Anti-Phospho-Raf1-S259 Rabbit Monoclonal Antibody for WB and ELISA - P04049

Core Product Specifications and Parameters

Biological Background: RAF1 Function and Localization

• RAF1 (also known as c-RAF or Raf-1) is a serine/threonine-protein kinase that serves as a critical regulatory link between membrane-bound Ras GTPases and the downstream MAPK/ERK signaling cascade, controlling cell fate decisions such as proliferation, differentiation, apoptosis, and survival.
• Upon activation, RAF1 phosphorylates and activates the dual-specificity kinases MEK1/2, which in turn phosphorylate ERK1/2, propagating signals that regulate gene expression and cellular responses.
• Phosphorylated RAF1 (e.g., at Ser-338 and Ser-339) directly phosphorylates the pro-apoptotic protein BAD, promoting cell survival. It also phosphorylates adenylyl cyclases (ADCY2/5/6) and modulates the phosphatase PPP1R12A.
• RAF1 influences multiple signaling pathways: it can promote NF-κB activation while inhibiting effectors like ROCK2 (motility), ASK1/MST2 (apoptosis), and RB1 (proliferation/angiogenesis).
• Under apoptotic stimuli, RAF1 can translocate to the mitochondria, where it binds BCL2 and displaces BAD, providing an additional anti-apoptotic mechanism.
• RAF1 plays a role in cell migration by regulating Rho signaling and is required for normal wound healing. It also contributes to oncogenic transformation through transcriptional repression of tight junction proteins like occludin (OCLN).
• The protein is predominantly localized in the cytoplasm and at the cell membrane, but can also be found in the mitochondria and nucleus under certain conditions.
• RAF1 is expressed in various tissues; in skeletal muscle, isoform 1 is more abundant than isoform 2.

Experimental Guidance and Technical Tips

• The immunogen is a synthetic phosphorylated peptide centered on Ser259 of human RAF1 (P04049). The resulting antibody is expected to specifically recognize RAF1 when phosphorylated at Ser259; confirm specificity by comparing signals between phosphatase-treated and untreated samples.
• For Western blot (WB), use a protein extract from human or rat cells known to express RAF1. A band at approximately 73 kDa should be observed under reducing conditions. Always include appropriate positive and negative controls (e.g., serum-starved vs. growth factor-stimulated lysates).
• For ELISA, titrate the antibody against the phosphorylated peptide immunogen and validate in a relevant biological matrix to ensure linear detection. Consider pairing with a total RAF1 antibody to normalize phosphorylation levels.
• Because the antibody is supplied on ice bags, avoid repeated freeze-thaw cycles and store at recommended conditions to preserve activity.

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