Cell Signaling Technology

Product Pathways - Protein Folding/Stability

ISG15 Antibody #2743

Applications Reactivity MW (kDa) Source
W F E-P H M Mk 15 Rabbit

Applications Key:  W=Western Blotting  F=Flow Cytometry  E-P=ELISA (Peptide)
Reactivity Key:  H=Human  M=Mouse  Mk=Monkey
Species enclosed in parentheses are predicted to react based on 100% sequence homology. Species cross-reactivity is determined by Western blot.

Specificity / Sensitivity

This antibody detects endogenous levels of both free and conjugated ISG15 protein. The antibody does not cross-react with other ubiquitin family members, including ubiquitin, SUMO1, SUMO2, SUMO3 and NEDD8.

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with a synthetic peptide (KLH-coupled) corresponding to amino acids from human ISG15 protein. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of lysates from HeLa, A549, RAW and COS cells, treated with or without IFN (1000 U/mL) for 24 hours, using ISG15 antibody.

Western Blotting

Western Blotting

Western blot analysis of NEDD8, Ubiquitin, ISG15 and SUMO-2/3 recombinant proteins (5 ng each), using NEDD8 (#2745) , Ubiquitin (#3936), ISG15 (#2743) and SUMO-2/3 (#4974) Antibodies.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of Hela cells, untreated (blue) or IFNa treated (green), using ISG15 antibody compared to a nonspecific negative control antibody (red).


ELISA-Peptide

ELISA-Peptide

The relationship between recombinant ISG15 protein concentration and assay optical density readings. Recombinant NEDD8 protein was used as a negative control.

Background

Interferon-stimulated 15 kDa protein (ISG15), also known as ubiquitin cross-reactive protein (UCRP), is a member of the ubiquitin-like protein family and functions in various biological pathways from pregnancy to innate immune responses (1). Expression of ISG15 is stimulated by cellular exposure to type 1 interferons α and β, in addition to infection with viruses such as influenza B (2,3). After exposure to type I interferons, both lymphocytes and monocytes, in addition to some fibroblasts and epithelial cells, release ISG15 into culture medium (1,4). ISG15 has been shown to function as a cytokine, stimulating interferon γ secretion by monocytes and macrophages, proliferation of natural killer cells, and chemotactic responses in neutrophils (4,5). ISG15 has also been shown to function intracellularly, being covalently conjugated to other proteins by E1 (Ube1L), E2 (UbcH8) and E3 ligases via a multi-step process analogous to ubiquitination (6,7). ISG15 is removed from proteins by the ubiquitin processing protease Ubp43 (8). ISG15-protein conjugation (ISGylation) is induced by type 1 interferons, and target proteins include the serine protease inhibitor Serpin 2A, PLCγ1, ERK1/2, Jak1 and Stat1 (9,10). Unlike ubiquitination, ISGylation does not target proteins for degradation, rather ISGylation increases Jak1 and Stat1 activity, enhancing the cellular response to interferons (11).

  1. Ritchie, K.J. and Zhang, D.E. (2004) Semin. Cell Dev. Biol. 15, 237-246.
  2. Korant, B.D. et al. (1984) J. Biol. Chem. 259, 14835-14839.
  3. Haas, A.L. et al. (1987) J. Biol. Chem. 262, 11315-11323.
  4. Knight, E. and Cordova, B. (1991) J. Immunol. 146, 2280-2284.
  5. D'Cunha, J. et al. (1996) Proc. Natl. Acad. Sci. USA 93, 211-215.
  6. Loeb, K.R. and Haas, A.L. (1992) J. Biol. Chem. 267, 7806-7813.
  7. Zhao, C. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 10200-10205.
  8. Malakhov, M.P. et al. (2002) J. Biol. Chem. 277, 9976-9981.
  9. Malakhov, M.P. et al. (2003) J. Biol. Chem. 278, 16608-16613.
  10. Hamerman, J.A. et al. (2002) J. Immunol. 168, 2415-2423.
  11. Malakhova, O.A. et al. (2003) Genes Dev. 17, 455-460.

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