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Analysis Biomolecular Interactions of Protein with Protein by BLI (CAT#: STEM-MB-0087-CJ)

Introduction

Proteins are the “workhorses” of the cell, participating in every structure and function involved in life. The human genome contains genes encoding 500,000 different types of proteins, of which 10,000 are expressed in the cell at any given time. It is estimated that almost 80% of these cellular proteins function in complexes rather than in isolation. Proteins interact with other proteins or biomolecules such as nucleic acids (DNA or RNA), metabolites, or lipids in order to serve as catalysts, defenses against foreign pathogens, mechanical supports, transporters, pumps, switches, growth factors, signal transduction receptors, and motors, among other functions. Characterization of protein-protein and protein-ligand interactions through analysis of real-time kinetic and binding affinity data can provide unique insights into specific molecular interactions that regulate cellular processes, and characterization of aberrant molecular interactions may provide insights into pathway dysregulation and disease.




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; Pharmacology; Metabolic; 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: Gator® Bio-Layer Interferometry (BLI)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules
• Optionals: Biotinylated Bait