Analysis Biomolecular Interactions of PHA-EVs with CD44-Fc by BLI (CAT#: STEM-MB-0217-CJ)

Introduction

Extracellular vesicles (EVs) are extracellular membrane vesicles (30 -- 200 nm in diameter) that are endogenous released by cells and are present in most biological fluids, including blood, urine, and saliva. Ev-mediated cellular communication is closely associated with normal physiological processes such as inflammation, homeostasis, and coagulation, as well as pathophysiological processes in refractory diseases such as rheumatoid arthritis (RA), atherosclerosis, stroke, and cancer. PHA was chosen as the representative part of the surface modification because it shows extended circulation in the blood and a specific binding affinity for CD44 overexpressed tissues. Due to the high in vivo CD44 targeting and stealth effect of PHA, PHA-EVs can accumulate in CD44-rich tissues through its in vivo active targeting mechanism after systemic administration.

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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

Oncology & Cancer; Immunology/Inflammation; 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: NIH/3T3 cell line, PC3 cell line, CT26 cell line, RAW264.7 cell line

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