Product Pathways - Cytoskeletal Signaling
Active Arf1 Detection Kit #8816
|GTP γS||50 µl|
|Arf1 Rabbit mAb||25 µl|
|Glutathione Resin||3 ml|
|SDS Sample Buffer||1.5 ml|
|Lysis/Binding/Wash Buffer||100 ml|
|Spin Cup and Collection Tubes||30 Ea|
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.
The Active Arf1 Detection Kit provides all reagents necessary for measuring activation of Arf1 GTPase in the cell. GST-GGA3-PBD fusion protein is used to bind the activated form of GTP-bound Arf1, which can then be immunoprecipitated with glutathione resin. Arf1 activation levels are then determined by western blot using an Arf1 Rabbit mAb.
Specificity / Sensitivity
Active Arf1 Detection Kit detects endogenous levels of GTP-bound (active) Arf1 as shown in Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Figure 1. NIH/3T3 cell lysates (500 µl at 1 mg/ml) were treated in vitro with GTPγS or GDP to activate or inactivate Arf1 (refer to optional step C in protocol). The lysates were then incubated with glutathione resin and GST-GGA3-PBD (lanes 2 and 3). GTPγS-treated lysate was also incubated without GST-GGA3-PBD in the presence of glutathione resin as a negative control (lane 4). Western blot analysis of cell lysate (20 µg, lane 1) or 20 µl of the eluted samples (lanes 2, 3, and 4) was performed using an Arf1 Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody. Detection was performed using 1X LumiGLO® reagent and peroxide (20X LumiGLO® Reagent and 20X Peroxide #7003), followed by exposure to x-ray film.
Figure 2. The GTP-bound GTPase pull-down process can be divided into 3 steps as shown. Step 1: Mix sample, binding protein, and glutathione resin in the spin cup and incubate at 4ºC to allow GTP-bound GTPase binding to the glutathione resin through GST-linked binding protein. Step 2: Remove unbound proteins by centrifugation. Step 3: Elute glutathione resin-bound GTPase with SDS buffer. The eluted sample can then be analyzed by western blot.
The Ras superfamily of small GTP-binding proteins (G proteins) comprise a large class of proteins (over 150 members) that can be classified into at least five families based on their sequence and functional similarities: Ras, Rho, Rab, Arf, and Ran (1-3). These small G proteins have both GDP/GTP-binding and GTPase activities and function as binary switches in diverse cellular and developmental events that include cell cycle progression, cell survival, actin cytoskeletal organization, cell polarity and movement, and vesicular and nuclear transport (1). An upstream signal stimulates the dissociation of GDP from the GDP-bound form (inactive), which leads to the binding of GTP and formation of the GTP-bound form (active). The activated G protein then goes through a conformational change in its downstream effector-binding region, leading to the binding and regulation of downstream effectors. This activation can be switched off by the intrinsic GTPase activity, which hydrolyzes GTP to GDP and releases the downstream effectors. These intrinsic guanine nucleotide exchange and GTP hydrolysis activities of Ras superfamily proteins are also regulated by guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound form and GTPase activating proteins (GAPs) that return the GTPase to its GDP-bound inactive form (4).
ADP-ribosylation factor (Arf) proteins are low molecular weight GTP binding proteins that belong to the Ras GTPase superfamily. Arf proteins are grouped into three distinct classes based on amino acid sequence and structural similarity. Arf1 is a class I Arf protein and is involved in the budding of vesicles from the Golgi (1).
- Takai, Y. et al. (2001) Physiol Rev 81, 153-208.
- Colicelli, J. (2004) Sci STKE 2004, RE13.
- Wennerberg, K. et al. (2005) J Cell Sci 118, 843-6.
- Vigil, D. et al. (2010) Nat Rev Cancer 10, 842-57.
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