Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length mouse IRGM protein (mIRGM-Myc/DDK; +), using IRGM Antibody (Rodent Specific) (upper) and Myc-Tag (71D10) Rabbit mAb #2278 (lower).
Western blot analysis of extracts from Raw 264.7 and J774A.1 cells, untreated (-) or treated with LPS #14011 (1 μg/ml, overnight; +), using IRGM Antibody (Rodent Specific) (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
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.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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 October 2016
Protocol Id: 10
IRGM Antibody (Rodent Specific) recognizes endogenous levels of total IRGM protein in mouse and rat.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg215 of mouse IRGM protein. Antibodies are purified by protein A and peptide affinity chromatography.
Immunity-related GTPase family M protein 1 (IRGM, LRG-47) belongs to the p47 family of immunity related guanosine triphosphatases (IRGs) that regulate innate immune responses to intracellular pathogens (1-3). Research studies indicate that IRGM plays a role in autophagy during clearance of intracellular bacteria (4). Expression of IRGM in mice, but not in humans, is induced by inflammatory signals that include interferon and LPS (2,3). Polymorphisms in the corresponding IRGM gene are associated with some cases of tuberculosis (5-7), Crohn’s disease (8,9), and severe sepsis (10). Additional studies indicate that IRGM functions through regulation of autophagy (4). Mitochondrial IRGM plays a role in mitochondrial fission, membrane polarization, and mitophagy (11). Knockout mice for IRGM show increased susceptibility to infection as well as intestinal inflammation and Paneth cell abnormalities (12,13). Knockout mice against IRGM are also resistant to neuronal autophagy following stroke (14). RNA viruses commonly target IRGM in order to suppress autophagy and enhance infection (15).
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