interaction. Therefore, it is imperative to establish an appropriate negative control group in Co-IP experiments to minimize the risk of obtaining false positive results and ensuring the reliability of the obtained information on interacting protein.

Figure 1. Three common schemes of Co-IP.

There are primarily three approaches for establishing negative control group: 1) Overexpression of the target protein using recombinant plasmid versus empty plasmid; 2) Utilization of target protein antibody versus Isotype nonfunctional control antibody; 3) Comparison between normal expression of the target protein and knockout or knockdown models.

Table1 The advantages and disadvantages of each scheme.

Our Co-IP/MS Service

Classically, utilizes antibody-conjugated agarose or magnetic beads to specifically extract and purify a target protein, along with its associated protein partners, from total cellular lysates. The eluted protein complexes can be verified by WB, and further to identified and quantified through shotgun proteomics technology.

Figure 2. Experimental workflow for Co-IP in MS applications ^[1].

In Creative Proteomics, we are professional to provide a feasible experimental design tailored to your specific requirements for Co-IP/MS project. Equipped with experienced technical team, stringent quality control system, and advanced LC-MS platform, we are confident to provide high-quality and reliable results for protein-proteins interactions analysis.

Our Co-IP/MS service contains

1) Experiment design based on your specific project needs: buffers, beads, antibodies, etc.;

2) Parameters optimization, such as incubating time, elution strength, etc.;

3) Cell lysis, Co-IP, washing & elution;

4) Western blot and/or LC-MS/MS, depending on the project needs;

5) Data analysis and report delivery.

How to place an order

Please feel free to contact us via email for a comprehensive discussion regarding your specific requirements. Our team of customer service representatives is available round the clock, seven days a week.

1. The difference between IP and Co-IP?

IP, immunoprecipitation, is a method of purification and enrichment of a specific protein by using specific reaction of antigen and antibody.

Co-IP, Co-Immunoprecipitation, is an extension of IP experiment, primarily employed for investigating protein-protein interactions and frequently utilized in the identification of known or unknown constituents within a specific protein complex.

In brief, IP primarily targets the antigen that binds to the antibody, while Co-IP focuses on identifying secondary protein interactions with the primary target proteins rather than antibody.

2. How to select tag for Co-IP experiments?

The commonly used tags of Co-IP in animal cells include HA, Flag, GFP and MYC, etc., while His and GST tags are often used for prokaryotic cell protein purification. Due to the high histidine background of proteins, His tag is rarely used in mammalian cells.

3. What are the characteristics of Co-IP/MS service?

1) Investigation of protein-protein interactions and protein complexes;

2) Monitor dynamics of proteins interaction;

3) Study protein-protein interactions in a non-denaturing condition, which is almost physiological;

4) Mapping the interacting domains of proteins.

4. What is the underlying mechanism that enables protein A to successfully IP protein B when studying protein interactions, while protein B fails to IP protein A?

If A can pull on B, it indicates that there is an interaction between protein A and protein B. However, if B cannot pull on A. At this time, the protein B antibody should be checked first to see if it can IP protein B.

1) If not, it indicates that the B antibody cannot be used for IP, and you can consider changing to another antibody or tagging the protein, using tag antibody to IP B protein;

2) If yes, it may be that B antibody preferentially occupies the binding site of protein B, so that when protein A comes to protein B, protein B no longer exists any binding site for protein A. At this time, it can be considered to change an antibody or add a tag on the protein and use the tag antibody for IP.

References

1. Jensen P, Patel B, Smith S, et al. Improved Immunoprecipitation to Mass Spectrometry Method for the Enrichment of Low-Abundant Protein Targets. Methods in Molecular Biology. 2021;2261:229-246.

TRIM67 drives tumorigenesis in oligodendrogliomas through Rho GTPase-dependent membrane blebbing

Journal: Neuro-Oncology

Published: 2023

Main Technology: RNA sequencing, total lysate-MS, and co-IP-MS with functional assays including immunofluorescence (IF) staining, and western blotting (WB)

Background: Isocitrate dehydrogenase (IDH) mutant gliomas are grouped into astrocytomas or oligodendrogliomas depending on the codeletion of chromosome arms 1p and 19q. Although the genomic alterations of IDH mutant gliomas have been well described, transcriptional changes unique to either tumor type have not been fully understood. Here, we identify Tripartite Motif Containing 67 (TRIM67), an E3 ubiquitin ligase with essential roles during neuronal development, as an oncogene distinctly upregulated in oligodendrogliomas.

Methods: We used several cell lines, including patient-derived oligodendroglioma tumorspheres, to knock down or overexpress TRIM67. We coupled high-throughput assays, including RNA sequencing, total lysate-mass spectrometry (MS), and coimmunoprecipitation (co-IP)-MS with functional assays including immunofluorescence (IF) staining, co-IP, and western blotting (WB) to assess the in vitro phenotype associated with TRIM67. Patient-derived oligodendroglioma tumorspheres were orthotopically implanted in mice to determine the effect of TRIM67 on tumor growth and survival.

Results: TRIM67 overexpression alters the abundance of cytoskeletal proteins and induces membrane bleb formation. TRIM67-associated blebbing was reverted with the nonmuscle class II myosin inhibitor blebbistatin and selective ROCK inhibitor fasudil. NOGO-A/Rho GTPase/ROCK2 signaling is altered upon TRIM67 ectopic expression, pointing to the underlying mechanism for TRIM67-induced blebbing. Phenotypically, TRIM67 expression resulted in higher cell motility and reduced cell adherence. In orthotopic implantation models of patient-derived oligodendrogliomas, TRIM67 accelerated tumor growth, reduced overall survival, and led to increased vimentin expression at the tumor margin.

Conclusions: Taken together, our results demonstrate that upregulated TRIM67 induces blebbing-based rounded cell morphology through Rho GTPase/ROCK-mediated signaling thereby contributing to glioma pathogenesis.