Investigation of protein binding properties using collagen suspensions in mobility shift affinity capillary electrophoresis

HARTUNG, Sophie, Christin SCHELLER, Taťána BRŽEZICKÁ a Wätzig HERMANN. Investigation of protein binding properties using collagen suspensions in mobility shift affinity capillary electrophoresis. In 29th International symposiumon electro- and liquid phase-separation techniques, ITP. 2023.

Základní údaje

Originální název

Investigation of protein binding properties using collagen suspensions in mobility shift affinity capillary electrophoresis

Autoři

HARTUNG, Sophie, Christin SCHELLER, Taťána BRŽEZICKÁ a Wätzig HERMANN

Vydání

29th International symposiumon electro- and liquid phase-separation techniques, ITP, 2023

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Další údaje

Typ výsledku

Konferenční abstrakt

Utajení

není předmětem státního či obchodního tajemství

Klíčová slova česky

mobilitní posun afinitní kapilární elektroforéza, konstantní výkon, kolagenní suspenze, fluorescenční detekce

Klíčová slova anglicky

mobility shift affinity capillary electrophoresis, constant power, collagen suspension, fluorescence detection

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Anotace

V originále

Mobility shift affinity capillary electrophoresis is an approach to study interactions of drugs with biomaterial. This technology can help to better understand the function and role of proteins in the human body and improves the development of drugs and medical devices. Therefore, various concentrations of ligand are dissolved in the BGE and an analyte and a noninteracting mobility marker are injected into the capillary. During electrophoresis, a ligand's presence can change the analyte's effective electrophoretic mobility. Thus, by fitting the data against a model function through non-linear regression, the strength of the interaction can be estimated. This project aims to investigate the binding properties of proteins with a collagen product constructed for wound healing to find out about its biocompatibility. As a BGE, a phosphate buffer with pH = 7.4 was chosen to mimic the natural conditions of the human body. Because of the poor solubility of collagen in a neutral medium, a procedure was established to make parts of the collagen product suspendable which was achieved by grinding and freeze-drying. Subsequently, a robust method will be developed to measure different proteins reproducibly with and without the presence of collagen. The development of such a method is challenging since differences in migration time and peak shape can occur due to adsorptive capillary effects. Therefore, an LPA-coated capillary (75 μm ID) is used to reduce protein adhesion. Another important aspect is, that the BGE containing collagen is a suspension and this results in an increase in current strength. Thus, we chose constant power as separation mode. The addition of large amounts of collagen changes the viscosity of the BGE and must be considered in later calculations. Because only small amounts of collagen material are available, we adapted and established the method by Allmendinger et al. [“High-throughput viscosity measurement using capillary electrophoresis instrumentation and its application to protein formulation”, Journal of pharm. and biomed. analysis, vol. 99, 2014] to determine the viscosity by using an Agilent 7100 capillary electrophoresis instrument. Furthermore, it is investigated whether, in our case, a mobility marker could also be used to calculate a correction factor for the effective mobilities of the protein. It is tested, if the previously used mobility marker is suitable or whether there is another adequate candidate that is visible with the fluorescence detector. Finally, mobility shift affinity capillary electrophoresis with collagen at various concentrations as ligand, the respective proteins, and a non-interacting marker is carried out and evaluated.