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Analysis Biomolecular Interactions of P-selectin and PSGL-1 to rhRod by BLI (CAT#: STEM-MB-0108-CJ)

Introduction

Leukocyte (WBC) capture on both platelets and vascular endothelium plays a key role in the initiation of acute inflammation. This early process is mediated through the interactions between adhesion molecules found on WBC, namely P-selectin glycoprotein ligand-1 (PSGL-1) and the β2-integrins (CD11a/CD18 [LFA-1] and CD11b/CD18 [Mac-1]) to those found on platelets and endothelial cells (e.g., P-selectin, GP1bα, E-selectin, and ICAM-1). These interactions will lead to WBC crawling and eventual transendothelial migration. Normally, inflammation is important in the maintenance of health, such as in infection and wound healing. But inflammation can also be pathologic, leading to tissue injury and organ dysfunction in both directly affected as well as distant sites, such as secondary acute lung injury and sepsis-associated liver injury.




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;

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: Escherichia Coli (Strain M15)