Western blot antibody-peptide competition and mutant analysis using a Phospho-c-Raf pSer621 polyclonal antibody (Product # PA1-26655). Immunoprecipitates of HEK293 cells overexpressing wild-type c-Raf and stimulated with 50 ng/ml EGF for 3 minutes (1-4) or c-Raf mutant S621A (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 overnight at 4°C, and incubated with c-Raf (pS621) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: the phosphopeptide immunogen (1), a generic phosphoserine-containing peptide (2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), or no peptide (4, 5). The inset shows the relative amount of total c-Raf protein in the wild-type vs. the S621A mutant extracts via c-Raf pan antibody. The data shows that only the phosphopeptide corresponding to c-Raf (pS621) blocks the antibody signal and that the antibody does not recognize the S621A mutant, demonstrating the specificity of the antibody.
|Tested species reactivity||Human, Mouse, Rat|
|Published species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic phosphopeptide corresponding to a portion of human c-Raf containing serine 621. The sequence is conserved in mouse and rat.|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS, pH 7.3, with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Western Blot (WB)||0.1-1 µg/ml|
* 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 2 publications below|
PA1-26655 detects c-Raf (phospho S621) from human samples. PA1-26655 is expected to cross react with mouse (100% conserved) and rat (100% conserved) due to sequence homology.
PA1-26655 has been successfully used in Western blot procedures.
The PA1-26655 immunogen is a synthetic phosphopeptide corresponding to a portion of human c-Raf containing serine 621. The sequence is conserved in mouse and rat.
This gene is the cellular homolog of viral raf gene (v-raf). The encoded protein 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, the cellular RAF1 protein 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. Mutations in this gene are associated with Noonan syndrome 5 and LEOPARD syndrome 2.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Nuclear Raf-1 kinase regulates the CXCR5 promoter by associating with NFATc3 to drive retinoic acid-induced leukemic cell differentiation.
PA1-26655 was used in ChIP assay, immunoprecipitation, and western blot to study the roles of NFATc3 and CXCR5 in the mechanism by which nuclear Raf-1 kinase drives retinoic acid-induced differentiation of leukemic cells
|Geil WM,Yen A||The FEBS journal (281:1170)||2014|
The Src-family kinase inhibitor PP2 rescues inducible differentiation events in emergent retinoic acid-resistant myeloblastic leukemia cells.
PA1-26655 was used in western blot to study the molecular mechanisms underlying emergent retinoic acid-resistance in two myeloblastic leukemia cell lines and the ability of a Src familily kinase inhibitor to restore cell cycle arrest
|Jensen HA,Styskal LE,Tasseff R,Bunaciu RP,Congleton J,Varner JD,Yen A||PloS one (8:null)||2013|