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Analysis Biomolecular Interactions of sybodies with RBD by BLI (CAT#: STEM-MB-0148-CJ)

Introduction

SARS-CoV-2 is heavily decorated by its surface Spike (S), a single-pass membrane protein that is key for the host-virus interactions. During the infection, S is cleaved by host proteases, yielding the N-terminal S1 and the C-terminal S2 subunit. S1 binds to angiotensin-converting enzyme 2 (ACE2) on the host cell membrane via its receptor-binding domain, causing conformational changes that trigger a secondary cleavage needed for the S2-mediated membrane fusion at the plasma membrane or in the endosome. Because of this essential role, RBD has been a hot spot for the development of therapeutic monoclonal antibodies (mAbs) and vaccine.




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

Immunology/Inflammation; Virology; Pharmacology

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: TES-high Buffer
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