Traditional Bead and Biotinylation (TBB)

Traditional Bead and Biotinylation Technology (TBB) is a well established, albeit dated method for target identification, and validation. This method has been used widely to establish the known protein-protein interactions and also, to identify the previously unknown protein-protein interactions. TBB is an affinity purification technology that utilizes the ‘bait-molecule-probe’ to enrich the protein(s) of interest, and thus helps in identifying of the most probable protein targets. 

Working Principle

This method relies on enriching target proteins of a ‘bait-molecule’ by preparing ’bait-molecule’ specific affinity matrix. Affinity isolation can be achieved by either immobilizing bait-molecule on solid-phase beads or by coupling the bait molecule to universal isolation agents such as biotin.

Workflow

In TBB, the ‘bait-molecule’ is appropriately derivatized and coupled to either solid-phase or biotin. Target proteins due to inherent affinity interaction with the bait-molecule and can be isolated by pre-immobilizing bait-molecule with solid-phase or biotin. These proteins are then identified and quantified using mass-spectrometry. Specific target proteins are de-convoluted by comparing the identity and quantity of identified protein with the proteins that were identified from control experiment.

Key Advantages

  • Optimum experiment design and high probability of success.
  • Target deconvolution can be completed within 3-4 weeks

Points of Consideration

  • ‘Bait-Molecule’ Structure-Activity Relationship (SAR) must be well defined.
  • ‘Bait-Molecule’ needs to be derivatized/modified to accommodate the coupling of bead/biotin.

References

Technical Note 1   This note explains various steps involved in de-convoluting the right target(s) of small molecule by immobilizing the bait-molecule on solid support.

(PDF, 484KB)

Application Note 1 This note describes the application of the technology in identifying/de-convoluting true positive targets of a test-molecule BIS-III.

(PDF, 172KB)

No, Currently Shantani do not have the expertise to assist in the SAR analysis of the compound 

Yes, Shantani can synthesize the compound on case to case basis. 

Typically molecule provided in the range of 5-10 mg is sufficient enough for the purpose. 

We prefer that ~15 mg of protein(cell-lysate) is provided for the purpose. In case not, we can culture the cells and generate the required amount of cell-lysate as long as either seed-culture of the cell and culture protocol is provided OR commercial source for the same is located.