Analysis Kinetics of Glycosylated Proteins by KinExA (CAT#: STEM-MB-0264-CJ)

Introduction

Protein glycosylation is a complex posttranslational modification that manipulates the biological activity and function of therapeutic glycoproteins. Glycan composition has been the target of modification to enhance the safety and efficacy of glycoprotein therapeutics. Glycans are the most complex and heterogeneous class of molecules due to their non-template-driven biosynthetic process, consequently making glycan characterization difficult. It is essential to apply several orthogonal methods to measure individual parameters such as glycosylation site analysis, oligosaccharide sequence, and monosaccharide content of a therapeutic glycoprotein. The importance of studying aberrant glycosylation in cancer is highlighted by the case of the prostate specific antigen (PSA). PSA is a clinical biomarker for prostate cancer, a disease estimated to have led to 375[thin space (1/6-em)]000 deaths and 1.4 million new cases worldwide in 2020.

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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

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: Bio Bio-Layer Interferometry (BLI)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules

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