• Affinity reagent for isolation of activated (GTP-bound) Arf1 and Arf6 proteins in cell and tissue lysates.

The Arf1 & Arf6 binding domain of Golgi-localized γ-ear containing Arf-binding protein 3 (GGA3) PBD has been overexpressed as a GST-tagged recombinant protein in a bacterial expression system and bound to colored glutathione Sepharose beads. The GGA3-PBD protein (amino acids 1-316) plus the GST-tag results in a protein of approximately 60 kD. GGA3-GST protein beads (500 µg protein per tube) are supplied as a pink/purple lyophilized powder. One tube of GGA07 is sufficient for approximately 25 assays.

GGA3-PBD beads selectively bind the active (GTP-bound) forms of Arf1 and Arf6, showing minimal affinity for their inactive (GDP-bound) forms. In a standard assay (20 µl beads, 500 µg lysate), lysates from serum-fed and serum-starved C2C12 cells are compared. After isolating activated Arf1/6, western blotting with Arf1- or Arf6-specific antibodies is performed. Serum-free conditions typically produce an Arf1 activation signal twice as strong as that from serum-containing cells.