The mammalian ADP-ribosylation factor (Arf) subfamily of Ras-related small G-proteins was originally named for the ability to stimulate cholera toxin mediated ADP-ribosylation of the Gsα subunit utilized by many GPCRs (1). The Arf GTPases have been grouped into three classes based on their size and amino acid similarity (2): class I (Arf1 and Arf3), class II (Arf4 and Arf5) and class III (Arf6). Arf1 functions in the anterograde (and retrograde) transport of proteins from the endoplasmic reticulum (ER) through the Golgi apparatus to the plasma membrane by activating lipid modifying enzymes and recruiting proteins needed to promote secretory vesicle development, scission, and transport between secretory compartments (3, 4, and reviewed in 5). Arf1 has also been shown to function in the maintenance of structural integrity of the Golgi and ER (6).
Arf1, like other small G-proteins, cycles between the inactive GDP-bound and the active GTP-bound states. The preferential association of effector proteins with the GTP-bound over the GDP-bound state of Arf1 provides the basis for Arf1’s function in the cell. This highly specific association of effector proteins with Arf1-GTP has been exploited to develop affinity precipitation assays to monitor Arf1 activation (7).
Cytoskeleton’s Arf1 Activation Assay Biochem Kit™ utilizes the Arf1 protein binding domain (PBD) of the effector protein GGA3 (Golgi-localized γ-ear containing, Arf-binding protein 3), which has been shown to specifically bind the GTP-bound form of Arf1 (7, 8). We have covalently conjugated purified GGA3-PBD (amino acids 1-316) expressed in E. coli to the colored sepharose beads provided in this kit (Figure 1). Using these beads, the researcher is able to “pull-down” Arf1-GTP and quantify the level of active Arf1 with a subsequent Western blotting step using the Arf1 specific antibody provided in this kit. This assay provides a simple means of analyzing cellular Arf1 activation levels in a variety of systems. A typical Arf1 pull-down assay is shown in Figure 2 below using either GTPgS and GDP loaded MDCK cell extracts or extracts from C2C12 myoblasts that have been induced to differentiate by serum starvation.
The kit contains sufficient materials for 20 assays, depending on assay setup, and includes reagents for positive and negative controls. The following components are included:
|Figure 1. The brightly colored glutathione agarose beads in BK032-S makes the kit easy to use.|
A typical Arf1 pull-down assay is shown in Figure 2 using either GTPγS and GDP loaded MDCK cell extracts or extracts from C2C12 myoblasts that have been induced to differentiate by serum starvation.
Figure 2. Arf1 Activation Assay Biochem Kit™ Pull-down Assay Results.
Top: MDCK cell lysates (500 µg) loaded with GTPγS or GDP using the method described in Section VI: Control Reactions (see product manual).
Bottom: C2C12 cell lysates (500 µg) from untreated cells (FBS) or cells that were serum starved for 1h (SF). All extracts were incubated with 20 μg of GGA3-PBD beads and processed as described in Section VI: Pull-down Assay. All bead samples were resuspended in 20 µl of 2x sample buffer and then separated on a 4-20% SDS-PAGE gel, transferred to PVDF, probed with a 1:250 dilution of anti-Arf1 antibody, and processed for chemiluminescent detection as described in Section VI: STEP 4 (see product manual).