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Analysis Biomolecular Interactions of rLmHtrA1-501 with ECM Molecules by BLI (CAT#: STEM-MB-0213-CJ)
Introduction
The extracellular matrix (ECM) consists of numerous macromolecules classified traditionally into collagens, elastin, and microfibrillar proteins, proteoglycans including hyaluronan, and noncollagenous glycoproteins. The extracellular matrix helps cells attach to, and communicate with, nearby cells, and plays an important role in cell growth, cell movement, and other cell functions. The extracellular matrix is also involved in repairing damaged tissue.The protease and ECM binding properties of HtrA from L. monocytogenes (LmHtrA) can be studied using recombinant proteins such as full length His-tagged rLmHtrA wild type (rLmHtrA1-501). And Full length His-tagged can active site mutant S343A (rLmHtrA1-501(S343A)) and His-tagged ∆PDZ (rLmHtrA1-394∆PDZ).
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
Microbiology
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.
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 expression strains (JRG6782, 6783 and 6784)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules
• Optionals: E. coli expression strains (JRG6782, 6783 and 6784)
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