Arf1 and Arf6 Activation Assays and
GTPase Affinity Beads
Cytoskeleton’s Arf1 and Arf6 activation assays are in two formats: G-LISAs and pull-downs. G-LISAs use a 96 well plate coated with an effector protein that selectively binds active Arf1 or Arf6, followed by quantitation with ELISA techniques. Typical Arf6 G-LISA data are shown (right). Arf pull-downs utilize the Arf1 and Arf6 protein binding domain (PBD) of the effector protein GGA3 (Golgi-localized γ-ear containing, Arf-binding protein 3), which specifically binds the GTP-bound form of Arf1 and Arf6. The GGA3-PBD is covalently conjugated to colored sepharose beads, enabling the “pull-down”of active Arf1-GTP and Arf6-GTP for quantification by Western blotting.
Right: Arf6 activation measured by G-LISA. Lysates for Arf6 G-LISA were prepared from MDCK cells that were either attached to tissue culture plates (activated) or kept in suspension for 2 hours (control). 12.5, 6.25, and 3.1 μgs of cell lysates were subjected to the G-LISA assay (Cat. # BK133). Absorbance was read at 490 nm. Data are background subtracted.
- Arf1 G-LISA Activation Assay Biochem Kit (Cat. # BK132)
- Arf1 Activation Assay Biochem Kit (Pull-down format) (Cat. # BK032-S)
- Arf6 G-LISA Activation Assay Biochem Kit (Cat. # BK133)
- Arf6 Activation Assay Biochem Kit (Pull-down format) (Cat. # BK033-S)
- GGA3-PBD Beads (Cat. # GGA05)
For more information about the Arf small G-protein click here.