Analysis Kinetics of Aptamer GO18-T-d Streptavidin-Targeting CIR (CIR1/CIR4) Reaction by BLI (CAT#: STEM-MB-0272-CJ)

Introduction

An emerging strategy in immunotherapy involves the use of synthetic small molecules to engage, redirect, or leverage the immune system for a therapeutic effect such as anticancer activity. The TBD contains a cancer receptor ligand such as glutamate urea lysine (GUL), which can bind to the highly expressed prostate-specific membrane antigen (PSMA) receptor on prostate tumors.

Antibody-labeling reaction kinetics were studied using CIRs (CIR1/CIR4) equipped with desthiobiotin (an analog of biotin) to serve as a model TBD with nearinfinite binding affinity for a target receptor. This enables sufficiently high CIR binding affinity to streptavidin-coated probes to efficiently monitor “on-probe” antibody-labeling kinetics without concern for CIR dissociation from the probe. The CIR4 only differs from CIR1 through a shorter and more rigid linker between the ALD and ABD.

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Principle Applications Procedure Materials

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

Oncology & Cancer; 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: CIR1, CIR4, NCIR5, Anti-DNP Antibody

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