Analysis Kinetics of Inhibitors of SSB PPIs by BLI (CAT#: STEM-MB-0293-CJ)

Introduction

Antibiotic-resistant bacterial infections are becoming increasingly common worldwide and there is an urgent need for new antibiotics that can overcome existing resistance mechanisms. One potential antibiotic target is bacterial single-stranded DNA binding proteins (SSBS), which can act as hubs for DNA repair, recombination, and replication. The eight highly conserved residues at the C-terminal of SSB can recruit more than a dozen important genome maintenance proteins to single-stranded DNA through direct protein-protein interactions (PPIs). Mutations of PPIs with C-terminal tails of SSBS are lethal, so small-molecule inhibitors of these key SSB PPIs may be effective antimicrobials.

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Principle Applications Procedure Materials Notes Related Products

Principle

Bio-Layer Interferometry (BLI) is an optical technique for measuring macromolecular interactions by analyzing interference patterns of white light reflected from the surface of a biosensor tip. BLI experiments are used to determine the kinetics and affinity of molecular interactions. In a BLI experiment, one molecule is immobilized to a Dip and Read Biosensor and binding to a second molecule is measured. A change in the number of molecules bound to the end of the biosensor tip causes a shift in the interference pattern that is measured in real-time.

Applications

Microbiology; Pharmacy

Procedure

1. Detect Buffers and prepare samples. BLI experiments are set up with one molecule immobilised on the surface of the biosensor (load sample) and a second molecule in solution (the analytical sample).
2. Fix the load sample on the biocompatible biosensor while the analytical sample is in solution.
3. The biosensor tip is immersed in the solution so that the target molecule begins to bind to the analysis sample.
4. Set up and run the BLI experiment. Molecules bound to or dissociated from the biosensor can generate response curves on the BLI system; unbound molecules, changes in the refractive index of the surrounding medium or changes in flow rate do not affect the interferogram pattern.
5. Collect and analyse data on the BLI's system.

Materials

• Equipment:Fortebio Bio-Layer Interferometry (BLI)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules

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