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#8815 Active Rac1 Detection Kit

  • Active Rac1 Detection Kit Data Sheet PDF
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希望納入価格 (円)
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2019年8月20日15時55分 現在
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#8815S1 Kit153,000
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RAC 製品一覧

キット内容 容量 Storage Temp
GST-Human PAK1-PBD 1 x 600 µg -20C
Rac1 Mouse Antibody 1 x 50 µl -20C
GDP 1 x 50 µl -80C
GTP γS 1 x 50 µl -80C
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Rac1 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。
MSDSはこちらから
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 Rac1 (refer to optional step C in protocol). The lysates were then incubated with glutathione resin and GST-PAK1-PBD (lanes 2 and 3). GTPγS-treated lysate was also incubated without GST-PAK1-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 a Rac1 Mouse mAb. Anti-mouse IgG, HRP-linked Antibody #7076 was used as the secondary antibody.

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).

Rac and Cdc42 are members of the Rho-GTPase family. In mammals, Rac exists as three isoforms, Rac1, Rac2, and Rac3, which are highly similar in sequence. Rac1 and Cdc42, the most widely studied of this group, are ubiquitously expressed. Rac2 is expressed in cells of hematopoietic origin, and Rac3, while highly expressed in brain, is also found in many other tissues. Rac and Cdc42 play key signaling roles in cytoskeletal reorganization, membrane trafficking, transcriptional regulation, cell growth, and development (5). GTP binding stimulates the activity of Rac/Cdc42, and the hydrolysis of GTP to GDP through the protein's intrinsic GTPase activity, rendering it inactive. GTP hydrolysis is aided by GTPase activating proteins (GAPs), while exchange of GDP for GTP is facilitated by guanine nucleotide exchange factors (GEFs). Another level of regulation is achieved through the binding of RhoGDI, a guanine nucleotide dissociation inhibitor, which retains Rho family GTPases, including Rac and Cdc42, in their inactive GDP-bound state (6,7).

使用文献

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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