Analysis Biomolecular Interactions of Vitamin K–dependent Factor X (FX), SR-AI, and PTX2 by BLI (CAT#: STEM-MB-0189-CJ)

Introduction

Vitamin K–dependent factor X (FX) is best known for its central role in hemostasis. Its functional deficiency is associated with severe bleeding, including epistaxis, hemarthrosis, and gastrointestinal bleeding. In addition, Nonhemostatic functions for FX include inhibition of angiogenesis, regulation of the immune response toward viral infections, and stimulation of proinflammatory and profibrotic responses in fibroblasts. SR-AI is known for its hyperendocytic ability to acetylate LDL, which binds to but does not internalize FX. The pentraxin-2 (PTX2) was identified as a hitherto unidentified FX binding protein. PTX2 forms complexes with FX in plasma and prevents the internalization of FX/SR-AI complexes. In addition, PTX2 contributes to the mechanism of maintaining normal FX plasma levels and allowing FX accumulation on the surface of macrophages.

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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

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: Human Blood and plasma

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