Effects of Chemical Fixatives on Kinetic Measurements of Biomolecular Interaction on Cell Membrane

Understanding the interaction between ligands and membrane proteins is important for drug design and optimization. Although investigation using live cells is desirable, it is not feasible in some circumstances and cell fixation is performed to reduce cell motion and degradation. This study compared...

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Published inThe Journal of membrane biology Vol. 257; no. 1-2; pp. 131 - 142
Main Authors Dong, Tianbao, Wan, Shengyang, Wang, Yanhui, Fu, Yaru, Wang, Pengcheng
Format Journal Article
LanguageEnglish
Published New York Springer US 01.04.2024
Springer Nature B.V
Subjects
Acetone
Antibodies
Binding
Biochemistry
Biomedical and Life Sciences
Cell Membrane
Cell membranes
Coagulants
Cytology
Design optimization
Drug development
ErbB-2 protein
Ethanol
Ethanol - pharmacology
Fixation
Fixatives
Fixatives - pharmacology
Glycan
Growth factors
Human Physiology
Kinetics
Lectins
Life Sciences
Membrane proteins
Methanol
Polysaccharides
Receptors
Cell fixation
Ligand binding
Membrane protein
LigandTracer
Kinetics
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Summary:Understanding the interaction between ligands and membrane proteins is important for drug design and optimization. Although investigation using live cells is desirable, it is not feasible in some circumstances and cell fixation is performed to reduce cell motion and degradation. This study compared the effects of five fixatives, i.e., formaldehyde vapor (FV), paraformaldehyde (PFA), acetone, methanol, and ethanol, on kinetic measurements via the LigandTracer method. We found that all five fixatives exerted insignificant effects on lectin-glycan interaction. However, antibody-receptor interaction is markedly perturbed by coagulant fixatives. The acetone fixation changed the binding of the anti-human epidermal growth factor receptor 2 (HER2) antibody to HER2 on the cell membrane from a 1:2 to a 1:1 binding model, while methanol and ethanol abolished the antibody binding possibly by removal of the HER2 receptors on the cell membrane. The capability of binding was retained when methanol fixation was performed at lower temperatures, albeit with a binding model of 1:1 instead. Moreover, whereas cell morphology does not exert a substantial impact on lectin-glycan interaction, it can indeed modify the binding model of antibody-receptor interaction. Our results provided insights into the selection of fixatives for cell-based kinetic studies. Graphical Abstract
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ISSN:0022-2631
1432-1424
DOI:10.1007/s00232-024-00305-4