The bulb-shaped invaginations on cell membrane surface, caveolae, are abundant in many mammalian cells and are involved in a number of cellular responses. The formation of the membrane invaginated caveolae has been shown to be mainly driven by two families of proteins, caveolins and cavins, and their coordinated interactions with lipids. All cavin proteins share a common pattern of possessing two highly conserved positively charged helical regions 1 (HR1) and 2 (HR2) interspersed with three poorly conserved and negatively charged disordered regions (DR1, DR2 and DR3). Here, I have investigated novel nanobodies as potent ligands against trimeric coiled-coil mouse Cavin1 HR1 domain, and tools for structural determination and cellular co-localisation. Two nanobody binders (A12 and B7) of the trimeric coiled-coil mouse Cavin1 HR1 were produced, and their interaction was validated by Isothermal Titration Calorimetry that both nanobodies bound with HR1 in nanomolar affinities with a 1 to 1 binding stoichiometry. Both nanobodies were found to bind primarily with N-terminal half of HR1. I have also validated the interaction of both nanobodies with other cavin proteins by ITC to test the cross-reactivity and binding specificity. To understand the molecular mechanism behind their interaction, the formation of nanobody in complex with mouse Cavin1 HR1 domain was confirmed by Western blot and mass photometry and X-ray crystallography was applied to observe the overall architecture. To understand the physiological impact of nanobodies on functional cavin coat complex in situ, I cloned both nanobodies in pEGFP-N1 vector and investigate co-localisation of nanobodies with Cavin1 in mammalian cells. B7 nanobody-eGFP appeared to be recruited to typical caveolae puncta, co-localising with endogenous Cavin1 at the plasma membrane whereas A12 nanobody-eGFP diffused to the cytosol. This analysis will provide a critical insight into the molecular underpinnings of cavin proteins using nanobodies as a tool.