Product Pathways - Protein Folding/Stability

Protein Folding and Stability Antibody Sampler Kit #4889

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Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry  E-P=ELISA (Peptide)
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  B=Bovine  Dr=Drosophila  X=Xenopus  Z=Zebra Fish

Specificity / Sensitivity

Skp1 Antibody detects endogenous levels of total Skp1 protein. Skp2 Antibody detects endogenous levels of total Skp2 protein (α, β and γ isoforms) and does not cross-react with other SKP proteins. ISG15 Antibody detects endogenous levels of both free and conjugated ISG15 protein, but does not cross-react with other ubiquitin family members. NEDD8 Antibody detects endogenous levels of both free and conjugated NEDD8 protein, but does not cross-react with other ubiquitin family members. Ubiquitin (P4D1) Mouse mAb detects ubiquitin, polyubiquitin and ubiquitinated proteins. UBC3 Antibody detects endogenous levels of total UBC3 and UBC3B protein. SUMO-1 Antibody detects recombinant SUMO-1 and endogenous levels of sumoylated proteins (e.g. SUMO-1-RanGAP, 90kD). SUMO-2/3 Antibody detects endogenous levels of SUMO-2/3, but does not cross-react with recombinant SUMO-1.

Western Blotting

Western blot analysis of extracts from PC12 and NIH/3T3 cells using Skp1 Antibody #2156.

Western Blotting

Western blot analysis of extracts from various cell types, untreated or IFN-α treated (1000 U/mL for 24 hours), using ISG15 Antibody #2743.

Western Blotting

Western blot analysis of extracts from various cell types using NEDD8 Antibody #2745.

Western Blotting

Western blot analysis of extracts from 293 and HeLa cells, untreated or MG-132 (a 26S proteosome inhibitor) treated (50 µM for 90 minutes), using Ubiquitin (P4D1) Mouse mAb #3936.

Western Blotting

Western blot analysis of extracts from HeLa, K562 and COS cells using Skp2 Antibody #4358.

Western Blotting

Western blot analysis of recombinant GST-SUMO-1 protein (38 kDa) and extracts from CAD, HeLa and PC-12 cells using SUMO-1 Antibody #4930.

Western Blotting

Western blot analysis of recombinant GST-SUMO-1 protein (38 kDa), recombinant SUMO-2, recombinant SUMO-3, and extracts from C6 and PC12 cells, using SUMO-2/3 (18H8) Rabbit mAb #4971 (upper) and SUMO-1 Antibody #4930 (lower).

Western Blotting

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

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with synthetic peptides (KLH-coupled) corresponding to human SUMO-1 (#4930), UBC3 (#4997), Skp2 (#4358), Skp1 (#2156), NEDD8 (#2745), and ISG15 (#2743). Polyclonal antibodies are purified by protein A and peptide affinity chromatography. SUMO-2/3 (18H8) Rabbit mAb (#4971) is produced by immunizing rabbits with a synthetic peptide (KLH-coupled) from the amino terminus of human SUMO-3. Ubiquitin (P4D1) Mouse monoclonal antibody (#3936) is produced by immunizing mice with 1-76 full-length ubiquitin of bovine origin.

Background

The small regulatory protein ubiquitin is often covalently linked to many cellular proteins, labeling these targeted proteins for proteasome-mediated degradation. Ubiquitin is first activated by forming a thiolester complex with the E1 activation component. Activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, and then to an E3 ubiquitin ligase for final delivery to the ε-NH2 of the target protein lysine residue (1). The ubiquitin-proteasome pathway has been implicated in a wide range of both normal biological processes and diseases (2,3).The ubiquitin-like protein family contains three small ubiquitin-related modifier proteins (SUMO-1, -2 and -3), neural precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) and interferon-stimulated 15 kDa protein (ISG15) (4-6). Their covalent attachment to target proteins is a reversible, multi-step process that is analogous to protein ubiquitination. Mature molecules are linked to the activating enzyme E1, conjugated to E2 and ligated to the target proteins by E3 (7-10). Ubiquitin is the predominant regulator for the degradation of a wide range of target proteins (8) while SUMO, NEDD8 and ISG15 modify a limited set of substrates to regulate various other biological processes (4, 11-18).During ubiquitination, the combinatorial interaction of different E2 and E3 proteins produces variable substrate specificity (4). UBC3 and UBC3B are E2 ubiquitin-carrier proteins (19, 20). The SCF (Skp1/CUL1/F-box) E3 ubiquitin ligase protein complex is composed of three protein components, including the S phase kinase associated protein 1 (Skp1), Cullin homolog 1 (CUL1) and the Skp2 F-box protein (21-23).

1. Ciechanover, A. (1998) EMBO J. 17, 7151-7160.
2. Bernardi, R. et al. (2000) Oncogene 19, 2447-2454.
3. Jesenberger, V. and Jentsch, S. (2002) Nat. Rev. Mol. Cell Biol. 3, 112-121.
4. Schwartz, D.C. and Hochstrasser, M. (2003) Trends Biochem. Sci. 28, 321-328.
5. Chiba, T. and Tanaka, K. (2004) Curr. Protein Pept. Sci. 5, 177-184.
6. Ritchie, K.J. and Zhang, D.E. (2004) Semin. Cell Dev. Biol. 15, 237-246.
7. Kim, K.I. et al. (2002) J. Cell Physiol. 191, 257-268.
8. Osaka, F. et al. (1998) Genes Dev. 12, 2263-2268.
9. Loeb, K.R. and Haas, A.L. (1992) J. Biol. Chem. 267, 7806-7813.
10. Zhao, C. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 10200-10205.
11. Matunis, M.J. et al. (1996) J. Cell Biol. 135, 1457-1470.
12. Duprez, E. et al. (1999) J. Cell Sci. 112 ( Pt 3), 381-393.
13. Gostissa, M. et al. (1999) EMBO J. 18, 6462-6471.
14. Rodriguez, M.S. et al. (1999) EMBO J. 18, 6455-6461.
15. Desterro, J.M. et al. (1998) Mol. Cell 2, 233-239.
16. Stickle, N.H. et al. (2004) Mol. Cell. Biol. 24, 3251-3261.
17. Xirodimas, D.P. et al. (2004) Cell 118, 83-97.
18. Hamerman, J.A. et al. (2002) J. Immunol. 168, 2415-2423.
19. Semplici, F. et al. (2002) Oncogene 21, 3978-3987.
20. Pagano, M. et al. (1995) Science 269, 682-685.
21. Yu, Z.K. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 11324-11329.
22. Pagano, M. (2004) Mol. Cell 14, 414-416.
23. Reed, S.I. (2003) Nat. Rev. Mol. Cell Biol. 4, 855-864.

Application References

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