PRODUCT CODE: ET1612-87

Recombinant Phospho Raf1 (S259) Monoclonal Antibody (ET1612-87)

  • Recombinant

Applications

  • WB

  • IHC-P

REACTIVITY

  • Human

  • Mouse

  • Rat

Western blot analysis of Phospho-Raf1 (S259) on Hela cell lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1612-87, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
  • Western blot analysis of Phospho-Raf1 (S259) on Hela cell lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1612-87, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
  • Immunohistochemical analysis of paraffin-embedded rat skeletal muscle tissue using anti-Phospho-Raf1 (S259) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1612-87, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Western blot analysis of Phospho-Raf1 (S259) on Hela cell lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1612-87, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.

Applications

  • WB

  • IHC-P

REACTIVITY

  • Human

  • Mouse

  • Rat

SPECIFICATIONS

Product Type

Recombinant Rabbit monoclonal primary

Product Name

Recombinant Phospho Raf1 (S259) Monoclonal Antibody (ET1612-87)

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding ser259 of human raf1

Host

Rabbit

Modification

Phospho

Modification Site

S259

Positive Control

Hela cell lysates, rat skeletal muscle tissue.

Conjugation

Unconjugated

Clonality

Monoclonal

Clone Number

SD85-07

PROPERTIES

Form

Liquid

Storage Condition

Store at +4C after thawing. Aliquot store at -20C or -80C. Avoid repeated freeze / thaw cycles.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Concentration

1 ug/ul

PURIFICATION

Protein A affinity purified.

MOLECULAR WEIGHT

73 kDa

Isotype

IgG

APPLICATION DILUTION

  • WB

  • 1:1,000-1:2,000

  • IHC-P

  • 1:50-1:200

TARGET

UNIPROT #

PROTEIN NAME

Phospho-Raf1 (S259)

SYNONYMS

c Raf antibody; C-raf antibody; C-Raf proto-oncogene, serine/threonine kinase antibody; CMD1NN antibody; Craf 1 transforming gene antibody; cRaf antibody; Craf1 transforming gene antibody; EC 2.7.11.1 antibody; kinase Raf1 antibody; Murine sarcoma 3611 oncogene 1 antibody; NS5 antibody; Oncogene MIL antibody; Oncogene RAF1 antibody; OTTHUMP00000160218 antibody; OTTHUMP00000207813 antibody; OTTHUMP00000209389 antibody; Protein kinase raf 1 antibody; Proto-oncogene c-RAF antibody; Raf 1 antibody; Raf 1 proto oncogene serine/threonine kinase antibody; RAF antibody; Raf proto oncogene serine/threonine protein kinase antibody; RAF proto-oncogene serine/threonine-protein kinase antibody; RAF-1 antibody; RAF1 antibody; RAF1_HUMAN antibody; Similar to murine leukemia viral (V-raf-1) oncogene homolog 1 antibody; TRANSFORMING REPLICATION-DEFECTIVE MURINE RETROVIRUS 3611-MSV antibody; v raf 1 murine leukemia viral oncogene homolog 1 antibody; v-raf murine sarcoma viral oncogene homolog 1 antibody; v-raf-1 murine leukemia viral oncogene-like protein 1 antibody; vraf1 murine leukemia viral oncogene homolog 1 antibody

SEQUENCE SIMILARITIES

Belongs to the protein kinase superfamily. TKL Ser/Thr protein kinase family. RAF subfamily.

TISSUE SPECIFICITY

In skeletal muscle, isoform 1 is more abundant than isoform 2.

POST-TRANSLATIONAL MODIFICATION

Phosphorylation at Thr-269, Ser-338, Tyr-341, Thr-491 and Ser-494 results in its activation. Phosphorylation at Ser-29, Ser-43, Ser-289, Ser-296, Ser-301 and Ser-642 by MAPK1/ERK2 results in its inactivation. Phosphorylation at Ser-259 induces the interaction with YWHAZ and inactivates kinase activity. Dephosphorylation of Ser-259 by the complex containing protein phosphatase 1, SHOC2 and M-Ras/MRAS relieves inactivation, leading to stimulate RAF1 activity. Phosphorylation at Ser-338 by PAK1 and PAK5 and Ser-339 by PAK1 is required for its mitochondrial localization. Phosphorylation at Ser-621 in response to growth factor treatment stabilizes the protein, possibly by preventing proteasomal degradation. Phosphorylation at Ser-289, Ser-296, Ser-301, Ser-338 and Ser-621 are somehow linked to the methylation potential of cells. Treatment of cells with HGF in the presence of the methylation inhibitor 5'-methylthioadenosine (MTA) results in increased phosphorylation at Ser-338 and Ser-621 and decreased phosphorylation at Ser-296, Ser-301 and Ser-338. Dephosphorylation at Ser-338 by PPP5C results in an activity decrease.; Methylated at Arg-563 in response to EGF treatment. This modification leads to destabilization of the protein, possibly through proteasomal degradation.

SUBCELLULAR LOCATION

Cytoplasm, Cell membrane, Mitochondrion, Nucleus.

FUNCTION

Raf-1 is a ubiquitously expressed cytoplasmic protein with intrinsic serine/threonine kinase activity. Raf-1, or c-Raf, is the cellular homolog of v-Raf, the product of the transforming gene of the 3611 strain of murine sarcoma virus. The unregulated kinase activity of the v-Raf protein is associated with cellular transformation and mitogenesis. Raf-1 is normally suppressed by its regulatory N-terminal domain. Raf-1 is activated in response to a variety of tyrosine kinase receptors as well as in response to pp60v-Src expression. Specifically, Raf-1 is phosphorylated in the catalytic domain at Ser 338 and, to a lesser extent, Ser 339. This phosphorylation requires the co-activation of PI 3-kinase and the Ras signaling pathway. Raf-1 is also phosphorylated on Tyr 340 and 341, which induces the phosphorylation of MEK. Phosphorylation of Ser 621 is essential for the catalytic activity of Raf-1 and downregulation by c-AMP-dependent protein kinase A (PKA). PKA also phosphorylates Raf-1 on Ser 43 and Ser 259. PKA phosphorylation of Ser 259 inhibits Raf-1 and decreases the phosphorylation necessary for Raf-1 activation at Ser 338.