Cell Signaling Technology

Product Pathways - Protein Stability

Ubiquitin E3 Ligase Complex Antibody Sampler Kit #12724

SCF Complex  

REACTIVITY

No. Size Price
12724T 1 Kit ( 8 x 20 µl ) ¥6,597.00 现货查询 购买询价
Kit Includes Quantity Applications Reactivity Homology† MW (kDa) Isotype
RBX1 (D3J5I) Rabbit mAb #11922 20 µl W,IP,IHC-P, H,M,R,Mk, 13 Rabbit IgG
β-TrCP (D12C8) Rabbit mAb #11984 20 µl W, H,M,R,Mk, 62 Rabbit IgG
Skp1 (D3J4N) Rabbit mAb #12248 20 µl W,IP, H,M,R,Mk, Z, 19 Rabbit IgG
CYLD (D6O5O) Rabbit mAb #12797 20 µl W, H,M,R,Hm,Mk, 109 Rabbit IgG
Skp2 (D3G5) XP® Rabbit mAb #2652 20 µl W,IP,IHC-P,IF-IC, H,Mk, 48 Rabbit IgG
CUL4A Antibody #2699 20 µl W,IP, H,Mk, 80, 82 Rabbit
DDB-2 (D4C4) Rabbit mAb #5416 20 µl W, H,M, R,Mk, 43 Rabbit IgG
DDB-1 (D4C8) Rabbit mAb #6998 20 µl W, H,M,R,Mk, C,B, 127 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl W, Goat

Specificity / Sensitivity

Each antibody in the Ubiquitin E3 Ligase Complex Antibody Sampler Kit recognizes endogenous levels of its respective target protein and does not cross-react with other family members. The Skp2 (D3G5) XP® Rabbit mAb is predicted to cross-react with Skp2a and Skp2B proteins.

Ubiquitin E3 Ligase Complex Antibody Sampler Kit中的每一个抗体都分别识别的内源性的相应目的蛋白,并且不与其他家族成员发生交叉反应。Skp2(D3G5) Rabbit mAb应与Skp2a和Skp2B蛋白存在交叉反应。

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a recombinant protein specific to human CYLD protein, a synthetic peptide corresponding to residues surrounding Gly832 of human DDB-1 protein, to residues surrounding Ala174 of human DDB-2 protein, to the carboxy terminus of human RBX1 protein, to the carboxy terminus of human Skp1 protein, to the amino terminus of human Skp2 protein, and with a recombinant protein specific to human β-TrCP protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser12 of human CUL4A protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

单克隆抗体由合成重组蛋白或肽段免疫动物产生,这些重组蛋白与合成肽与人CYLD蛋白的重组蛋白,人源DDB-1蛋白Gly832,人源DDB-2蛋白Ala174,人源RBX1蛋白羰基末端,人源Skp1蛋白羰基末端,人源Skp2蛋白氨基末端周围的残基,以及人源β-TrCP蛋白的重组蛋白相一致。多克隆抗体通过合成肽免疫动物制备,这些合成肽与人源CUL4A的Ser12周围残基相一致。该多克隆抗体由蛋白A和肽亲和层析纯化。

Description

The Ubiquitin E3 Ligase Complex Antibody Sampler Kit provides an economical means to study multiple protein components of ubiquitin E3 ligase complexes. The kit includes enough antibody to perform four western blot experiments per primary antibody.

The Ubiquitin E3 Ligase Complex Antibody Sampler Kit提供了一种研究E3泛素酶的多蛋白组分的经济方法。该试剂盒包含了4次western blots的所需抗体量。

Western Blotting

Western Blotting

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO* is added and emits light during enzyme catalyzed decomposition.

Western Blotting

Western Blotting

Western blot analysis of extracts from COS and SK-MEL-28 cell lines using CUL4A Antibody.

Western Blotting

Western Blotting

Western blot analysis of extracts from Raji and HL-60 cells using DDB-2 (D4C4) Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using DDB-1 (D4C8) Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with a Myc-tagged cDNA expression construct encoding full-length human DDB-1 (+), using DDB-1 (D4C8) Rabbit mAb.

IF-IC

IF-IC

Confocal immunofluorescent analyis of HeLa cells expressing either a non-targeting shRNA (shNT; left) or shSkp2 (right) using Skp2 (D3G5) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red) (Cell lines kindly provided by Dr. Wenyi Wei, Harvard Medical School.)

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Skp2 (D3G5) XP® Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Skp2 (D3G5) XP® Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with a Myc/DDK-tagged cDNA expression construct encoding full-length human Skp2 (hSkp2-Myc/DDK, +), using Skp2 (D3G5) XP® Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells expressing either non-targeting shRNA (shNT) or shSkp2, using Skp2 (D3G5) XP® Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lymphoma using Skp2 (D3G5) XP® Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Skp2 (D3G5) XP® Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using β-TrCP (D12C8) Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using RBX1 (D3J5I) Rabbit mAb.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using RBX1 (D3J5I) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded mouse prostate using RBX1 (D3J5I) Rabbit mAb.

Also 11922 on normal mouse lung.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Skp1 (D3J4N) Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human Skp1 isoform b (hSkp1-Myc/DDK; +), using Skp1 (D3J4N) Rabbit mAb.

IP

IP

Immunoprecipitation of Skp1 from 293T cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Skp1 (D3J4N) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Skp1 (D3J4N) Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using CYLD (D6O5O) Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using β-TrCP (D12C8) Rabbit mAb #11984.Western blot检测多种细胞提取物,使用的抗体是β-TrCP (D12C8) Rabbit mAb #11984。

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using CYLD (D6O5O) Rabbit mAb #12797.Western blot检测多种细胞提取物,使用的抗体是CYLD (D6O5O) Rabbit mAb #12797。

Western Blotting

Western Blotting

Western blot analysis of extracts from COS-1 and SK-MEL-28 cells using CUL4A Antibody #2699.Western blot检测多种细胞提取物,使用的抗体是CUL4A Antibody #2699。

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using DDB-1 (D4C8) Rabbit mAb #6998.Western blot检测多种细胞提取物,使用的抗体是DDB-1 (D4C8) Rabbit mAb #6998。

Western Blotting

Western Blotting

Western blot analysis of extracts from Raji and HL-60 cells using DDB-2 (D4C4) Rabbit mAb #5416.Western blot检测多种细胞提取物,使用的抗体是DDB-2 (D4C4) Rabbit mAb #5416。

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using RBX1 (D3J5I) Rabbit mAb #11922.Western blot检测多种细胞提取物,使用的抗体是RBX1 (D3J5I) Rabbit mAb #11922。

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Skp1 (D3J4N) Rabbit mAb #12248.Western blot检测多种细胞提取物,使用的抗体是Skp1 (D3J4N) Rabbit mAb #12248。

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Skp2 (D3G5) XP® Rabbit mAb #2652.Western blot检测多种细胞提取物,使用的抗体是Skp2 (D3G5) XP® Rabbit mAb #2652。

Background

Ubiquitin can be covalently linked to many cellular proteins by the ubiquitination process, which targets proteins for degradation by the 26S proteasome. Ubiquitin is first activated by forming a thiolester complex with the activation component E1. The activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, and then from E2 to ubiquitin ligase E3 for final delivery to the ε-NH2 of the target protein lysine residue (1-3). Research studies suggest that activated E2 associates transiently with E3, and the dissociation is a critical step for ubiquitination (4).

S phase kinase-associated protein 1 (Skp1) is a critical scaffold protein of the Skp1/CUL1/F-box (SCF) E3 ubiquitin ligase protein complex. Various F-box proteins (e.g. β-TrCP, Skp2) mediate an interaction with Skp1 via their defining and conserved domain of 40 amino acids and with substrates to be ubiquitinated (5). RING-box protein 1 (RBX1 or ROC1) is another essential component of the SCF complex (6). RBX1 mediates the neddylation of CUL1, which activates SCF E3 ligase by facilitating the ubiquitin transfer from E2 to substrates (7-9). The RING finger domain of RBX1 is required for ubiquitin ligation (10).

Cullin-4 (CUL4) is a member of the cullin family of related ubiquitin ligases (11). The carboxy-terminal domain of CUL4 interacts with Rbx1 and E2 enzyme while the amino-terminal CUL4 domain interacts with BPB domain of UV-damaged DNA binding protein DDB-1 to form a CUL4-DDB1 ubiquitin ligase complex (12). Damaged DNA-Binding Protein (DDB) consists of a 127 kDa subunit (DDB-1) and a 48 kDa subunit (DDB-2) that contribute to the formation of the UV-damaged DNA-binding protein complex (UV-DDB) (13-15). In conjunction with CUL4A and RBX1, the UV-DDB complex forms an E3 ubiquitin ligase that recognizes a broad spectrum of DNA lesions. The complex polyubiquitinates components of the nucleotide excision repair pathway (16-18).

泛素能与许多细胞蛋白共价连接,并被26S蛋白酶降解。3种组分包含在靶蛋白泛素共价结合过程。泛素首先在具有泛素活化酶E1的硫酯复合物的形成中被活化。活化的泛素随后被转运到泛素载体蛋白(共价结合酶)E2,随后为了最后的靶蛋白赖氨酸残基的epsilon-NH2,从E2到泛素连接酶E3(1-3)。研究表明,激活的E2与E3迅速结合然后分离是一个关键步骤(4)。S期激酶蛋白1(Skp1)是Skp1/CUL1/F-box(SCF)E3泛素化连接酶蛋白复合体中一个关键的支架蛋白。各种F-box蛋白质(如β-TrCP Skp2) 通过其保守的40个氨基酸和泛素化的底物介导他们与Skp1的相互作用 (5)。RING-box蛋白1(RBX1或ROC1)是SCF复合体中另一个重要组成部分(6)。RBX1介导CUL1类泛素化neddylation, 通过促进泛素从E2转移到底物激活SCF E3连接酶 (7-9)。RBX1的RING finger结构域需要泛素连接(10)。Cullin-4(CUL4)是相关的泛素连接酶cullin家庭成员之一(11)。CUL4的羰基末端与Rbx1和E2酶相互作用,此时CUL4氨基末端与UV损坏的DNA结合蛋白DDB-1的BPB结构域相互作用,形成CUL4-DDB1泛素连接酶复合体(12)。损伤DNA结合蛋白(DDB)由127 kDa亚基(DDB-1)和48 kDa亚基(DDB-2)组成,形成UV损伤DNA结合蛋白质复合体(UV-DDB)(13 - 15)。与CUL4A和RBX1结合后,UV-DDB复杂体形成一个E3泛素连接酶,可识别广泛的DNA损伤。这个复合体多泛素化核苷酸切除修复通路的组分(16-18)。

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