 |
|
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 |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| 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 |
Notes:
No need to perform antigen retrieval on frozen sections |
References: