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p-Met Antibody Staining Protocol for Immunohistochemistry
Description: Met, a high affinity receptor for hepatocyte growth factor (HGF; also known as scatter factor), is a disulfide-linked heterodimer made of 45 kDa a- and 145 kDa b-subunits. The a-subunit and the amino-terminal region of the b-subunit form the extracellular domain. The remainder of the b-chain spans the plasma membrane and contains a cytoplasmic region with tyrosine kinase activity. Interaction of Met with HGF results in autophosphorylation at multiple tyrosines, which recruit several downstream signaling components, including Gab1, c-Cbl and PI3 kinase and is critical for all of its known biological functions. Phosphorylation of Tyr1234/1235 in the Met kinase domain is critical to kinase activation. Phosphorylation of Tyr1349 in the Met cytoplasmic domain provides a direct binding site for Gab1. Altered Met levels and/or tyrosine kinase activities were found in different tumors, especially in renal, colon and breast cancers. Thus, Met is an attractive cancer therapeutic and diagnostic target.
Primary Antibody
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Name: Phospho-Met (Tyr1349) Antibody |
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Clone: Rabbit polyclonal |
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Supplier: Cell Signaling Technology |
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Catalog Number: 3121 |
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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: Membrane |
| Images: Search image |
Additional Information:
| Species Reactivity: Human, mouse, monkey, rat |
| Fixation: Formalin fixed paraffin sections |
| Positive Control: Human breast cancer, mouse xenograft tumor |
| 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: