Investigation of protein-protein interactions with Agrisera anti-TurboID antibodies

Protein-protein interactions in crop plants can be studied using transient TurboID-based proximity labeling.

In the recent publication "Protocol to identify protein-protein interaction networks in Solanum tuberosum using transient TurboID-based proximity labeling" from the Laboratory of Plant Breeding, Wageningen University & Research, The Netherlands, authors Li Shi et al. described the methodology of transient expression of constructs of a protein under investigation, fused to TurboID, and the identification of protein-protein interactions in potato leaves.

The publication describes a detailed protocol for preparation of plant material and transgene desing, as well as protein identification, using Agrisera's anti-TurboID antibody (AS20 4440). The authors also discuss limitations of the protocol, and provide suggestions on how to adress them.

Steps of the protocol include:

Design and Construction of TurboID Fusion Protein with the protein of interest (POI).
The TurboID enzyme biotinylates nearby proteins within a certain radius (typically tens of nanometers) when activated by the addition of biotin-tyramide and hydrogen peroxide.
Expression of POI with TurboID. After expressing the TurboID fusion proteins, cells are treated with biotin-tyramide and hydrogen peroxide to activate TurboID. TurboID then catalyzes the covalent attachment of biotin to nearby proteins within its labeling radius.
Protein Extraction: Biotinylated proteins are enriched using streptavidin beads or other methods, isolating the proteins that were in close proximity to the TurboID-fused protein.
Protein Identification by Mass Spectrometry. The enriched protein sample is subjected to to mass spectrometry for identification. This helps identify the proteins that were labeled by TurboID.
Validation. Identified protein-protein interactions are often validated using additional experimental techniques, such as Western blot.

Investigation of protein-protein interactions with Agrisera anti-TurboID antibodies

Agrisera blog

Latest

Archive