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Analysis Biomolecular Interactions of Immobilized 3′-biotinylated VA2/VA22 with VEGF165 by BLI (CAT#: STEM-MB-0200-CJ)

Introduction

Vascular Endothelial Growth Factor (VEGF) is a potent growth and angiogenic cytokine. It stimulates proliferation and survival of endothelial cells, and promotes angiogenesis and vascular permeability. VEGF165 functions as a dimer and promotes the formation of VEGFR2 dimers resulting in activation of the kinase domains via the phosphorylation of its tyrosine residues. Robust tyrosine phosphorylation results in the activation of angiogenic signaling pathways. VA2 and VA22 are RNA aptamers.




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

Biochemistry

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: HEK293 cell line, T7 polymerase, Pierce™ RNA 3′ end biotinylation kit