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.
WB 1:500 - 1:2000
IF 1:50 - 1:200
IP 1:50 - 1:100
A phospho specific peptide corresponding to residues surrounding S621 of human RAF1
Western blot analysis of extracts of HeLa and 293T cells, using Phospho-RAF1-S621 antibody (144-50087) at 1:1000 dilution. HeLa cells were treated by EGF (100ng/ml) for 30 minutes after serum-starvation overnight. 293T cells were treated by PMA/TPA (200nM) for 30 minutes or treated by EGF (25ug/ml) for 30 minutes after serum-starvation overnight.
Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L) at 1:10000 dilution.
Lysates/proteins: 25ug per lane.
Blocking buffer: 3% BSA.
Immunofluorescence analysis of U2OS cells using Phospho-RAF1-S621 antibody (144-50087). Blue: DAPI for nuclear staining.
Immunoprecipitation analysis of 200ug extracts of HeLa cells, using 3 ug Phospho-RAF1-S621 pAb (144-50087). Western blot was performed from the immunoprecipitate using Phospho-RAF1-S621 pAb (144-50087) at a dilition of 1:1000. HeLa cells were treated by EGF (100 ng/ml) at 37? for 30 minutes after serum-starvation overnight.
12 months from the date of shipment when stored properly.