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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 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 June 2020
Protocol Id: 263
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro298 of human eIF3D protein.
Translation initiation requires a set of factors to facilitate the association of the 40S ribosomal subunit with mRNA. The eIF4F complex, consisting of eIF4E, eIF4A, and eIF4G, binds to the 5' cap structure of mRNA. eIF4F and eIF4B unwind the secondary structure of mRNA at its 5' untranslated region. The 40S ribosomal subunit, along with some initiation factors including eIF3, then binds to the 5' mRNA cap and searches along the mRNA for the initiation codon. eIF3 is a large translation initiation complex with 10 to 13 different subunits. eIF3A, eIF3B, eIF3C, eIF3E, eIF3F, and eIF3H are the core subunits critical for the function of this complex. eIF3 physically interacts with eIF4G, which may be responsible for the association of the 40S ribosomal subunit with mRNA (1). eIF3D is another subunit of the eIF3 protein complex. It binds to the 5' cap structure of a subset of mRNAs. This eIF3D-mRNA interaction is required for the translation initiation complex formation on eIF3-specialized mRNAs (2). Studies found that eIF3D is activated under metabolic stress by decreased phosphorylation near its cap-binding pocket. The translation adaptation mediated by eIF3D is necessary for cell survival during glucose deprivation (3).
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