Fig. 1.
Isolation of mAb16 and its binding profile to each component. a Analytical SEC of rhodopsin/Gi1 with each antibody. Rhodopsin/Gi1 runs at 8.2 mL and each mAb alone runs 8.4–9 mL (c). Rhodopsin/Gi1 bound to mAb makes higher molecular weight product and migrates at the elution volume of 6–7 mL depending on the mAb. b Analytical SEC of rhodopsin/Gi1 with each antibody following to GTPγS treatment. Intact complex remains at 6–7 mL only in the mAb16 condition. c Analytical SEC of individual component of rhodopsin/Gi1 or heterotrimeric Gi1 with each antibody. Top left: Binding experiment with Gαi1 subunit and each mAb. The peak of Gαi1 at 11 mL stays intact indicating there is no binding with each mAb. Top right: Binding experiment with opsin. Both mAb peaks and Opsin peak (at 9.5 mL) stays intact. Bottom left: Binding experiment with Gβγ subunit. The peak of Gβγ at 11.2 mL disappears upon incubating with mAbs except mAb16 and each mAb peak shifts towards left compared to the ones with Gαi1 or opsin indicating those mAbs recognize Gβγ subunit as an epitope. Bottom right: Binding experiment with heterotrimeric Gi1