Raf Family Antibody Sampler Kit

• 产品编号：CST-2330S      品牌：Cell Signaling Technology       原厂货号：2330S
• 产品分类：抗体 > 一抗 > 复合抗体试剂盒
• 应用分类：

其它组分：

描述：

Raf Family Antibody Sampler Kit中的每个抗体都能检测它特异靶向的蛋白水平。该抗体不能与其他Raf家族成员发生交叉反应。该磷酸特异性多克隆抗体通过采用合成的与人源A-Raf蛋白丝氨酸（299位点）和人源B-Raf蛋白丝氨酸（445位点）、c-Raf蛋白丝氨酸（259/289/296 /301位点）周围残基相一致的磷酸化肽段免疫动物而获得。多克隆抗体通过使用与人源A-Raf蛋白联合结构域和人源c-Raf蛋白脯氨酸（302位点）附近区域残基相一致的合成肽段免疫兔后获得。该抗体经蛋白A和肽亲和层析纯化。兔单克隆抗体通过使用与人源c-Raf蛋白丝氨酸（338位点）周围残基相一致的合成磷酸化肽段、与人源B-Raf蛋白残基相一致的合成肽段免疫动物而获得。

A-Raf, B-Raf, c-Raf (Raf-1)是由GTP结合蛋白-Ras蛋白所募集的主要效应因子，能激活MEK-MAPK途径(1)。目前对c-Raf的活化机制了解的最清楚，它涉及到许多活化位点的磷酸化，包括Ser338,Tyr341,Thr491,Ser494,Ser497和Ser499位点(2)。p21活化的蛋白激酶(PAK)能磷酸化c-Raf蛋白的Ser338位点 ，Src家族蛋白能磷酸化c-Raf蛋白的Tyr341位点，进而诱导c-Raf的活化(3,4)。c-Raf蛋白的Ser338位点，与A-Raf 的Ser299位点 、 B-Raf 的Ser445位点相对应，尽管B-Raf蛋白的Ser445位点已经被组成性的磷酸化 (5)。Akt和AMPK可以分别磷酸化c-Raf蛋白的Ser259和Ser621位点，从而抑制14-3-3与c-Raf的结合(6,7)。虽然A-Raf, B-Raf, c-Raf的序列和功能相似，但是调节方式各不相同(8)。值得关注的是，B-Raf蛋白包含三个Akt的磷酸化位点(Ser364,Ser428和Thr439位点)，但缺少与c-Raf蛋白的Tyr341位点相对应的磷酸化位点(8,9)。研究表明，B-Raf蛋白的突变体V600E，会导致激酶活性的调高，而且这种突变在恶性黑色素瘤中普遍存在(10)。c-Raf蛋白的六个位点 (Ser29, Ser43, Ser289, Ser296, Ser301和Ser642位点)会以某种方式过度磷酸化，这与c-Raf蛋白的失活有关。这六个位点的过度磷酸化依赖于下游的MEK信号转导，并使c-Raf不应答后续的活化事件(11)。

1. Avruch, J. et al. (1994) Trends Biochem Sci 19, 279-83.
2. Chong, H. et al. (2001) EMBO J 20, 3716-27.
3. King, A.J. et al. (1998) Nature 396, 180-3.
4. Fabian, J.R. et al. (1993) Mol Cell Biol 13, 7170-9.
5. Mason, C.S. et al. (1999) EMBO J 18, 2137-48.
6. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
7. Sprenkle, A.B. et al. (1997) FEBS Lett 403, 254-8.
8. Marais, R. et al. (1997) J Biol Chem 272, 4378-83.
9. Guan, K.L. et al. (2000) J Biol Chem 275, 27354-9.
10. Davies, H. et al. (2002) Nature 417, 949-54.
11. Dougherty, M.K. et al. (2005) Mol Cell 17, 215-24.

原厂资料：

Specificity / Sensitivity

Each antibody in the Raf Family Antibody Sampler Kit recognizes only its specific target and does not cross react with other Raf family members.

Source / Purification

The phospho-specific polyclonal antibodies are produced by immunizing rabbits with a synthetic phosphopeptide corresponding to residues surrounding Ser299 of human A-Raf, Ser445 of human B-Raf and Ser259, 289, 296 and 301 of c-Raf. The total polyclonal antibodies are produced by immunizing rabbits with a synthetic peptide corresponding to residues close to the linker domain of human A-Raf and a region surrounding Pro302 of human c-Raf. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. The rabbit monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser338 of human c-Raf. The mouse monoclonal antibody is produced by immunizing animals with a recombinant fragment of B-Raf protein.

Background

A-Raf, B-Raf and c-Raf (Raf-1) are the main effectors recruited by GTP-bound Ras to activate the MEK-MAP kinase pathway (1). Activation of c-Raf is the best understood and involves phosphorylation at multiple activating sites including Ser338, Tyr341, Thr491, Ser494, Ser497 and Ser499 (2). p21-activated protein kinase (PAK) has been shown to phosphorylate c-Raf at Ser338 and the Src family phosphorylates Tyr341 to induce c-Raf activity (3,4). Ser338 of c-Raf corresponds to similar sites in A-Raf (Ser299) and B-Raf (Ser445), although this site is constitutively phosphorylated in B-Raf (5). Inhibitory 14-3-3 binding sites on c-Raf (Ser259 and Ser621) can be phosphorylated by Akt and AMPK, respectively (6,7). While A-Raf, B-Raf and c-Raf are similar in sequence and function, differential regulation has been observed (8). Of particular interest, B-Raf contains three consensus Akt phosphorylation sites (Ser364, Ser428 and Thr439) and lacks a site equivalent to Tyr341 of c-Raf (8,9). The B-Raf mutation V600E results in elevated kinase activity and is commonly found in malignant melanoma (10). Six residues of c-Raf (Ser29, Ser43, Ser289, Ser296, Ser301 and Ser642) become hyperphosphorylated in a manner consistent with c-Raf inactivation. The hyperphosphorylation of these six sites is dependent on downstream MEK signaling and renders c-Raf unresponsive to subsequent activation events (11).

1. Avruch, J. et al. (1994) Trends Biochem Sci 19, 279-83.
2. Chong, H. et al. (2001) EMBO J 20, 3716-27.
3. King, A.J. et al. (1998) Nature 396, 180-3.
4. Fabian, J.R. et al. (1993) Mol Cell Biol 13, 7170-9.
5. Mason, C.S. et al. (1999) EMBO J 18, 2137-48.
6. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
7. Sprenkle, A.B. et al. (1997) FEBS Lett 403, 254-8.
8. Marais, R. et al. (1997) J Biol Chem 272, 4378-83.
9. Guan, K.L. et al. (2000) J Biol Chem 275, 27354-9.
10. Davies, H. et al. (2002) Nature 417, 949-54.
11. Dougherty, M.K. et al. (2005) Mol Cell 17, 215-24.

注意事项：

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM
NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not
aliquot the antibodies.