Western blot analysis of extracts from various cell lines using EPRS Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
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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 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
EPRS Antibody recognizes endogenous levels of total EPRS protein.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro978 of human EPRS protein. Antibodies are purified by protein A and peptide affinity chromatography.
EPRS (Glutamatyl-prolyl-tRNA synthetase) is a bifunctional enzyme in the aminoacyl-tRNA ligase family that attaches the cognate amino acid to the corresponding tRNA for protein translation (1,2). EPRS usually resides in the tRNA multisynthetase complex (MSC) that may facilitate the delivery of aminoacylated tRNAs to the ribosome during protein synthesis (3,4). In monocytic cells, upon interferon (IFN)-gamma activation, EPRS becomes phosphorylated and is released from the MSC to form the so-called GAIT (IFN-Gamma-Activated Inhibitor of Translation) complex with NS1-associated protein (NSAP1), ribosomal protein L13a, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The GAIT complex binds to a defined RNA element through EPRS in the 3’ untranslated region (UTR) to inhibit translation of target transcripts, including vascular endothelial growth factor (VEGF)-A, ceruloplasmin, and several cytokines and their receptors. Thus, EPRS plays an important role in inflammation regulation (5-9).
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