Phospho-Smad2-S465/467 + Smad3-S423/425 Rabbit Polyclonal Antibody for WB, IF/ICC, IHC-P, ELISA - Q15796 / P84022

Core Product Specifications and Parameters

Biological Background: SMAD2 and SMAD3 Function and Localization

• SMAD2 is a receptor-regulated SMAD (R-SMAD) that acts as an intracellular signal transducer for TGF-beta and activin pathways.
• Upon ligand binding, type 1 receptor kinases phosphorylate SMAD2 at the C-terminal SSXS motif (Ser465 and Ser467), triggering its activation, complex formation with SMAD4, and nuclear translocation.
• In the nucleus, the SMAD2/SMAD4 complex binds the TGF-beta responsive element (TRE) in target gene promoters and regulates transcription.
• SMAD2 may function as a tumor suppressor in colorectal carcinoma. Related references: PMID:8752209
• It is highly expressed in skeletal muscle, endothelial cells, heart, and placenta.
• SMAD3 is a close homolog of SMAD2 and shares similar activation mechanisms. The antibody detects phosphorylation at the corresponding sites Ser423/425 in SMAD3.
• Key post-translational modifications include phosphorylation, acetylation, and ubiquitination; SMAD2 also contains a zinc-binding site.

Experimental Guidance and Technical Tips

• The immunogen is a synthetic phosphopeptide targeting the dual phosphorylation motif, providing specificity for the activated forms of SMAD2 and SMAD3. Verify phospho-specificity using phosphatase-treated samples.
• For Western blot, include phosphatase inhibitors during lysis to preserve phosphorylation, and consider using fresh samples. The predicted molecular weights of SMAD2 and SMAD3 are around 48-52 kDa, but multiple bands may appear due to phosphorylation and isoforms.
• For immunofluorescence and immunohistochemistry, standard fixation with 4% PFA and permeabilization with Triton X-100 may be suitable; optimize antigen retrieval for IHC-P.
• ELISA may be used for high-throughput screening of TGF-beta pathway activation; cross-reactivity testing is advisable for multi-species studies.
• Always include appropriate positive and negative controls (e.g., TGF-beta-treated vs. untreated cells) to confirm phospho-specificity.

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