Western blot analysis of extracts from various cell lines using cGAS (D1D3G) Rabbit mAb.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc-tagged full-length human cGAS protein (hcGAS-Myc; +), using cGAS (D1D3G) Rabbit mAb.
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
cGAS (D1D3G) Rabbit mAb recognizes endogenous levels of total cGAS protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala19 of human cGAS protein.
Cyclic GMP-AMP synthase (cGAS, MB21D1) is an antiviral enzyme that produces the second messenger cyclic-GMP-AMP (cGAMP) in response to cytoplasmic DNA (1,2). The cGAS protein acts as a cytosolic DNA sensor that binds DNA and produces the cGAMP second messenger from ATP and GTP (1,2). cGAMP binds to and activates STING, a transmembrane adaptor protein that is a critical component of the cellular innate immune response to pathogenic cytoplasmic DNA (1-4). STING is ubiquitously expressed and found predominantly in the ER (3). Following activation, STING translocates with TBK1 to perinuclear endosomes (5). The TBK1 kinase phosphorylates and activates interferon regulatory factors (IRFs) and NF-κB, which leads to the induction of type I interferon and other immune response genes (3-5).
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