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Simple route of caspase-3 FRET sensor synthesis using “click chemistry”
Lišková, Marcela ; Křenková, Jana ; Klepárník, Karel ; Pazdera, P. ; Foret, František
Programmed cell death or apoptosis is regulated process of cell suicide. The central role in apoptosis play cysteine proteases called caspases. Caspases recognize tetra-peptide sequences Asp-Glu-Val-Asp (DEVD) on their substrates and hydrolyze peptide bonds after aspartic acid residues. Various techniques for the determination of caspase-3 are commercially available e.g. Enzyme Linked Immuno-Sorbent Assay (ELISA), Western blotting or flow cytometric analysis. The products of the cleavage can be detected by spectrophotometry, fluorimetry, chemiluminescence (CL) or ELISA. In this work, we suggested fluorescent sensor based on easily prepared Förster Resonance Energy Transfer (FRET). We use very simple chemistry called “click”. This type of chemistry takes advantages of quickness, simplicity and cheapness. “Click chemistry” is based on usage of various functional group and cross-linkers to combine individual molecules together.
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