Table 1.

Commonly used methodologies and peptide tag sequences for labeling of membrane‐embedded proteins like G protein‐coupled receptors and receptor tyrosine kinases in live mammalian cell systems.

Labeling approach	Tag sequence	Labeling conditions for live cell application
Biotin ligase [60]	GLNDIFEAQKIEWHE (AP‐tag)	Biotinylation: 0.5 μM BirA, 10 μM biotin and 2 mM ATP in PBS (reaction time: 20 min at room temperature). [61] Biotin detection requires reporter‐equipped streptavidin. Ketone probe: 0.2 μM BirA, 1 mM ketone probe, and 1 mM ATP in DPBS (reaction time: 10–60 min at 32 °C). Subsequent ketone conversion requires 1 mM hydrazine probe (e. g., incubation for 10–60 min at 16 °C). [62]
Lipoic acid ligase[ 63 , 64 ]	DEVLVEIETDKAVLEVPGGEEE (LAP‐tag) GFEIDKVWYDLDA (LAP2‐tag)	High concentrations of enzyme (10 μM LplA) and lipoic acid derivative (350 μM azide substrate) and additional co‐factors (1 mM ATP) required for labeling within 1 h. [63] Probe detection depends on installed reactive handle.
Phosphopantetheinyl transferase[ 65 , 66 , 67 , 68 , 69 ]	DSLEFIASKLA (ybbR‐tag) GDSLSWLLRLLN (S6‐tag) GDSLDMLEWSLM (A1‐tag)	Labeling is performed within 30 min, using 5 μM CoA substrate, 1 μM enzyme, and 10 μM Mg2+).[ 65 , 68 ]
Transglutaminase[ 70 , 71 , 72 ]	PNPQLPF (Q1‐tag) PKPQQFM (Q2‐tag) GQQQLG (Q3‐tag)	Micromolar concentrations of the enzyme (∼1 μM) and the labeling agent (04–0.5 mM) in the presence of 12 mM CaCl2 (reaction time: ∼30 min at 4 or 37 °C). [72]
Transpeptidation by sortase A[ 73 , 74 , 75 , 76 , 77 , 78 ]	LPETGGn (n depending on the POI)	For protein labeling in live cells: 30 μM SrtA and 10 μM labeling agent (reaction time up to 1 h at 37 °C). [75]
Tubulin tyrosine ligase [79]	VDSVEGEGEEEGEE (Tub‐tag)	For protein modification, 1 μM tubulin tyrosine ligase, 1 mM tyrosine derivative, and 2.5 mM ATP (reaction time 1–3 h). [79] Subsequent reporter transfer onto the reactive handle depends on the bio‐orthogonal chemistry.
splitNluc complementation [80]	VSGWRLFKKIS (HiBiT‐tag)	Luminophore reassembly: addition of 10 nM LgBiT (15 min at 37 °C) prior to luminescence readout after furimazine addition. [81]
splitGFP reassembly [82]	NHYLSTQTVLSKDPNEKRDHMVLHEYVNAAGIT (GFP10‐11; shown without flanking Gly4)	Reporter reassembly: 2 μM of GFP 1–9 (reaction time: 20 min at 37 °C in PBS). [82]
SpyTag/SpyCatcher [83]	AHIVMVDAYKPTK	5 μM SpyCatcher‐Alexa Fluor 555 in PBS, supplemented with 5 mM Mg2+ (reaction time: 15 min at 25 °C). [83]
Heterodimerization of coiled‐coil peptides[ 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 ]	see Table 2
Fluorescein arsenical helix binder (FIAsH)[ 92 , 93 ]	CCXXCC (Tetracysteine motif)	Extracellular labeling: incubation with 5 mM MES and 0.5 mM TCEP (for 20–30 min) prior to addition of FlAsH reactant (2–5 μM). [92]
Ni2+ loaded NTA[ 94 , 95 ]	His6 or His10	15 nM of dye‐conjugated, Ni2+‐loaded NTA for noncovalent labeling in <15 min. [96] Covalent modification: pre‐incubation with 0.5 mM TCEP prior to addition of the labeling probe (0.5 μM) at room temperature. [95]
Split intein [97]	Gp41‐1 system: N‐intein, 88 aa; C‐intein, 37 aa	For live cell labeling: 0.5 μM of N‐intein, equipped with biotin, and 20 mM reduced glutathione (reaction time: 8 h). [98] Biotin detection requires reporter‐equipped streptavidin.