Protein-Protein Interaction Analysis Service

Protein-protein interaction (PPI) refers to the process where two or more protein molecules bind to form a protein complex. Proteins typically do not function in isolation but rather collaborate with multiple protein molecules to achieve complex cellular functions. Therefore, focusing on the study of protein interactions is advantageous for exploring the occurrence and development of diseases, identifying specific disease prediction and treatment methods, and providing new insights into drug development. Various methods are available for protein interaction analysis. MtoZ Biolabs offers protein interaction analysis services, including mass spectrometry identification analysis based on LC-MS/MS for IP, Co-IP samples, and GST-fusion protein pull-down purified samples.

 

1. Co-IP PPI Analysis

Co-IP is a method that utilizes the specificity between antigen and antibody to detect physiological PPIs. Initially, non-ionic detergents are used in experiments to maximize the preservation of protein interactions within cells. During cell lysis, protein interactions are retained, and specific antibodies are added to bind to target proteins within the cell. Subsequently, protein A/G is added to form antibody-protein complexes, followed by centrifugation and separation to precipitate the antibody-protein complexes. The supernatant is discarded, and the precipitated proteins are identified using MS to demonstrate their interaction. This method is suitable for analyzing stable or strong PPIs. It is typically used to identify interactions between a target protein and another protein or to identify unknown proteins that interact with a target protein.

 

MtoZ Biolabs provides optimized solutions for the extraction, expression, and purification of sample proteins, ensuring the acquisition of high-quality bait and prey proteins. We offer high-quality Co-IP protein interaction analysis services and MS analysis for proteins obtained from Co-IP.

 

(1) Advantages

Enables the separation of protein complexes in their natural state.

 

(2) Disadvantages

Lower sensitivity, unable to analyze low-affinity and transient PPIs.

 

2. PPI Analysis using GST Fusion Protein Pull-Down Technique

The GST fusion protein pull-down technique involves inserting a vector carrying a GST tag into the target protein A using genetic recombination. Magnetic beads coated with glutathione are bound to the target fusion protein. After adding cell lysis buffer, proteins with interactions are adsorbed by the fusion protein. Excess glutathione is added for elution, followed by analysis using MS. This method is mainly used to study strong or stable PPIs in vitro. It can identify direct interactions between two known proteins of interest and search for unknown proteins that may interact with a target protein.

 

As a technology-driven research service company, MtoZ Biolabs has years of experience in protein interaction analysis services and MS-based identification analysis of interacting proteins. We offer GST pull-down protein interaction analysis services and pull-down target protein MS identification services. With stringent quality control and short detection cycles, MtoZ Biolabs provides customers with customizable one-stop services covering every step from gene synthesis to data reporting in your project.

 

(1) Advantages

Eliminates interference from other conditions within cells; can directly detect PPIs; simple method with convenient operation.

 

(2) Disadvantages

Conducted extracellularly, unable to accurately reflect PPI relationships within cells.

 

3. PPI Analysis using SILAC Combined with Co-IP-MS

The SILAC method involves labeling experimental and control cell groups separately, followed by Co-IP experiments. The specificity of the reaction between antigen and antibody is relied upon to separate immunocomplexes. Qualitative/quantitative detection of proteins in immunocomplexes is achieved using LC-MS/MS. When the protein content of a specific protein differs statistically between the experimental and control groups, it is determined that this protein interacts with the studied protein, greatly reducing the possibility of false-positive results in protein interaction.

 

MtoZ Biolabs utilizes Thermo Fisher's Q Exactive HF, Orbitrap Fusion, and Orbitrap Fusion Lumos mass spectrometry platforms combined with Nano-LC to provide SILAC-based Co-IP-MS protein interaction analysis service packages. Just inform us of your experimental objectives and send us your cells, and we will take care of all aspects of the project, including cell culture, cell labeling, protein extraction, antibody IP, efficiency detection, protein digestion, peptide separation, mass spectrometry analysis, raw data analysis, and bioinformatics analysis.

• • Label Transfer Protein Interaction Analysis Service

  The label transfer method, in conjunction with crosslinking, facilitates the study of protein interactions by marking proteins that bind to target proteins. This technique is invaluable for discovering new interactions, validating proteins identified by other methods, and analyzing protein complexes. It is particularly effective for detecting weak and transient interactions, which often elude detection by co-immunoprecipitation or pull-down methods.

• • Far-Western Blot Analysis Service

  Far-western blot is a molecular biology method based on western blot technology that can be used to detect protein-protein interactions in vitro, especially interactions that do not require the natural structure of the target protein.

• • Crosslinking Protein Interaction Analysis Service

  Under physiological conditions, most protein interactions are transient, posing significant challenges for their study. Crosslinking reagents resolve this by covalently bonding interacting proteins, thereby capturing protein-protein complexes and preserving brief, weak interactions for easier separation and characterization. Researchers can perform crosslinking both in vivo and in vitro, choosing based on project needs.

• • MS-Based Protein-Protein Interaction Analysis Service

  Over recent decades, proteomics driven by mass spectrometry (MS) has emerged as a critical methodology for analyzing protein-protein interactions (PPIs). Proteins exert their biological roles by forming macromolecular complexes through interactions with other proteins or molecules. Investigating these interactions provides deeper insights into protein functionalities.