GRB-2 Antibody Staining Protocol for Immunohistochemistry


Description: The superfamily of GTP binding proteins, of which Ras proteins are prototypes, has been implicated in a broad range of biological activities. Studies have identified a family of guanine nucleotidereleasing factors (GRFs) that activate Ras in mammalian cells and an "adapter" protein (Sem 5/GRB2) that appears to mediate the interaction of GRFs with activated receptor molecules. Ras-GRF p140 promotes nucleotide exchange on Ras p21s but not on other members of the Ras gene superfamily. In addition, three mammalian homologs of the Drosophila Ras-GRF, son of sevenless (Sos), have been described. These include two from mouse, m Sos 1 and m Sos 2, and one from human, h Sos. Subsequent to activation, Ras appears to functionally interact with Raf, thereby activating the MAP kinase phosphorylation pathway.


Primary Antibody

Name: GRB-2 Antibody

Clone: Rabbit anti-Human

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-255

Dilution: 1:500 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 minutes/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 ºC - 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:
Tissue Type: Colon carcinoma
Fixation: Formalin fixed paraffin sections, acetone fixed frozen sections
Positive Control: Hela Cells, human colon carcinoma.
Negative Control: Omit primary antibody, isotype control or 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


No need to perform antigen retrieval on frozen sections