Il18 (37-194) Mouse Monoclonal Antibody [Clone ID: 91D8]

Interleukin 18 (IL-18) is an 18-kDa cytokine which identified as a costimulatory factor for production of interferon-γ (IFN-γ) in response to toxic shock and shares functional similarities with IL-12. IL-18 is synthesized as a precursor 24-kDa molecule without a signal peptide and must be cleaved to produce an active molecule. IL-1 converting enzyme (ICE, Caspase-1) cleaves pro-IL-18 at aspartic acid in the P1 position, producing the mature, bioactive peptide that is readily released from the cells. It is reported that IL-18 is produced from Kupffer cells, activated macrophages, keratinocytes, intestinal epithelial cells, osteoblasts, adrenal cortex cells and murine diencephalon. IFN-γ is produced by activated T or NK cells and plays critical roles in the defense against microbiral pathogens. IFN-γ activates macrophages and enhances NK activity and B cell maturation, proliferation and Ig secretion. IFN-γ also induces expression of MHC class I and II antigens and inhibits osteoclast activation. IL-18 acts on T helper type-1 (Th1) T cells and in combination with IL-12 strongly induces them to produce IFN-γ. Pleiotropic effects of IL-18 have also been reported, such as, enhancement production of IFN-γ and GM-CSF in peripheral blood mononuclear cells, production of Th1 cytokines, IL-2, GM-CSF and IFN-γ in T cells, enhancement of Fas ligand expression by Th1 cells.

This product was originally produced by MBL International.

Immunoprecipitation
1) Add primary antibody as suggest in the APPLICATIONS into the recombinant protein. Mix well and incubate with gentle agitation for 30-120 minutes at 4oC.
2) Add 10 µL of 50% protein A agarose resuspended in the cold Lysis buffer. Mix well and incubate with gentle agitation for 60 minutes at 4oC.
3) Wash the beads 3-5 times with the cold Lysis buffer (centrifuge the tube at 2,500 x g for 10 seconds).
4) Resuspend the beads in 20 µL of Laemmli’s sample buffer, boil for 3-5 minutes, and centrifuge for 5 minutes.
5) Load 10 µL of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis.
6) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for precise transfer procedure.
7) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4oC.
8) Incubate the membrane with 1 µg/mL of anti-rat IL-18 monoclonal antibody as primary antibody diluted with PBS, pH 7.2 containing 1% skimmed milk for 1 hour at room temperature. (The concentration of antibody will depend on condition.)
9) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (5 minutes x 3 times).
10) Incubate the membrane with the 1:1,000 mouse True Blot diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature.
11) Wash the membrane with PBS-T (5 minutes x 6 times).
12) Wipe excess buffer on the membrane, then incubate it with appropriate chemiluminescence reagent for 1 minute.
13) Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap.
14) Expose to an X-ray film in a dark room for 2 minutes.
15) Develop the film as usual. The condition for exposure and development may vary.