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#8818 Active Rap1 Detection Kit

  • Active Rap1 Detection Kit Data Sheet PDF
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希望納入価格 (円)
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2019年12月5日15時20分 現在
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#8818S1 Kit153,000
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RAP 製品一覧

キット内容 容量 Storage Temp
GST-RalGDS-RBD 1 x 600 µg -80C
GDP 1 x 50 µl -80C
GTP γS 1 x 50 µl -80C
Rap1 Rabbit mAb 1 x 50 µl -20C
Glutathione Resin 1 x 3 ml 4C
SDS Sample Buffer 1 x 1.5 ml 4C
Lysis/Binding/Wash Buffer 1 x 100 ml 4C
Spin Cup and Collection Tubes 1 x 30 ea 4C
特異性・感度
細胞内Rap1 GTPase の活性を測定するのに必要な全ての試薬を含んでいます。
種交差性
ヒト、マウス
※輸送温度毎に梱包が分かれています。輸送温度と保管温度が異なることがありますので、本製品受領後は速やかに製品記載温度での保管をお願いいたします。

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社内データ

Western Blotting

Western Blotting

Figure 1. NIH/3T3 cell lysates (500 µl at 1 mg/ml) were treated in vitro with GTPγS or GDP to activate or inactivate Rap1 (refer to optional step C in protocol). The lysates were then incubated with glutathione resin and GST-RalGDS-RBD (lanes 2 and 3). GTPγS-treated lysate was also incubated without GST-RalGDS-RBD 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 a Rap1 Rabbit Antibody. 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.

Schematic

Schematic

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.

バックグラウンド

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).

Rap1 and Rap2 belong to the Ras subfamily of small GTPases and are activated by a wide variety of stimuli through integrins, receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCR), death domain associated receptors (DD-R), and ion channels (5,6). Like other small GTPases, Rap activity is stimulated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase activating proteins (GAPs). A wide variety of Rap GEFs have been identified: C3G connects Rap1 with RTKs through adaptor proteins such as Crk, Epacs (or cAMP-GEFs) transmit signals from cAMP, and CD-GEFs (or CalDAG-GEFs) convey signals from either or both Ca2+ and DAG (5). Rap1 primarily regulates multiple integrin-dependent processes such as morphogenesis, cell-cell adhesion, hematopoiesis, leukocyte migration and tumor invasion (5,6). Rap1 may also regulate proliferation, differentiation and survival through downstream effectors including B-Raf, PI3K, RalGEF and phospholipases (PLCs) (5-8). Rap1 and Rap2 are not fuctionally redundant as they perform overlapping but distinct functions (9). Recent research indicates that Rap2 regulates Dsh subcellular localization and is required for Wnt signaling in early development (10).

使用文献

LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

本製品は試験研究用です。

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