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Analysis Biomolecular Interactions of IGF1R Immunoprecipitated Lysates with rSIRPα Protein by BLI (CAT#: STEM-MB-0196-CJ)

Introduction

The IGF-1R is a tyrosine kinase that undergoes autophosphorylation and catalyzes the phosphorylation of multiple cellular proteins, including members of the IRS family. On phosphorylation, IRSs interact with signaling molecules, including Akt, Ras/Raf, and Rac. Diseases associated with IGF1R include Insulin-Like Growth Factor I and Nk-Cell Enteropathy. Among its related pathways are Apoptotic Pathways in Synovial Fibroblasts and GPCR Pathway. The RsiRp-α protein is a recombinant Sirp-α protein. SIRPA plays a protective role in cardiac hypertrophy through negative regulation of the Toll-like receptor 4/nuclear factor-kappaB pathway.




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;

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: E. coli Rosetta (DE3) pLysS cells
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