Extracts of HEK293 cells overexpressing c-Raf unstimulated (1) or stimulated with active Src (2-5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer for one hour at room temperature, then incubated with the c-Raf [pYpY 340/341] antibody for two hours at room temperature in a 1% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphotyrosine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate (Prod # ALI4404) and signals were detected using the Pierce SuperSignal™ method.
|Tested species reactivity||Human, Mouse, Rat|
|Published species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antibody was produced against a chemically synthesized phosphopeptide derived from a region of human c-Raf that contains tyrosines 340 and 341. The sequence is conserved in mouse and rat.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 50% glycerol, 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Immunohistochemistry (IHC)||Assay Dependent|
|Western Blot (WB)||Assay Dependent|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
|Western Blot (WB)||See 1 publications below|
Raf-1 is a MAP kinase kinase kinase (MAP3K) which functions downstream of the Ras family of membrane associated GTPases to which it binds directly. Once activated Raf-1 can phosphorylate to activate the dual specificity protein kinases MEK1 and MEK2 which in turn phosphorylate to activate the serine/threonine specific protein kinases ERK1 and ERK2. Activated ERKs are pleiotropic effectors of cell physiology and play an important role in the control of gene expression involved in the cell division cycle, apoptosis, cell differentiation and cell migration.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Human||Not Cited||CNK1 is a scaffold protein that regulates Src-mediated Raf-1 activation.||Ziogas A,Moelling K,Radziwill G||The Journal of biological chemistry (280:24205)||2005|