Analysis Biomolecular Interactions of Anti-human IgM Nanobodies9 (Nbs9) with IgM by BLI (CAT#: STEM-MB-0146-CJ)

Introduction

IgM antibodies are secreted by B-cells in the early stages of humoral immune responses. They are present in serum as pentameric high avidity antibodies (900 kDa), comprising five monomeric IgMs (180 kDa) linked by disulfide bridges and by one molecule of the polypeptide J chain, or as monomeric transmembrane proteins on B cell surfaces. Transmembrane and secreted IgM are target molecules for the diagnosis of different diseases.

Anti-human IgM polyclonal or monoclonal antibodies are commonly used. Nanobodies are recombinant antibody fragments (15 kDa) derived from the variable domain (VHH) of a subtype of antibodies that are devoid of light chains and found in camelids. They possess salient properties as reagents for diagnostic methods, notably solubility and stability at extreme conditions of temperatures and high solvent concentrations.

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Principle Applications Procedure Materials Notes Related Products

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

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

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