Cell Signaling Technology

Product Pathways - Protein Stability

Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit #8645

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Ubiquitin Antibody #3933 40 µl W IHC-P H M R Mk Dm (X) (Z) (B) (Pg) (Hr) Rabbit
UBE1a Antibody #4890 40 µl W IP IHC-P IF-IC F H M R 117 Rabbit
UBC3 Antibody #4997 40 µl W F H M R 32 Rabbit
UbcH5C (D60E2) Rabbit mAb #4330 40 µl W IP H M R Mk 14 Rabbit IgG
UBE2L3 (D5G1) Rabbit mAb #8721 40 µl W IP H M R Mk (X) (Z) (B) (Dg) (Hr) 18 Rabbit IgG
UBE2N (D2A1) Rabbit mAb #6999 40 µl W IP H M R Mk (C) (X) (B) (Dg) (Hr) 17 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  C=Chicken  Dm=D. melanogaster  X=Xenopus  Z=Zebrafish  B=Bovine  Dg=Dog  Pg=Pig  Hr=Horse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Ubiquitin Antibody recognizes endogenous levels of ubiquitin, polyubiquitin, and ubiquitinated proteins. This antibody may cross-react with recombinant NEDD8. UBE1a Antibody recognizes endogenous levels of total UBE1a protein. UBC3 Antibody recognizes endogenous levels of total UBC3 and UBC3B proteins. UbcH5C (D60E2) Rabbit mAb recognizes endogenous levels of total UbcH5C protein. UBE2L3 (D5G1) Rabbit mAb recognizes endogenous levels of total UBE2L3 protein. UBE2N (D2A1) Rabbit mAb recognizes endogenous levels of total UBE2N protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated (-) or treated with the proteasome inhibitor MG132 (10 µM, 90 min; +), using Ubiquitin Antibody #3933.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using UbcH5C (D60E2) Rabbit mAb #4330.

Western Blotting

Western Blotting

Western blot analysis of extracts from K562 and SK-N-MC cells using UBE1a Antibody #4890.


Western Blotting

Western Blotting

Western blot analysis of extracts from Jurkat, CAD, and C6 cells using UBC3 Antibody #4997.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using UBE2N (D2A1) Rabbit mAb #6999.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using UBE2L3 (D5G1) Rabbit mAb #8721.


Description

The Ubiquitin Activation (E1, E2 Enzymes) Antibody Sampler Kit provides an economical means to study ubiquitin activation and conjugation. This kit contains enough primary antibody to perform four western blots per primary.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of the human ubiquitin protein, the amino terminus of human UBE1 protein, or the sequence of human UBC3 protein. Polyclonal antibodies are purified by protein A and peptide affinity chromotography.Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp135 of human UbcH5C protein, residues surrounding Leu125 of human UBE2L3 protein, or residues near the carboxy terminus of human UBE2N protein.

Background

Ubiquitin is a conserved polypeptide unit that plays an important role in the ubiquitin-proteasome pathway. Ubiquitin can be covalently linked to many cellular proteins for degradation by the 26S proteasome. Three components are involved in the target protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thioester complex with the ubiquitin-activating enzyme (UBE1 or E1). The activated ubiquitin is subsequently transferred to the ubiqutin-carrier protein (conjugating enzyme) E2, and then from E2 to ubiqutin ligase E3 for final delivery to the epsilon-NH2 of the target protein lysine residue (1-3). The ubiquitin-proteasome pathway has been implicated in a wide range of normal biological processes and in disease-related abnormalities. Several proteins such as IκB, p53, cdc25a, and Bcl-2 have been shown to be targets for the ubiquitin-proteasome process as part of the regulation of cell cycle progression, differentiation, cell stress response, and apoptosis (4-7). UBC3, the mammalian ortholog of yeast cdc34, and UBC3B, a UBC3 family member, are E2 ubiquitin-carrier proteins. UBC3, in concert with SCF-Skp2 (Skp1, Cullin, F-box protein/Skp2) complex, mediates cell cycle progression from G1 to S phase by targeting the CDK inhibitor p27 for proteolysis (8). UBC3B, in concert with SCFb-Trcp (Skp1, Cullin and F-box protein/b-Trcp) complex, mediates degradation of β-catenin (9). UbcH5C is a universally expressed E2 ubiquitin conjugating enzyme and member of the UbcH5 family that also includes UbcH5A and UbcH5B (10). Evidence suggests that UbcH5 plays an important role in regulating a number of signaling pathways by catalyzing the ubiquitination of key target proteins, including p53, PCNA, the IκB kinase proein NEMO, and the apoptosis inhibitor BRUCE (11-14). UBE2L3, also commonly referred to as UBCH7, is a ubiquitin-conjugating enzyme that has been linked to the ubiquitination of numerous substrates via its interaction with protein-ubiquitin E3 ligases, such as NEDD4 (15), E6AP (16), Parkin (17), c-Cbl (18), and Triad1 (19,20). UBE2N/Ubc13 is a ubiquitin-E2-conjugating enzyme that catalyzes K63-linked polyubiquitin chain formation (21,22). UBE2N forms a heterodimer with MMS2 or Uev1A to exert its E2 ligase function. The UBE2N/MMS2 and UBE2N/Uev1A heterodimers catalyze different modes of target protein ubiquitination to mediate various signaling pathways (23-25) including DNA damage and recombination, p53 and check point control, cell cycle (26-30), immunoreceptor signaling (31,32), and endocytosis (33).

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