Applications

Please Note: Optimal dilutions should be determined by each laboratory for each application.General Protocolsare available in the Technical Information section on our website.

Data Example

ImmunoprecipitationView Larger

Immunoprecipitation of Human ISG15/UCRP.A549 cells transfected with HA-ISG15 were treated briefly with IFN gammaor carrier only. 500 µg of cell lysates were subjected to immunoprecipitation (IP) with 20 µg A-830 using Protein G Sepharose. IP’d material was resolved on reducing SDS-PAGE gels and Western Blots were developed using PVDF membranes and anti-HA epitope (R&D Systems MAB060) at 0.5 µg/ml followed by HRP-labeled anti-mouse (R&D Systems # HAF007) secondary antibody at 1:2000 dilution. Free (unconjugated) HA-ISG15 was visualized in both treated and untreated cells, while ISGylated proteins were clearly recovered (IP’d) from the interferon-treated cell lysates.

Preparation and Storage

Background: ISG15/UCRP

Interferon­stimulated Gene 15 (ISG15), also known as Ubiquitin Cross­reacting Protein (UCRP), is a Ubiquitin­like protein that is covalently coupled to target proteins in a process termed ISGylation. It is a 165 amino acid (aa) polypeptide with a predicted molecular weight of 18 kDa. ISG15/UCRP exhibits 66% aa sequence identity with its mouse ortholog. Structurally, ISG15/UCRP consists of two tandem Ubiquitin­like domains that share a similar 3­dimensional structure with Ubiquitin and other Ubiquitin­like modifiers including NEDD8 and SUMO1. Modification of targets by ISG15/UCRP occurs in a stepwise enzymatic process similar to that of Ubiquitin. Enzymes regulating ISGylation include the activating (E1) enzyme UBE1L, the conjugating (E2) enzyme UbcH8, and ligases (E3) such as HERC5. Removal of ISG15/UCRP is catalyzed by the deconjugating enzyme UBP43/USP18. Functionally, ISG15/UCRP has putative roles in the immune response and tumorigenesis. This is reflected by intracellular ISG15/UCRP targets that include Cyclin D1, tumor suppressor p63, IRF3, and a range of viral proteins. It is induced by type 1 interferons and microbial infection, and knockout mice exhibit an increased sensitivity to infection by some viruses. ISG15/UCRP can also be secreted by cells of the immune system and may act in a cytokine­like manner. For instance, it is produced by human granulocytes in response to mycobacterium exposure, and natural killer cells and T cells respond to extracellular ISG15/UCRP with IFN­gamma production. Further supporting a role in immune function, ISG15/UCRP mutations are associated with MSMD, an inherited disorder characterized by increased susceptibility to mycobacterial infection.

Product Datasheets

Related Research Areas

• Ubiquitin-like Modifiers (UBLs) and Regulators