Proximity-dependent Biotin Identification (BioID) Service

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  • Case Study

Introduction of Proximity-dependent Biotin Identification (BioID)

Many methods for studying protein-protein interactions usually fail to capture low-affinity interactions, and after cell lysis and protein complex purification, some interactions have disappeared, which has caused difficulties for researchers. At present, Proximity-dependent biotin identification (BioID) has become a powerful tool for identifying candidate protein-protein interactions in living cells. This method uses a promiscuous biotin ligase fused with the target protein to covalently biotin label the protein, and capture and identify the interacting and neighboring proteins without keeping the protein complex intact. Compared with other technologies, BioID allows the detection of transient and weak interactions in a specific biological environment within a specific time.

Schematic of BioID protein purificationFigure 1. Schematic of BioID protein purification (Batsios, P.; et al. 2016)

BioID Service in Creative Proteomics

Creative Proteomics has established a stable and advanced BioID technology platform, aiming to provide accurate and efficient protein-protein interaction determination services for scientific researchers all over the world. Our platform is composed of a large number of sophisticated instruments and well-trained technicians to provide guarantees for customers' experimental results. The experimental process of BioID in our platform is as follows:

Workflow of BioID in Creative Proteomics - Creative ProteomicsFigure 2. Workflow of BioID in Creative Proteomics - Creative Proteomics

Customers can choose different technology platforms according to project requirements, or contact us directly for consultation, and our expert team will provide you with customized experimental procedures.

Feature of BioID

Dynamic Interaction Monitoring: Real-time tracking of weak or transient interactions in vivo, enabling effective study of dynamic protein complexes.

Versatile Protein Structure Analysis: Facilitates examination of soluble or typically inaccessible protein structures.

Reliable Kinetic Data: Provides valid kinetic data for a comprehensive understanding of molecular interactions.

Low False Positive Rate: Rigorous washing with harsh detergents eliminates most non-specific binding, ensuring reliable results with a minimal false positive rate.

Short Experiment Period: BioID ensures efficient experimentation with a short duration, facilitating quicker data acquisition and analysis.

High Sensitivity: The affinity between biotin and avidin (Kd=10-15 mol/L) in BioID is exceptionally high, surpassing antibody-mediated interactions by over 1,000 times. This heightened sensitivity enables the more effective and stable capture of protein complexes.

High Specificity: BioID achieves high specificity by avoiding the use of antibodies, significantly reducing non-specific binding. The remarkable stability of the biotin-avidin interaction allows for stringent purification, eliminating protein contaminants.

High Adaptability: The biotin-avidin interaction is rapid and specific, even under conditions where many other proteins have denatured, such as high temperature or exposure to harsh agents like 6 M guanidine hydrochloride or 1% sodium dodecyl sulfate (SDS). Biotinylated proteins can be efficiently purified directly from crude extracts in a single-step procedure, in contrast to commonly used affinity tags that require multiple purification steps before affinity binding.

Application Areas of BioID Service

Structural Biology: Explore the structural intricacies of proteins, especially those challenging to access, shedding light on their functions and interactions.

Systems Biology: Uncover the dynamic networks of protein interactions within living systems, providing valuable insights into cellular processes.

Drug Discovery: Accelerate drug development by identifying and validating potential drug targets through the precise mapping of protein-protein interactions.

Functional Proteomics: Gain a deeper understanding of protein function by elucidating proximity-based associations, enabling the characterization of protein complexes and pathways.

Cell Signaling Studies: Investigate signaling cascades and regulatory pathways by capturing the spatial organization of proteins involved in cellular communication.

Disease Mechanisms: Illuminate the molecular mechanisms underlying diseases, aiding in the identification of diagnostic markers and therapeutic targets.

Targeted Protein Analysis: Efficiently study specific proteins or protein complexes in their native cellular context, enhancing the accuracy of your analyses.

Biomarker Discovery: Identify potential biomarkers associated with specific cellular processes or diseases, offering novel targets for diagnostic and prognostic applications.

Creative Proteomics is an international biotechnology company dedicated to the study of intermolecular interactions and other related fields. We have established an advanced proximity-dependent biotin identification (BioID) technology platform to help customers more accurately study protein-protein interactions. Our one-stop service is designed to save customers time and money.


  1. Batsios, P.; et al. Proximity-dependent biotin identification (BioID) in Dictyostelium Amoebae. Methods in Enzymology. 2016.
  2. Sears, R.M.; et al. BioID as a tool for protein-proximity labeling in living cells. Methods in Molecular Biology. 2012.

Case Proteomic Analysis of Epstein-Barr Virus LMP1 Interactome Reveals Novel Insights into Cellular Pathways


Epstein-Barr Virus (EBV) is associated with various cancers, and its latent membrane protein 1 (LMP1) plays a pivotal role in oncogenesis. Traditional approaches have elucidated LMP1 interactions, but a comprehensive understanding necessitates advanced methodologies. This study employs a proximity-based biotin ligase system coupled with mass spectrometry for an in-depth exploration of the LMP1 interactome.


Replicate BioID and GFP-AP samples, along with Significance Analysis of INTeractome (SAINT) examination, were utilized. A total of 485 proteins with high SAINT scores were identified, with 13 common to all conditions. BioID experiments exhibited more proteins with high confidence scores compared to GFP-AP datasets, emphasizing the utility of the BioID method.

Technical Methods

BioID Approach:

Principle: The BioID method leverages proximity-dependent biotinylation to identify proteins in close proximity to a target protein (LMP1, in this case). A fusion protein of LMP1 and the bacterial biotin ligase BirA* was expressed in cells, allowing biotinylation of neighboring proteins over time.

Experimental Setup: Replicate BioID samples were generated, and for comparison, GFP-AP (GFP fused with the Avidin protein) samples were included. The latter served as a negative control for non-specific protein interactions.

Significance Analysis of INTeractome (SAINT):

Algorithm Overview: SAINT examination was employed to assess the confidence of identified protein interactions. This algorithm considers spectral count and reproducibility data, comparing them to negative control runs (GFP-AP). The output is a score that reflects the likelihood of a true interaction.

Criteria for High SAINT Scores: Proteins with high SAINT scores (485 in total) were considered potential interacting partners, with 13 proteins common to all three conditions.

Data Analysis and Comparison:

Comparison of BioID and GFP-AP Datasets: The study observed a higher number of proteins with high SAINT scores in BioID experiments compared to GFP-AP datasets. This discrepancy is attributed to non-specific protein pull-down with control GFP-AP beads under gentler lysis conditions.

Interaction Network Analysis using FunRich:

Objective: To understand how proteins with high SAINT scores are interconnected, an interaction network analysis was performed using FunRich.

Identified Pathways and Proteins: Clusters of proteins involved in known LMP1 pathways (e.g., TRAF6, EGFR, NF-κB, MAPK/ERK) were identified. Additionally, novel proteins and pathways potentially influenced by LMP1, such as MCC, GRB2, ARRB1, VHL, YWHAG, PRKAB1, MAP3K14, MAPK13, and MAP3K3, were uncovered.

Validation of Protein Interactions:

Experimental Validation: Selected proteins identified in the unbiased profiling experiments were validated by expressing LMP1 BioID constructs in cells. Streptavidin pull-downs of biotinylated proteins confirmed interactions with both previously known LMP1 interacting proteins and novel proteins identified in the dataset.

Spatial Considerations: Some proteins were exclusively biotinylated by the C-terminal tagged LMP1 construct, suggesting potential spatial specificity of these interactions.

Functional Analysis:

Functional Significance Testing: To test the functional significance of two identified interacting partners (syntenin-1 and ALIX), stable cells with inducible shRNA constructs against these proteins were generated. The study demonstrated that decreased levels of syntenin-1 and ALIX significantly affected LMP1 exosomal packaging, highlighting the functional relevance of identified interactions.


  • Proteomic analysis using BioID identified over 1000 proteins as direct, transient, weak, or proximal interactors of LMP1.
  • Pathway analysis revealed enrichment in biological processes linked to LMP1, including protein degradation, endocytosis, cell proliferation, cell cycle control, cell-cell adhesion, mRNA stability, MAPK signaling, metabolic pathways, and protein/vesicle transport.
  • Identified proteins suggested connections to exosome biogenesis, with components of ESCRT-dependent and -independent mechanisms present, including ALIX and syntenin-1.
  • The study highlighted potential crosstalk between autophagy and endo-lysosomal pathways, implicating HSC70 in LMP1 trafficking to multivesicular bodies.
  • Rab GTPases (Rab7a and Rab10) and SNARE proteins associated with endosomal trafficking and membrane fusion were identified as potential LMP1 interactors.
  • Known interactions (e.g., TRAF2, vimentin, HSC70) were validated, and novel interactions linked to NF-κB, MAPK, PI3K, STAT, mTOR, insulin, and integrin signaling were uncovered.
  • Spatial considerations indicated distinct protein profiles based on the tag's location, emphasizing spatial information in the nature of interactions.
  • Experimental validation, including streptavidin pull-downs, confirmed both known and novel LMP1 interactions, with functional testing revealing the importance of syntenin-1 and ALIX in LMP1 exosomal packaging.

Functional analysis of BioID constructs.Functional analysis of BioID constructs.

Mass spectrometry analysis of affinity purified proteins.Mass spectrometry analysis of affinity purified proteins.


  1. Rider, Mark A., et al. "The interactome of EBV LMP1 evaluated by proximity-based BioID approach." Virology 516 (2018): 55-70.
* This service is for RESEARCH USE ONLY, not intended for any clinical use.