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Analysis Biomolecular Interactions of Pba and PG Sacculi by BLI (CAT#: STEM-MB-0234-CJ)

Introduction

The cell wall of Gram-negative bacteria consists of a lipopolysaccharide (LPS)-covered outer membrane (OM) and an inner phospholipid bilayer membrane that surrounds the periplasmic space. The peptidoglycan (PG) layer is contained within the periplasmic space. The PG layer gives the bacterial cell stability through its elastic semi-rigid structure of linear glycan strands and cross-linked pentapeptides. And the PG attachment has been characterized in greatest depth in Escherichia coli and Acinetobacter baumannii. Peptidoglycan binding anchor (Pba) was named as the protein encoded at PA1041, and the gene was named Pba.




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

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
• Optionals: E.coil BL21