Western blot analysis of recombinant human IL-18 protein (hIL-18) at indicated levels using IL-18 (D2F3B) Rabbit mAb.
Western blot analysis of extracts from various cell lines using IL-18 (D2F3B) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
IL-18 (D2F3B) Rabbit mAb recognizes endogenous levels of total IL-18 protein. This antibody detects 110 and 250 kDa bands of unknown origin in some cell lines.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with human IL-18 recombinant protein.
Interleukin-18 (IL-18), originally known as interferon-gamma inducing factor (IGIF), is part of the IL-superfamily of cytokines (1,2). This proinflammatory cytokine is synthesized as an inactive precursor which requires cleavage by Caspase-1 to become an active, mature molecule, which is secreted by monocytes and macrophages (3,4,6). It induces IFNγ production in T-helper-1 (Th1) cells and natural killer (NK) cells, and can regulate innate immunity via both Th1 and Th2 responses (4,5). Elevated IL-18 levels are associated with autoimmune disease, but may be balanced by binding to IL-18 binding protein (IL-18BP), which along with IL-18 neutralizing antibodies, are being examined in clinical trials (6,7).
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