Which amino acids are often target of protein labelling?
How can the labels be introduced?
Trp and Tyr
Cys
Lys
Name bioorthogonal labelling strategies.
What kinds of enzyme and peptide tags do you know?
SNAP/CLIP tags
mutants of O6-alkylguanine-DNA-alkyltransferase
both react with specific molecules
SNAP: benzyl guanine
CLIP: O2-benzyl cytosine
SNAP and CLIP are ligated with protein of interest and can be labelled very easily with labels that have a benzyl guanine/O2-benzyl cytosine handle
Halo-tags
Coiled-coil tags
Protein of interest and label have coiled-coil interaction that leads to covalent ligation
What is affinity-based labelling?
tag consists of suicide-inhibitor for target enzyme, a linker region, and a reporter group (e.g. fluorophor)
What is the problem with fluorogenic probes and how can it be solved?
fluorescence needs to be quenched reliably when not activated by target group
otherwise: background fluorescence due to unspecific interactions, unsufficient contrast
Therefore, additional quencher (not just fluorescence label and substrate part) is needed
Bioorthogonal labeling: (some) fluorophores are quenched by the active groups (azide, tetrazine)
Peptide and protein tags:
FlAsH labelling
Snap-tag: guanine is modified with quencher
Compare the advantages and disadvantages of different labelling strategies.
Last changeda year ago
