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Analysis Biomolecular Interactions of TNF-α and Recombinant Monoclonal IgG Antibody by BLI (CAT#: STEM-MB-0210-CJ)

Introduction

Tumour Necrosis Factor alpha (TNF α), is an inflammatory cytokine produced by macrophages/monocytes during acute inflammation and is responsible for a diverse range of signalling events within cells, leading to necrosis or apoptosis. The protein is also important for resistance to infection and cancers.<br /><br />Immunoglobulin G (IgG) is the main immunoglobulin in natural human serum. It constitutes 70 to 75 percent of all immunoglobulins. Monoclonal antibodies have many applications in diagnosis, treatment and purification. The conjugated monoclonal antibodies against human IgG are used in most diagnostic kits.




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: Recombinant human TNF-α, Alexa Fluor 488 antibody labeling kit