Lysates from Hek293 cells overexpressing c-Raf al one (1) or immunoprecipitates prepared from cells co-transfected with c-Raf, Ras[V12], and Rac1 (2-6) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4°C, and then incubated with the c-Raf [pSpY 338/340] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphoserine-containing peptide (4), a generic phosphotyrosine-containing peptide (5), or the phosphopeptide immunogen (6). After washing, the membrane was incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase (Prod # ALI4405) and signals were detected using the Tropix WesternStar™ method.
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human c-Raf that contains serine 338 and tyrosine 340. The sequence is conserved in mouse and rat.|
|Purification||Antigen affinity chromatography|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA, 50% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|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.
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.