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Phospho-mTOR (Ser2448) Antibody Staining Protocol for Immunohistochemistry
Description: The mammalian target of rapamycin mTOR, also know as FRAP or RAFT(1–3), is a Ser/Thr protein kinase. mTOR acts as a sensor for ATP and amino acids, balancing the availability of nutrients and cell growth (4,5). When sufficient nutrients are available, mTOR responds to a phosphatidic acid-mediated signal (6), and transmits a positive signal to p70 S6 kinase, and participates in the inactivation of the eIF4E inhibitor, 4E-BP1. 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 cellular growth and homeostasis, and its regulation is frequently altered in tumors. For these reasons, mTOR is currently under investigation as a potential target for anti-cancer therapy.
Primary Antibody
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Name: Phospho-mTOR (Ser2448) Antibody |
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Clone: 49F9, Rabbit monoclonal |
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Supplier: Cell Signaling Technology |
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Catalog Number: 2976 |
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Dilution: 1:50 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed. |
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Incubation Time/Temp: 60 min/room temperature |
Antigen Retrieval
| Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102) |
| Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100) |
| Heat/Cool Temperature: 95-100 ºC/room temperature |
| Heat/Cool Time: 20 minutes/20 minutes |
Detection Methods
| Standard Method: ABC Method or LSAB Method |
| Enhanced Method: Polymeric Methods |
Chromogen Substrate
| Reagent: DAB |
| Incubation Time/Temperature: 1-3 minutes/room temperature |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Cytoplasmic |
| Images: Search image |
Additional Information:
| Tissue Type: Human breast carcinoma, colon carcinoma, bladder carcinoma, prostate carcinoma. |
| Fixation: Formalin fixed paraffin sections, or acetone-fixed frozen sections (no antigen retrieval needed) |
| Positive Control: Human breast carcinoma, colon carcinoma, bladder carcinoma, prostate carcinoma. |
| Negative Control: Omit primary antibody, isotype control, absorption control |
| Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary |
References: