Western blot analysis of extracts from HT29 cells using eIF2B-epsilon 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 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 June 2020
Protocol Id: 10
eIF2B-epsilon Antibody detects endogenous levels of total eIF2B-epsilon protein.
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to amino acids near the middle of human eIF2B-epsilon. Antibodies are purified by protein A and peptide affinity chromatography.
Phosphorylation of the eukaryotic initiation factor 2 (eIF2) α subunit is a well-documented mechanism to downregulate protein synthesis under a variety of stress conditions. eIF2 binds GTP and Met-tRNAi and transfers Met-tRNA to the 40S subunit to form the 43S preinitiation complex (1,2). eIF2 promotes a new round of translation initiation by exchanging GDP for GTP, a reaction catalyzed by eIF2B (1,2). Kinases that are activated by viral infection (PKR), endoplasmic reticulum stress (PERK/PEK), amino acid deprivation (GCN2), or heme deficiency (HRI) can phosphorylate the α subunit of eIF2 (3,4). This phosphorylation stabilizes the eIF2-GDP-eIF2B complex and inhibits the turnover of eIF2B. Induction of PKR by IFN-γ and TNF-α induces potent phosphorylation of eIF2α at Ser51 (5,6).
eIF2B, a guanine nucleotide exchange factor, is composed of 5 subunits, the largest of which is eIF2B-epsilon (7). Multiple in vivo phosphorylation sites have been identified on eIF2B-epsilon (8). Casein Kinase II can phosphorylate eIF2B-epsilon at Ser717/718 to allow for association with its substrate eIF2. Phosphorylation at Ser544 allows GSK-3 to phosphorylate the key regulatory site Ser540. A fifth eIF2B-epsilon phosphorylation site, Ser466, can be phosphorylated by casein kinase I.
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