Analysis Biomolecular Interactions of IgG by Protein A by BLI (CAT#: STEM-MB-0131-CJ)

Introduction

Protein A and G are popular choices for antibody purification, because they are both stable and target selective. IgG class antibodies from multiple species bind to protein A and/or G, allowing antibody to be captured on protein-bound beads. Protein A and G bind IgG subtypes with varying affinities, determined by species and the properties of the heavy chain. Protein A and protein G bind with high affinity and specificity to Fc region of immunoglobulins from many of mammalian species, most notably IgGs. IgGs from most species bind to Protein G at near physiological pH and ionic strength with a higher affinity than IgG binding to Protein A.

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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; 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: CHO Cell Lines

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