Workflow of Label Transfer Protein Interaction Analysis

The core mechanism of the label transfer method involves treating bait proteins with labeling reagents, followed by crosslinking these reagents to prey proteins through light exposure or other activation methods. The detailed steps are as follows:

 

1. Labeling of Bait Proteins

Initially, bait proteins are reacted with label transfer reagents (LTR) that are tagged with radioactive, fluorescent, or biotin labels, forming a labeled complex. These reagents typically contain photosensitive or other reactive groups, which will form stable covalent bonds with prey proteins upon subsequent light exposure or chemical activation.

 

2. Binding of Bait and Prey Proteins

The labeled bait proteins are then incubated with target proteins in vitro, forming stable or transient protein complexes. This step enables the label transfer reagent to specifically label prey proteins that interact directly with the bait protein.

 

3. Crosslinking Reaction

Once the protein complex forms, the photosensitive groups of the label transfer reagent are activated by ultraviolet light or specific conditions, leading to covalent bonding with the amino acid residues of prey proteins. The key to this step is ensuring specificity and efficiency, accurately capturing the transient states of protein interactions.

 

4. Label Transfer and Separation

Following crosslinking, the spacer arm of the crosslinker is cleaved, releasing the labeled prey proteins. These labeled prey proteins can then be separated and identified through radioactive detection, fluorescence analysis, or affinity capture methods. The separation and identification of these labeled prey proteins are crucial for determining their interactions with the bait protein.

 

5. Analysis and Detection

Finally, the labeled prey proteins are analyzed using mass spectrometry, Western blotting, or other protein analysis techniques. 

 

Applications and Advantages of Label Transfer

Compared to traditional co-immunoprecipitation (Co-IP) or pull-down techniques, this method captures a broader range of protein interactions. Furthermore, it enables precise analysis of the components within specific protein complexes. MtoZ Biolabs provides integrate label transfer protein interaction analysis service.