Immunohistochemical analysis of paraffin-embedded human transitional epithelial carcinoma of the bladder, using Phospho-mTOR (Ser2448) (49F9) Rabbit mAb (IHC Specific) (#2976) in the presence of control peptide (left) or Phospho-mTOR (Ser2448) 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: 305
This peptide is used to specifically block Phospho-mTOR (Ser2448) (49F9) Rabbit mAb (IHC Specific) #2976 and #2971 Phospho-mTOR (Ser2448) Antibody reactivity.
The quality of the peptide was evaluated by reversed-phase HPLC and by mass spectrometry. The peptide blocks Phospho-mTOR (Ser2448) (49F9) Rabbit mAb (IHC Specific) (#2976) signal in immunohistochemistry and #2971 Phospho-mTOR (Ser2448) Antibody in western blotting.
Use as a blocking reagent to evaluate the specificity of antibody reactivity in immunohistochemistry protocols. For immunohistochemistry, add twice the volume of peptide as volume of antibody used in 100 ul 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.
The mammalian target of rapamycin (mTOR, FRAP, RAFT) is a Ser/Thr protein kinase (1-3) that functions as an ATP and amino acid sensor to balance nutrient availability and cell growth (4,5). When sufficient nutrients are available, mTOR responds to a phosphatidic acid-mediated signal to transmit a positive signal to p70 S6 kinase and participate in the inactivation of the eIF4E inhibitor, 4E-BP1 (6). These events result in the translation of specific mRNA subpopulations. mTOR is phosphorylated at Ser2448 via the PI3 kinase/Akt signaling pathway and autophosphorylated at Ser2481 (7,8). mTOR plays a key role in cell growth and homeostasis and may be abnormally regulated in tumors. For these reasons, mTOR is currently under investigation as a potential target for anti-cancer therapy (9).
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