Analysis Biomolecular Interactions of the Glycolipid with B-subunits of Shiga Toxin (Stx) Variants by BLI (CAT#: STEM-MB-0223-CJ)

Introduction

Glycolipids are a type of complex lipids comprising carbohydrates, fatty acids, sphingolipids or a glycerol group. Glycolipids are found on the outer leaflet of cellular membranes where it plays not only a structural role to maintain membrane stability but also facilitates cell-cell communication acting as receptors, anchors for proteins and regulators of signal transduction. Shiga toxin (Stx) producing E. coli (STEC) is a major cause of foodborne illnesses. Themajor virulence factor of STEC is Stx, an AB5 toxin that consists of a single A-subunit with ribosomal RNA-cleaving activity, surrounded by a receptor-binding B-penta mer. Two major isoforms, Stx1 and Stx2, and Stx2 variants (Stx2a-h) significantly differ in toxicity.STEC colonize the gastrointestinal tract and produce Stx.

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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

Toxicology

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

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