Studying synaptic proteins, which are regulating synaptic transmission and organization, is a challenging task as traditional localization and staining methods face limitations in mapping proteins in living cells. For example, traditional antibodies are too large to freely diffuse into the extracellular protein-enriched synaptic cleft and the use of detergents for improving cell penetration cannot be applied in living cells and damages synaptic membrane integrity. We are particularly interested in understanding the mechanism of calcium-regulated neurotransmitter release and synapse formation by voltage-gated calcium channels (CaV) and their auxiliary subunits α2δ. Mutations in genes encoding α2δ isoforms are linked to neurological and neuropsychiatric disorders such as epilepsy, schizophrenia, and autism spectrum disorders. Up to now, the proteins interacting with α2δ inside the synaptic cleft are still not well known. Within this project, we are aiming to use single domain antibodies (nanobodies) to target synaptic proteins without using detergents in live cell conditions. We have successfully cloned an epitope tag (ALFA) into an extracellularly exposed position within α2δ isoforms and confirmed highly specific immunostaining in hippocampal neurons, using purified anti-ALFA-mCherry nanobodies. Most importantly, patch-clamp analysis of tsA‑201 transfected with CaV2.1 and β subunits revealed similar current modulation by ALFA-tagged α2δ isoforms when compared to untagged α2δ-proteins. Ultimately, we are aiming to fuse an APEX2 enzyme to the anti-ALFA nanobody to biotin label extracellular proteins in close proximity to α2δ in live cell conditions. This approach will pave the way to identify and characterize new protein complexes involved in synapse formation and modulation.