Immunostaining of PFA-fixed COS7 cells with an anti-Tubulin primary antibody (Synaptic Systems, Cat. No. 302211, 1:1000). The primary antibody was pre-incubated with sdAb anti-mouse IgG1 Halo fusion (N2041, NanoTag Biotechnologies) and the corresponding Halo ligand Atto488 . Subsequently, the sample was stained with FluoTag®-X4 anti-Halo Alexa Fluor647 (N5004-AF647, NanoTag Biotechnologies, 1:1000) and imaged by confocal microscopy. A Staining with FluoTag®-X4 anti-Halo Alexa Fluor647 (N5004-AF647, NanoTag Biotechnologies, 1:1000), false color representation: magenta. B Staining with a Halo ligand Atto488, false color representation: green. C Merge of A and B with the corresponding DAPI signal (blue). Scale bar: 10 µm.

Immunostaining of PFA-fixed HeLa cells stably expressing a non-fluorescent mRuby-ALFA fusion protein with mitochondrial localization. A Staining with the FluoTag®-X2 anti-mRuby Alexa Fluor647 (Cat. No. N3302-AF647, dilution 1:500), false color representation magenta. B Co-staining performed with sdAb anti-ALFA Halo fusion (Cat. No. N1541, dilution 1:500), detected with FluoTag®-X4 anti-Halo Atto488 (N5004-At488, dilution 1:500), false color green. C Merge of A and B with the corresponding DAPI signal (blue). Scale bar: 10µm D Control staining of mRuby-ALFA positive-HeLa cells without N1541.

Kinetic analysis of the interaction between immobilized sdAb anti-Halo clone 1A12 (biotinylated, 5 µg/mL) and a Halo-tagged analyte (20–0.3125 nM), measured by Bio-Layer Interferometry (BLI) using Streptavidin biosensors. Association and dissociation phases (colored traces) were globally fitted (black lines) using a 1:1 binding model. The resulting kinetic parameters indicate high-affinity binding with an association rate constant kon = (2.96 ± 0.17) × 10⁵ M⁻¹ s⁻¹ and a dissociation rate constant koff = (5.21 ± 0.01) × 10⁻⁵ s⁻¹ corresponding to an equilibrium dissociation constant KD = 176.9 ± 0.2 pM.

Kinetic analysis of the interaction between immobilized sdAb anti-Halo clone 1C3 (biotinylated, 5 µg/mL) and a Halo-tagged analyte (20–0.3125 nM), measured by Bio-Layer Interferometry (BLI) using Streptavidin biosensors. Association and dissociation phases (colored traces) were globally fitted (black lines) using a 1:1 binding model. The resulting kinetic parameters indicate high-affinity binding with an association rate constant kon = (2.52 ± 0.02) × 10⁵ M⁻¹ s⁻¹ and a dissociation rate constant koff = (6.21 ± 0.01) × 10⁻⁵ s⁻¹, corresponding to an equilibrium dissociation constant KD = 246.1 ± 0.4 pM.