The Thermo Scientific Pierce Active Arf1 Pull-Down and Detection Kit is a complete kit for selective enrichment and detection of GTP-bound Arf1 GTPase through specific protein interaction with the GGA3 protein-binding domain.
The Active Arf1 Pull-Down and Detection Kit includes purified GST-GGA3 protein-binding domain (PBD), glutathione agarose resin, positive and negative controls (GTPγS and GDP, respectively), lysis/binding/wash buffer, anti-Arf1 primary antibody, sample buffer, spin columns and collection tubes. The kit was validated using lysates from MDCK cells, a cell line that is known to have robust Arf1 activity.
- Highly sensitive and accurate – optimized reagents, specific anti-Arf1 antibody and Western blot procedure ensure accurate controls and semi-quantitative results
- Validated – functionally tested for Arf1 detection to ensure quality and performance
- Compatible – effective with a variety of cell types from mouse, rat and human sources
- Follow activation of Arf1 GTPase during cell differentiation, migration, division and cytoskeletal rearrangement
- Study the activation of Arf1 during the assembly of coat proteins onto budding vesicles or trans-golgi network and endosomes
- Monitor Arf1 activity after stimulation with growth factors
- Monitor Arf1 activity after small molecule inhibitor treatment
The Active Arf1 Pull-Down and Detection Kit was validated for function and specificity of the active Arf1 enrichment method using cell lysates treated with GTPγS to activate endogenous Arf1 and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment traps Arf1 in the GTP-bound form (active), resulting in a strong signal when endogenous Arf1 is present. GDP treatment pushes Arf1 into the GDP-bound state (inactive), resulting in minimal or no signal, regardless of Arf1 protein levels. The kit is optimized for Western blot detection using an HRP-conjugated secondary antibody (Goat Anti-rabbit IgG, Part No. 31460) and Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080). The kit contains sufficient components for 30 pull-down assays.
|Arf1 response after hepatocyte growth factor (HGF) stimulation. MDCK cells (canine) were stimulated with 50ng/mL of HGF after 24-hour serum starvation. Panel A: Western blots of active Arf1 isolated using the Active Arf1 Pull-Down and Detection Kit (top) and total Arf1 levels (bottom) during 6 hours of HGF stimulation. Non-treated cells were used as a control (c). Panel B: Densitometry of the active Arf1 Western blot bands reveals the biphasic activation of Arf1 in response to HGF stimulation.
|Arf1 is active during serum starvation and associated with actin filaments. Panel A: C2C12 mouse muscle cells were differentiated by serum starvation, fixed in 4% paraformaldehyde and stained using anti-Arf1 antibody and BODIPY* 558/568 phalloidin to detect actin filaments. Arf1 was detected using Thermo Scientific DyLight 488-conjugated Goat Anti-rabbit IgG. Panel B: C2C12 cells were serum-starved then stimulated with 10% serum. The Western blot compares active Arf1 protein isolated by pull-down assays (top panel) to total Arf1 levels (bottom panel) during 1 hour of serum stimulation. Non-starved cells were used as a control (c).
ADP ribosylation factor proteins 1-6 (Arfs) are members of the Ras family of small GTPases. Although structurally similar, the cellular roles of Arf1-6 are different from the other Arf family members; their endogenous roles are not ADP ribosylation, but rather regulation of heterotrimeric G proteins. The Arf proteins can be divided into three classes: Class I – Arf 1-3; Class II – Arf 4,5; Class III – Arf6. Class I and II Arfs are associated with trans-Golgi network (TGN) and are involved in recruiting effector proteins to the Golgi membrane and forming vesicles. Unlike other GTPases, Arf GTPases are modified by myristoylation at the amino-terminus to allow insertion into the membrane. The Class III protein Arf6 is associated with the plasma membrane and is involved in vesicle formation at the plasma membrane, vesicle recycling and remodeling of the actin cytoskeleton.
Although the Arf GTPases are expressed in all cells, Arf1 is the most abundant, active and best-characterized. Arf1 recruits its effectors, including coatomer and clathrin adaptor complex (AP), to the Golgi for vesicle formation and budding. After recruitment, Arf1 maintains the coat proteins on the membrane until the vesicle is formed. Arf1 then interacts with its effector resulting in the release of Arf1 from the membrane and vesicle budding. The golgi-localized γ-ear-containing Arf (GGA) effector binding proteins contain an amino-terminal VHS domain, a GGA homology domain (GGAH), a proline-rich linker region and a carboxy-terminal γ-ear adaptin homology domain (AGEH). The VHS domain interacts with cytoplasmic domains for receptor sorting, the GGAH domain binds activated Arf, the proline-rich region interacts with clathrin and the AGEH domain interacts with cytosolic proteins. Through mutational studies, the GGAH domain has been shown to be sufficient to bind GTP-bound Arf, and Arf1 binds all three GGA effector proteins. The association of the GGA proteins with the TGN and with the plasma membrane is regulated by the Arf.
- Boulay, P.L., et al. (2008). ADP-ribosylation factor 1 controls the activation of the phosphatidylinositol 3-kinase pathway to regulate epidermal growth factor-dependent growth and migration of breast cancer cells. J Biol Chem 283:36425-34.
- Gillingham, A.K. and Munro S. (2007). The small G proteins of the Arf family and their regulators. Annu Rev Cell Dev Biol 23:579-611.
- Donaldson, J.G. and Honda A. (2005). Localization and function of Arf family GTPases. Biochem Soc Trans 33:639-42.
- Yoon, H.Y., et al. (2005). In vitro assays of Arf1 interaction with GGA proteins. Methods Enzymol 404:316-32.
- Takatsu, H. et al. (2002). GGA proteins associate with golgi membranes through interaction between their GGAH domains and APD-ribosylation factors. Biochem J 365:369-78.
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