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

Name: Phospho-Met (Tyr1349) Antibody

Clone: Rabbit polyclonal

Supplier: Cell Signaling Technology

Catalog Number: 3121

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.

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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

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