Product Uses Include
- Analysis of in vivo Arf1 activation.
IntroductionThe 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.
Kit contentsThe kit contains sufficient materials for 20 assays, depending on assay setup, and includes reagents for positive and negative controls. The following components are included:
- GGA3-PBD beads (Cat. # GGA05)
- Arf1 monoclonal antibody (Cat. # ARF01)
- His-tagged Arf1 protein
- GTPγS: (non-hydrolyzable GTP analog) (Cat. # BS01)
- GDP
- Cell lysis Buffer
- Wash Buffer
- Loading Buffer
- STOP Buffer
- Protease inhibitor cocktail (Cat. # PIC02)
- DMSO
- Manual with detailed protocols and extensive troubleshooting guide
| Figure 1. The brightly colored glutathione agarose beads in BK032-S makes the kit easy to use. |
Example results
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).
References
- 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.
- 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.
- Wang, G., and Wu, G. (2012) Small GTPase regulation of GPCR anterograde trafficking. Trends Pharmacol. Sci. 33, 28-34.
- 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.
- D’Souza-Schorey, C., and Chavrier, P. (2006) ARF proteins: Roles in membrane traffic and beyond. Nat. Rev. Mol. Cell Biol. 7, 347-358.
- 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.
- Yoon, HY, Bonifacino, J. S., and Randazzo, P. A. (2005) In Vitro Assays of Arf1 Interaction with GGA Proteins. Methods Enzymol. 404, 316-332.
- 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.
Yu, Q. et al. A NAV2729-sensitive mechanism promotes adrenergic smooth muscle contraction and growth of stromal cells in the human prostate. J. Biol. Chem. 294, 12231–12248 (2019). Abdul-Salam, V. B. et al. CLIC4/Arf6 Pathway: A New Lead in BMPRII Inhibition in Pulmonary Hypertension. Circ. Res. 124, 52–65 (2019). RIOS, A. et al. Participation of Rho, ROCK-2, and GAP activities during actin microfilament rearrangements in Entamoeba histolytica induced by fibronectin signaling. Cell Biol. Int. 32, 984–1000 (2008). |
Coming soon! If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
Cytoskeleton公司成立于1993年，专注于生物化学和细胞过程研究中的纯化蛋白和便捷试剂盒开发与生产。公司提供药物筛选、信号转导、细胞骨架研究相关的系列试剂盒和产品，尤其以细胞骨架相关研究见长，既能满足于样品较少的科学研究，也可以用于小规模筛选研究和高通量大规模筛选研究。此外，公司还提供微管蛋白，肌动蛋白，小G蛋白，GAPs，GEFs等现有产品的药物筛选服务。
本网站将在规定时间内给予删除等相关处理。
