Click Chemistry (CuAAC) and Detection of Tagged de novo Synthesized Proteins in Drosophila

Abstract

Copper-catalyzed azide-alkyne-cycloaddition (CuAAC), also known as 'click chemistry' serves as a technique for bio-orthogonal, that is, bio-compatible labeling of macromolecules including proteins or lipids. Click chemistry has been widely used to covalently, selectively, and efficiently attach probes such as fluorophores or biotin to small bio-orthogonal chemical reporter groups introduced into macromolecules. In bio-orthogonal non-canonical amino acid tagging (BONCAT) and fluorescent noncanonical amino acid tagging (FUNCAT) proteins are metabolically labeled with a non-canonical, azidebearing amino acid and subsequently CuAAC-clicked either to an alkyne-bearing biotin (BONCAT) for protein purification, Western blot, or mass spectrometry analyses or to an alkyne-bearing fluorophore (FUNCAT) for immunohistochemistry. In combination with mass spectrometry, these kinds of labeling and tagging strategies are a suitable option to identify and characterize specific proteomes in living organisms without the need of prior cell sorting. Here, we provide detailed protocols for FUNCAT and BONCAT click chemistry and the detection of tagged de novo synthesized proteins in Drosophila melanogaster.

Interactive nanocluster compaction of the ELKS scaffold and Cacophony Ca 2+ channels drives sustained active zone potentiation

“…We then used sptPALM to follow functional Cac channels in AZs over time. For this, we modified a preparation described previously ( 21 ), allowing for the use of the oblique illumination profiles of a total internal reflection fluorescence (TIRF) microscope to induce stochastic blinking of individual CacmEOS4b molecules in a focal plane across several boutons of a given NMJ (Fig. 1, F to J, and fig.…”

An active zone state switch concentrates and immobilizes voltage-gated Ca2+ channels to promote long-term plasticity

“…We then employed sptPALM to follow functional Cac channels in AZs over time. For this, we modi ed a preparation described previously 20 , allowing for the use of the oblique illumination pro les of a TIRF microscope to induce stochastic blinking of individual CacmEOS4b molecules in a focal plane across several boutons of a given NMJ (Fig. 1F-J, Suppl.…”

“…All live sptPALM experiments were conducted on male third instar larvae. Larval body walls designated for single particle tracking were prepared according to Ramachandran and Budnik 2010 and Marter et al, 2019 20,63 and imaging experiments were performed using an inverted total internal re ection uorescence (TIRF) setup. The microscope (Nikon Eclipse Ti) was equipped with a 100x NA 1.49 Apo TIRF oil objective (Nikon).…”

“…Larvae were placed dorsal side up into the chamber and clips consisting of stainless pins with tinmade holders on the magnetic foil were used to clamp the animals at either end. Dissections were then carried out as described in 20,63 Ca 2+ and Mg 2+ free HL3.1 saline (in mM: NaCl 70, KCl 5, NaHCO3 10, Sucrose 115, Trehalose 5, HEPES 5). Subsequently, the larvae were washed brie y in HL3.1 imaging buffer containing 4 mM Mg 2+ and 1.5 mM Ca 2+ for all control situations and/or 0.2 mM for the experiment in Suppl.…”

Rapid Cell Type-Specific Nascent Proteome Labeling in Drosophila

“…The protocol is modified from Marter et al (45). Brains were thawed on ice and homogenized in 100 µL of homogenization buffer (0.5%SDS/PBS containing 2X Complete EDTA-free Protease Inhibitor Cocktail).…”