Arf1 Pulldown Activation Assay Kit

Product Uses Include

• Analysis of in vivo Arf1 activation.

Introduction
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 G_s? 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 ?-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.

Kit contents
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:

1. GGA3-PBD beads (Cat. # GGA05)
2. Arf1 monoclonal antibody (Cat. # ARF01)
3. His-tagged Arf1 protein
4. GTP?S: (non-hydrolyzable GTP analog) (Cat. # BS01)
5. GDP
6. Cell lysis Buffer
7. Wash Buffer
8. Loading Buffer
9. STOP Buffer
10. Protease inhibitor cocktail (Cat. # PIC02)
11. DMSO
12. Manual with detailed protocols and extensive troubleshooting guide

Example results

A typical Arf1 pull-down assay is shown in Figure 2 using either GTP?S 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 ug) loaded with GTP?S or GDP using the method described in Section VI: Control Reactions (see product manual).

Bottom: C2C12 cell lysates (500 ug) from untreated cells (FBS) or cells that were serum starved for 1h (SF). All extracts were incubated with 20 ?g of GGA3-PBD beads and processed as described in Section VI: Pull-down Assay. All bead samples were resuspended in 20 ul 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).

References

1. Kahn, R. A., and Gilman, A. G. (1986) The Protein Cofactor for ADP-ribosylation of Gs by Cholera Toxin is Itself a GTP Binding Protein. J. Biol. Chem. 261, 7906-7911.
2. Tsuchiya, M., Price, S. R., Tsai, S-C, Moss, J. and Vaughan, M. (1991) Molecular Identification of ADP-Ribosylation Factor mRNAs and Their Expression in Mammalian Cells. J. Biol. Chem. 266, 2772-2777.
3. Wang, G., and Wu, G. (2012) Small GTPase regulation of GPCR anterograde trafficking. Trends Pharmacol. Sci. 33, 28-34.
4. Volpicelli-Daley, L. A., Li, Y., Zhang, C-J, and Kahn, R. A. (2005) Isoform-selective Effects of the Depletion of ADP-Ribosylation Factors 1-5 on Membrane Traffic. Mol. Biol. Cell. 16, 4495-4508.
5. D'Souza-Schorey, C., and Chavrier, P. (2006) ARF proteins: Roles in membrane traffic and beyond. Nat. Rev. Mol. Cell Biol. 7, 347-358.
6. Lippincott-Schwartz, J., Cole, N. B., and Donaldson, J. G. (1998) Building a secretory apparatus: role of ARF1/COP1 in Golgi biogenesis and maintenance. Histochem. Cell Biol. 109, 449-462.
7. Yoon, HY, Bonifacino, J. S., and Randazzo, P. A. (2005) In Vitro Assays of Arf1 Interaction with GGA Proteins. Methods Enzymol. 404, 316-332.
8. Takatsu, H., Yoshino, K., Toda, K., and Nakayama, K. (2002) GGA proteins associate with Golgi membranes through interaction between their GGAH domains and ADPribosylation factors. Biochem. J. 365, 369-378.

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