Protein Interactions & BioID

Mapping the "Neighborhood"

Proteins rarely act alone. To truly understand a protein's function, you must know who its neighbors are.

Standard Immunoprecipitation (IP) is excellent for strong, stable complexes ("lock and key"), but it often fails to capture the dynamic reality of the cell. Weak interactions, transient signaling events, and insoluble membrane contacts are frequently lost during cell lysis.

To solve this, we specialize in Proximity Labeling (BioID/TurboID)—a powerful technique that labels proteins based on spatial proximity rather than physical binding strength.

How BioID & TurboID Work

BioID turns your protein of interest into a "molecular paintbrush." Here is the mechanism:

1. Fusion: We express your protein of interest fused to a promiscuous biotin ligase (BirA* or TurboID).
2. Labeling: When biotin is added to the culture media, the ligase creates a cloud of reactive biotin-AMP. This covalently tags any protein within a ~10nm radius (the "neighborhood").
3. Capture: Because the biotin bond is covalent, we can use harsh lysis buffers to solubilize membranes and chromatin without losing the label. The tagged proteins are then purified using Streptavidin beads.
4. Identification: The enriched "neighbors" are identified by high-resolution Mass Spectrometry.

TurboID vs. BioID: While classic BioID requires 18-24 hours of labeling, TurboID is a faster, engineered variant that can label a neighborhood in as little as 10 minutes, allowing for dynamic "snapshots" of cellular processes.

The ligase biotinylates lysine residues on any protein within the immediate vicinity.

Success Story: The Deretic Lab

Uncovering Novel Roles for ATG5

Our facility collaborates closely with the Vojo Deretic Lab (University of New Mexico) to map the interactomes of key autophagy proteins.

In a recent high-impact study, we utilized mass spectrometry to define the interaction landscape of ATG5. This work revealed a surprising "non-canonical" role for ATG5 and membrane Atg8ylation in retromer assembly and vesicular trafficking, completely distinct from their traditional roles in degradative autophagy.

Supported Workflows

• BioID / TurboID: Full support for biotin enrichment and MS analysis.
• AP-MS (Affinity Purification): For stable complexes (FLAG, HA, GFP pulldowns).
• Cross-Linking (XL-MS): For structural mapping of direct binding interfaces using chemical crosslinkers (DSS, BS3).

Filtering the Noise

Interaction data is complex. We can use advanced scoring like (SAINTq) to distinguish true interactors from "sticky" background proteins.

• Contaminant Removal: Using our internal contaminant database of common background binders.
• Visualization: Dot plots and Cytoscape like network like maps can be included using R or other freely avaliable software