Revision 1

#12724Store at -20C

1 Kit

(8 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
CUL4A Antibody 2699 20 µl 80, 82 kDa Rabbit 
CYLD (D6O5O) Rabbit mAb 12797 20 µl 109 kDa Rabbit IgG
DDB-1 (D4C8) Rabbit mAb 6998 20 µl 127 kDa Rabbit IgG
DDB-2 (D4C4) Rabbit mAb 5416 20 µl 43 kDa Rabbit IgG
RBX1 (D3J5I) Rabbit mAb 11922 20 µl 13 kDa Rabbit IgG
Skp1 (D3J4N) Rabbit mAb 12248 20 µl 19 kDa Rabbit IgG
Skp2 (D3G5) XP® Rabbit mAb 2652 20 µl 48 kDa Rabbit IgG
β-TrCP (D12C8) Rabbit mAb 11984 20 µl 62 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

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 two western blot experiments per primary antibody.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

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).

  1. Ciechanover, A. (1998) EMBO J 17, 7151-60.
  2. Hochstrasser, M. (2000) Nat Cell Biol 2, E153-7.
  3. Hochstrasser, M. (2000) Science 289, 563-4.
  4. Deffenbaugh, A.E. et al. (2003) Cell 114, 611-22.
  5. DeSalle, L.M. and Pagano, M. (2001) FEBS Lett 490, 179-89.
  6. Zheng, N. et al. (2002) Nature 416, 703-9.
  7. Kamura, T. et al. (1999) Genes Dev 13, 2928-33.
  8. Morimoto, M. et al. (2003) Biochem Biophys Res Commun 301, 392-8.
  9. Pan, Z.Q. et al. (2004) Oncogene 23, 1985-97.
  10. Sun, Y. et al. (2001) Antioxid Redox Signal 3, 635-50.
  11. Petroski, M.D. and Deshaies, R.J. (2005) Nat Rev Mol Cell Biol 6, 9-20.
  12. Lee, J. and Zhou, P. (2007) Mol Cell 26, 775-80.
  13. Reardon, J.T. et al. (1993) J Biol Chem 268, 21301-8.
  14. Keeney, S. et al. (1993) J Biol Chem 268, 21293-300.
  15. Hwang, B.J. and Chu, G. (1993) Biochemistry 32, 1657-66.
  16. Chu, G. and Chang, E. (1990) Proc Natl Acad Sci U S A 87, 3324-7.
  17. Hirschfeld, S. et al. (1990) Mol Cell Biol 10, 2041-8.
  18. Payne, A. and Chu, G. (1994) Mutat Res 310, 89-102.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP is a registered trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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    Revision 1
    #12724

    Ubiquitin E3 Ligase Complex Antibody Sampler Kit

    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 1 Expand Image
    Western Blot analyis of GST-Tagged recombinant Β-TrCP1 and Β-TrCP2 using Β-TrCP (D12C8) Rabbit mAb #11984 (upper) or GST-Tag (91G1) Rabbit mAb #2625.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from various cell lines using RBX1 (D3J5I) Rabbit mAb.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from various cell lines using β-TrCP (D12C8) Rabbit mAb.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using Skp1 (D3J4N) Rabbit mAb.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from various cell lines using CYLD (D6O5O) Rabbit mAb.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various cell lines using Skp2 (D3G5) XP® Rabbit mAb.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from COS and SK-MEL-28 cell lines using CUL4A Antibody.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from Raji and HL-60 cells using DDB-2 (D4C4) Rabbit mAb.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from various cell lines using DDB-1 (D4C8) Rabbit mAb.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 10 Expand Image
    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.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 11 Expand Image
    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).
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 12 Expand Image
    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.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 13 Expand Image
    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.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 14 Expand Image
    Immunoprecipitation of CUL4A from HT-29 extracts. Lane 1 is CUL4A Antibody, lane 2 is Normal Rabbit IgG #2729, and lane 3 is 10% input. Western blot analysis was perfomed using CUL4A Antibody. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 15 Expand Image
    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.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 16 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse prostate using RBX1 (D3J5I) Rabbit mAb.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 17 Expand Image
    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.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 18 Expand Image
    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).
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 19 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Skp2 (D3G5) XP® Rabbit mAb.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 20 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lymphoma using Skp2 (D3G5) XP® Rabbit mAb.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 21 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Skp2 (D3G5) XP® Rabbit mAb.
    Ubiquitin E3 Ligase Complex Antibody Sampler Kit: Image 22 Expand Image
    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.)