Dual labelling with a modified α-bungarotoxin binding site

Labelling of multiple proteins for visualisation using microscopy can be challenging. Here we describe a method for dual labelling proteins using a modified α-bungarotoxin binding site method

To develop a dual labelling strategy using α-bungarotoxin, we substituted two vicinal serines in the α-bungarotoxin (BTX) binding site (BBS) of R1^BBS for cysteines (R1a^BBS-CC).

By covalent labeling of the Cys residues using a sulfhydryl reagent (MTSES), the binding of BTX to R1 was prevented, whereas binding to the wild-type BBS on R2 could proceed unhindered. Subsequent removal of MTSES and exposure to a second flourophore-conjugated BTX allowed labelling of R1 subunits.

Using this strategy we found that R1 and R2 subunits are internalized as heterodimers.

For more details please visit the publication:

S Hannan, ME Wilkins, E Dehghani-Tafti, P Thomas, SM Baddeley & TG Smart (2011). γ-Aminobutyric Acid Type B (GABA_B) Receptor Internalization Is Regulated by the R2 Subunit. Journal of Biological Chemistry 286, 24324-24335.