Western blot analysis of extracts from various cell lines using eRF3 Antibody.
Western blot analysis of extracts from THP-1 cells, untreated (-) or treated with Etoposide #2200 (25 μM, 24 hr; +), using eRF3 Antibody (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Etoposide treatment of THP-1 cells induces cleavage of full-length eRF3.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human eRF3a protein, isoform 3 (heRF3a-Myc/DDK; +), using eRF3 Antibody.
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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 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
eRF3 Antibody recognizes endogenous levels of total eRF3 protein. This antibody recognizes eRF3a and eRF3b proteins.Species Reactivity:
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro180 of human eRF3a protein, isoform 3. Antibodies are purified by protein A and peptide affinity chromatography.
Eukaryotic release factor 3 (eRF3, GSPT) is an evolutionarily conserved class II release factor and member of the GTPase superfamily that cooperates with eRF1 in polypeptide translation termination (1). Paralogous genes encode a pair of eRF3 proteins (eRF3a/GSPT1, eRF3b/GSPT2) that share a conserved carboxy-terminal GTPase/eRF1-binding domain and a non-conserved amino-terminal PABP1 binding site (2). The eRF3 carboxy-terminal region is involved in translation termination through binding and activation of the eRF1 release factor (1). The amino-terminal region of eRF3 is not required for eRF1 binding and activation, but is implicated in control of mRNA stability (3,4). Expression of eRF3 proteins vary, with eRF3a ubiquitously expressed and proliferation-dependent, while eRF3b expression is more restricted to brain tissue (2,5,6). Research studies demonstrate that eRF3 undergoes caspase-mediated cleavage and degradation related to reduced protein synthesis during DNA damage-induced apoptosis (7). Additional studies indicate that polyglycine expansion of the eRF3a amino terminus is associated with an increased susceptibility to breast and gastric cancer (8,9). It is likely that the polyglycine expansions of amino-terminal eRF3a may affect the ability of eRF3a to undergo caspase-mediated cleavage (9).
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