The Thermo Scientific Pierce Active Arf6 Pull-Down and Detection Kit is a complete kit for selective enrichment and detection of GTP-bound Arf6 GTPase through specific protein interaction with the GGA3 protein-binding domain.
The Active Arf6 Pull-Down and Detection Kit includes purified GST-GGA3 protein-binding domain (PBD), glutathione agarose resin, positive and negative controls (GTPyS and GDP, respectively), lysis/binding/wash buffer, anti-Arf6 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 Arf6 activity.
- Highly sensitive and accurate – optimized reagents, specific anti-Arf6 antibody and Western blot procedure ensure accurate controls and semi-quantitative results
- Validated – functionally tested for Arf6 detection to ensure quality and performance
- Compatible – effective with a variety of cell types from mouse, rat and human sources
- Follow activation of Arf6 GTPase during cell differentiation, migration, division and cytoskeletal rearrangement
- Study the activation of Arf6 during membrane trafficking
- Monitor Arf6 activity after stimulation with growth factors
- Monitor Arf6 activity after small molecule inhibitor treatment
The Active Arf6 Pull-Down and Detection Kit was validated for function and specificity of the active Arf6 enrichment method using cell lysates treated with GTPγS to activate endogenous Arf6 and compared to lysates treated with GDP to inactivate the small GTPase. GTPγS treatment traps Arf6 in the GTP-bound form (active), resulting in a strong signal when endogenous Arf6 is present. GDP treatment pushes Arf6 into the GDP-bound state (inactive), resulting in minimal or no signal, regardless of Arf6 protein levels. The kit is optimized for Western blot detection using an HRP-conjugated secondary antibody (Goat Anti-mouse IgG, Part No. 31430) and Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080). The kit contains sufficient components for 30 pull-down assays.
||Biphasic activity of Arf6 in MDCK cells. MDCK cells (canine) were stimulated with 50ng/mL of HGF after 24-hour serum starvation. The Western blots compare active Arf6 isolated using the Active Arf6 Pull-Down and Detection Kit (top) to total Arf6 levels (bottom) during 6 hours of HGF stimulation. Non-starved cells were used as a control (c).
|Arf6 is highly active and associated with actin in differentiating muscle cells. Panel A: C2C12 mouse muscle cells were differentiated by serum-starvation for 48 hours and assayed for Arf6 and Rac1 activity by pull-down assays. Total protein (1mg) was used per pull-down reaction and half of the elution fraction was analyzed by Western blot (top) and compared to the levels of total Arf6 and Rac1 from cell lysate (bottom) for both control and differentiated cell samples. Panel B: Arf6 localization was compared to Rac1 using the anti-Arf6 antibody and an anti-Rac1 antibody supplied in the kit and detected using Thermo Scientific DyLight 488-conjugated Goat Anti-mouse IgG. Actin filaments were stained with BODIPY* 558/568 phalloidin. Nuclei were stained with Hoechst 33347.
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 ubiquitously in all cells, Arf6 is less abundant and its cellular localization is cell type-dependent. Arf6 is differentiated from the other Arf proteins due to its plasma membrane association and its involvement in the regulation of receptor mediated endocytosis and remodeling of the actin cytoskeleton. Arf6 interacts with the Arf GAP effectors ACAP1, ACAP2, Git1, Git2, ASAP1 and ASAP2 for plasma membrane rearrangement. Arf6 is also involved the phospholipid metabolism. Another class of effector proteins is the Golgi-localized γ-ear-containing Arf (GGA) binding proteins. The GGA 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 is sufficient to bind GTP-bound Arf, and in vitro Arf6 binds GGA1 with the highest affinity.
- 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 ADP-ribosylation factors. Biochem J 365:369-78.
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