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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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Iron oxide nanoparticle modified monolithic pipette tips for selective enrichment of phosphopeptides
Křenková, Jana ; Foret, František
We have developed iron oxide nanoparticle modified monolithic pipette tips for selective and efficient enrichment of phosphopeptides. Iron oxide nanoparticles were synthesized using a co-precipitation method and stabilized by citrate ions. A stable coating of nanoparticles was obtained via multivalent interactions of citrate ions on the nanoparticle surface with a quaternary amine functionalized surface of the methacrylate based monolithic tips. The performance of the developed and commercially available tips was demonstrated with the enrichment of phosphorylated peptides from tryptic digests of caseins followed by MALDI/MS detection.
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Oriented immobilization of PNgase F on a porous polymer monolith for rapid N-glycan release
Szekrényes, A. ; Křenková, Jana ; Keresztessy, Z. ; Foret, František ; Guttman, A.
We demonstrate a simple and rapid method for the oriented immobilization of peptide-N4-(Nacetyl- glucosaminyl) asparagine amidase F (PNGase F) on a porous polymer monolith. The oriented immobilization is based on the affinity of glutathione-S-transferase (GST) tagged PNGase F towards glutathione modified monolith prepared in the capillary format. This approach allows the oriented and easily replaceable immobilization of PNGase F for rapid and efficient release of N-linked glycans. The reactor was tested by deglycosylation of several native glycoproteins such as ribonuclease B, fetuin or human immunoglobulin G. The proteins were effectively deglycosylated in several minutes and the released N-linked glycans were analyzed using off-line MALDI/MS or CE/LIF.
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