Cell Signaling Technology

Product Pathways - Protein Folding

Protein Folding and Stability Antibody Sampler Kit #4889

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Skp1 Antibody #2156 40 µl W IF-IC F H M R 19 Rabbit
Skp2 (D3G5) XP® Rabbit mAb #2652 40 µl W IP IHC-P IF-IC H Mk 48 Rabbit IgG
ISG15 (22D2) Rabbit mAb #2758 40 µl W IP H Hr 15 Rabbit IgG
NEDD8 (19E3) Rabbit mAb #2754 40 µl W IP IHC-P F H M R Mk (X) (Z) (B) 9 Rabbit IgG
Ubiquitin (P4D1) Mouse mAb #3936 40 µl W IHC-P All Mouse IgG1
UBC3 Antibody #4997 40 µl W F H M R 32 Rabbit
SUMO-1 Antibody #4930 40 µl W IP IHC-P IF-IC H M R Mk Rabbit
SUMO-2/3 (18H8) Rabbit mAb #4971 40 µl W IF-IC H R (M) Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat
Anti-mouse IgG, HRP-linked Antibody #7076 100 µl Horse

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  Dm=D. melanogaster  X=Xenopus  Z=Zebrafish  B=Bovine  Hr=Horse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Skp1 Antibody detects endogenous levels of total Skp1 protein. Skp2 (D3G5) XP® Rabbit mAb recognizes endogenous levels of total Skp2 protein. This antibody is predicted to cross-react with Skp2α and Skp2ß. ISG15 (22D2) Rabbit mAb detects endogenous levels of the uncleaved precursor form of ISG15 protein. This antibody does not recognize the activated (cleaved) or conjugated forms of ISG15. The antibody does not cross-react with other ubiquitin family members, including ubiquitin, SUMO-1, SUMO-2, SUMO-3 and NEDD8. NEDD8 (19E3) Rabbit mAb detects endogenous levels of both free and conjugated NEDD8 protein. The antibody does not cross-react with other ubiquitin family members, including ubiquitin, SUMO-1, SUMO-2, SUMO-3 and ISG15. 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 Blotting

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

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Skp2 (D3G5) XP® Rabbit mAb #2652.

Western Blotting

Western Blotting

Western blot analysis of lysates from HeLa, RAW, C6 and COS cells, using NEDD8 (19E3) Rabbit mAb #2754.


Western Blotting

Western Blotting

Western blot analysis of lysates from HeLa and A549 cells, untreated (-) or treated with IFN-α (1000 U/mL) (+) for 24 hours, using ISG15 (22D2) Rabbit mAb #2758.

Western Blotting

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 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 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 Blotting

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

Description

This sampler kit provides an economical means to investigate protein folding and stability. The kit contains primary and secondary antibodies to perform four Western blots with each antibody.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to human SUMO-1 (#4930), UBC3 (#4997), and Skp1 (#2156). 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 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. ISG15 (22D2) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to amino acids at the amino terminus of human ISG15 protein. NEDD8 (19E3) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to amino acids at the amino terminus of human NEDD8 protein. Skp2 (D3G5) XP® Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human Skp2 protein.

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).

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  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.

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