Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855 in the presence of control peptide (left) or Phospho-4E-BP1 (Thr37/46) Blocking Peptide (right).Learn more about how we get our images
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
posted February 2010
revised March 2016
Protocol Id: 283
This peptide is used to block Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855 reactivity.
The quality of the peptide was evaluated by reversed-phase HPLC and by mass spectrometry. The peptide blocks Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855 signal in immunohistochemistry and both Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855 and Phospho-4E-BP1 (Thr37/46) Antibody #9459 in Western blotting.
Use as a blocking reagent to evaluate the specificity of antibody reactivity in Western blotting and immunohistochemistry protocols. For immunohistochemistry, add twice the volume of peptide as volume of antibody used in 100 μl total volume. Incubate for a minimum of 30 minutes prior to adding the entire volume to the slide. For Western blotting, add an equal volume of peptide as volume of antibody used in 10 ml total volume, and incubate at room temperature for 30 minutes before allowing to react with the blot. Recommended antibody dilutions can be found on the product data sheet.
Supplied in 20 mM potassium phosphate (pH 7.0), 50 mM NaCl, 0.1 mM EDTA, 1 mg/ml BSA, 5% glycerol and 1% DMSO. Store at –20°C.
Translation repressor protein 4E-BP1 (also known as PHAS-1) inhibits cap-dependent translation by binding to the translation initiation factor eIF4E. Hyperphosphorylation of 4E-BP1 disrupts this interaction and results in activation of cap-dependent translation (1). Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase regulate 4E-BP1 activity (2,3). Multiple 4E-BP1 residues are phosphorylated in vivo (4). While phosphorylation by FRAP/mTOR at Thr37 and Thr46 does not prevent the binding of 4E-BP1 to eIF4E, it is thought to prime 4E-BP1 for subsequent phosphorylation at Ser65 and Thr70 (5).
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