Western blot analysis of extracts from RAW 264.7 cells, untreated (-) or treated with LPS #14011 (5 μg/ml, 6 hr; +), using IRG1 Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).Learn more about how we get our images.
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
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
IRG1 Antibody recognizes endogenous levels of total IRG1 protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of mouse IRG1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
IRG1 (Immune-responsive gene 1) is one of the most up-regulated genes in macrophages under proinflammatory conditions (1). It is also highly expressed in the pregnant uterus during implantation (2,3). IRG1 is a cis-aconitate decarboxylase that produces itaconic acid by decarboxylating cis-aconic acid, an intermediate of the tricarboxylic acid cycle (4). Itaconic acid is an endogenous inhibitor of succinate dehydrogenase, linking macrophage metabolic rewiring and regulation of inflammation (5,6).
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|17805S||100 µl||$ 260|