ION Thallos-HTL: a fluorescent thallium indicator that enables cell-selective and localizable thallium flux assays.

Ion channels are essential proteins for all organisms. Electrophysiology is a useful and commonly employed method to study ion channels, however there is a need for operationally simpler, cost-effective and higher throughput techniques to study ion channel functions in their native environments. Fluorescent ion indicators, such as Fluo-4 and Thallos, have been used for decades to study ion channel activity by measuring the flux of ions through channels of interest.

SNAP-tagging live cells chelation-assisted copper-catalyzed azide-alkyne cycloaddition.

SNAP-tag is a single-turnover enzyme that has become a powerful tool, hence a popular choice, of targeted cellular protein labeling. Three SNAP-tag substrates that carry the copper-chelating 2-picolyl azide moiety are prepared, one of which has an unconventional 5-pyridylmethyl-substituted guanine structure, rather than the usual benzylguanine that is optimized to be accepted by SNAP-tag. All three substrates are effective in transferring a 2-picolyl azide moiety to SNAP-tag in live cells under conventional labeling conditions (30-minute incubation of cells with labeling reagents at 37 °C under 5% CO).

Orthogonal Versatile Interacting Peptide Tags for Imaging Cellular Proteins.

Genetic tags are transformative tools for investigating the function, localization, and interactions of cellular proteins. Most studies today are reliant on selective labeling of more than one protein to obtain comprehensive information on a protein's behavior in situ. Some proteins can be analyzed by fusion to a protein tag, such as green fluorescent protein, HaloTag, or SNAP-Tag.

Expanding the substrate selectivity of SNAP/CLIP-tagging of intracellular targets.

SNAP-tag belongs to a class of genetic tools of protein labeling that complements fluorescent proteins. This single-turnover enzyme is a mutant of human DNA repair protein O-alkylguanine-DNA alkyltransferase (hAGT). It accepts, in most cases, label-carrying O-benzylguanines or benzyl-2-chloro-6-aminopyrimidines as suitable substrates.

SNAP/CLIP-Tags and Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC)/Inverse Electron Demand Diels-Alder (IEDDA) for Intracellular Orthogonal/Bioorthogonal Labeling.

Labeling a protein of interest (POI) with a fluorescent reporter is a powerful strategy for studying protein structures and dynamics in their native environments. Compared to fluorescent proteins, synthetic dyes provide more choices in photophysical or photochemical attributes to microscopic characterizations. The specificity of bioorthogonal reactions in conjunction with the fidelity of subcellular destinations of genetically encoded protein tags can be employed to label POIs in live and fixed cells in a two-step process.

Beyond O-Benzylguanine: O-(5-Pyridylmethyl)guanine as a Substrate for the Self-Labeling Enzyme SNAP-Tag.

SNAP-tag is a genetically encoded label for tracking proteins of interest that is known to utilize O-benzylguanine derivatives as substrates. In this work, mass spectrometric analysis revealed that SNAP-tag also accepts O-(5-pyridylmethyl)guanine derivatives as substrates. A fluorescently conjugated O-(5-pyridylmethyl)guanine was synthesized and used to selectively label intracellular compartments.

Progressive structural modification to a zinc-actuated photoinduced electron transfer (PeT) switch in the context of intracellular zinc imaging.

Photoinduced electron transfer (PeT)-type fluorescent molecular switches are often applied in ion-selective sensors. Zinc-targeting sensors that contain an anilino-based electron donor (aka, the PeT 'switch') have multiple advantages over those with an aliphatic amino switch. In addition to the lower pK value of an aniline than that of a comparably substituted aliphatic amine, which reduces the interference of pH on the spectral properties of the attached fluorophore, the oxidation potentials of anilino groups are lower than those of aliphatic amino counterparts, which make them better electron donors in PeT.