Phospho-SMAD2 (S465) Antibody

What is the biological significance of SMAD2 phosphorylation at S465/S467?

SMAD2 phosphorylation at serine residues 465 and 467 represents a critical regulatory step in the TGF-β signaling pathway. Following TGF-β stimulation, the receptor kinase TGF-β R1 phosphorylates these specific serine residues at the carboxy-terminus of SMAD2. This phosphorylation enables SMAD2 to form heteromeric complexes with SMAD4, triggering nuclear translocation where these complexes regulate gene transcription. This post-translational modification is essential for propagating TGF-β signals from the cell membrane to the nucleus, ultimately controlling numerous cellular processes including proliferation, differentiation, and extracellular matrix production . Understanding this phosphorylation event is crucial for studying developmental processes, fibrotic diseases, and cancer progression where TGF-β signaling plays significant roles.

How does SMAD2 phosphorylation relate to SMAD3 phosphorylation?

SMAD2 and SMAD3 are highly homologous proteins that undergo similar phosphorylation events in response to TGF-β stimulation. While SMAD2 is phosphorylated at Ser465/467, SMAD3 is phosphorylated at the corresponding residues Ser423/425 by the same receptor kinase TGF-β R1 . These phosphorylation events enable both proteins to participate in signaling complexes with SMAD4. Despite their similarities, SMAD2 and SMAD3 can regulate distinct and sometimes opposing gene expression programs. Some antibodies are designed to detect phosphorylation at both SMAD2 (S465/S467) and SMAD3 (S423/S425) due to the conserved phosphorylation motifs, while others are specific to phospho-SMAD2 only . Researchers should carefully select antibodies based on whether they need to distinguish between phosphorylated SMAD2 and SMAD3 or detect both simultaneously.

What are the key differences between monoclonal and polyclonal phospho-SMAD2 antibodies?

Monoclonal phospho-SMAD2 antibodies, like the rabbit monoclonal antibody 138D4 (Cell Signaling #3108), offer high specificity by detecting SMAD2 only when dually phosphorylated at serines 465 and 467 . These antibodies provide consistent lot-to-lot reproducibility and reduced background, making them particularly valuable for quantitative applications. Polyclonal phospho-SMAD2 antibodies, such as those from Proteintech (29129-1-AP), recognize multiple epitopes around the phosphorylation sites, potentially offering higher sensitivity but with possible batch-to-batch variation . When selecting an antibody, researchers should consider their specific experimental needs. For precise quantification of phospho-SMAD2 levels across multiple experiments, monoclonal antibodies typically provide more consistent results. For maximum sensitivity in detecting low levels of phosphorylated protein, polyclonal antibodies may offer advantages. Always validate new antibody lots against positive controls (e.g., TGF-β-treated cells) to ensure consistent performance.

How can I determine the cross-reactivity of phospho-SMAD2 antibodies with other species?

Cross-reactivity information is typically provided in product documentation and should be carefully reviewed before selecting an antibody for non-human samples. Based on the search results, several commercial phospho-SMAD2 antibodies demonstrate cross-reactivity across multiple species. For example, Cell Signaling's 138D4 rabbit monoclonal antibody (#3108) shows reactivity with human, mouse, rat, and mink samples . R&D Systems' antibodies have been tested with human and mink samples . Proteintech's antibody (29129-1-AP) has tested reactivity with human samples and cited reactivity with mouse samples . When working with a species not explicitly listed in the documentation, researchers should perform their own validation studies. This could involve comparing signals from the species of interest with known positive controls, or using recombinant proteins or phospho-peptides of the target species. Sequence alignment of the phosphorylation site region across species can also provide preliminary information about potential cross-reactivity.

What are the optimal conditions for detecting phospho-SMAD2 by Western blot?

For optimal detection of phospho-SMAD2 (S465/S467) by Western blot, multiple factors need careful consideration. Based on the search results, a standardized protocol would include:

Sample preparation:

• Treat cells with 10 ng/mL of recombinant TGF-β1 for 30 minutes to induce SMAD2 phosphorylation as a positive control

• Lyse cells in appropriate buffer (manufacturers typically provide recommended buffer groups)

• Use phosphatase inhibitors in lysis buffers to prevent dephosphorylation

Antibody conditions:

• Use antibody at manufacturer-recommended dilutions (typically 1:1000 for Western blot as seen with Cell Signaling's antibody)

• R&D Systems recommends 2 μg/mL concentration for their antibody

• Incubate membranes with primary antibody overnight at 4°C for optimal sensitivity

Detection:

• Use appropriate HRP-conjugated secondary antibodies (e.g., Anti-Rabbit IgG for rabbit primary antibodies)

• Perform all blotting under reducing conditions

• Expect to detect phospho-SMAD2 at approximately 60-68 kDa

For reproducible results, researchers should optimize blocking conditions, antibody concentrations, and incubation times for their specific experimental systems. Phosphorylation signals can be ephemeral, so careful timing of cell stimulation and rapid processing of samples is crucial for consistent results.

How can I optimize immunofluorescence staining for phospho-SMAD2?

Optimizing immunofluorescence staining for phospho-SMAD2 requires attention to fixation, permeabilization, and antibody incubation conditions. Based on search result , a successful protocol includes:

• Cell treatment: Stimulate cells with TGF-β (e.g., 10 ng/mL of Recombinant Human TGF-β2) to induce SMAD2 phosphorylation

• Fixation: Use immersion fixation (typically 4% paraformaldehyde for 15 minutes at room temperature)

• Permeabilization: Carefully permeabilize cells (0.1-0.5% Triton X-100 for 10 minutes) to allow antibody access to nuclear phospho-SMAD2

• Antibody dilution: Use phospho-SMAD2 antibodies at dilutions of 1:50-1:100 as a starting point

• Incubation conditions: Incubate with primary antibody for 3 hours at room temperature or overnight at 4°C

• Detection: Use appropriate fluorophore-conjugated secondary antibodies (e.g., NorthernLights™ 557-conjugated Anti-Rabbit IgG)

• Counterstaining: Include DAPI nuclear counterstain to visualize nuclear localization of phospho-SMAD2

The pattern of phospho-SMAD2 staining should show significant nuclear accumulation after TGF-β treatment, with minimal cytoplasmic staining. Always include both unstimulated controls and TGF-β-stimulated positive controls. Since phosphorylation is dynamic, standardizing the time between stimulation and fixation is critical for reproducible results. If background is problematic, increasing blocking time and optimizing antibody dilutions may improve signal-to-noise ratio.

How can I differentiate between specific and non-specific bands when analyzing phospho-SMAD2 Western blots?

Distinguishing specific phospho-SMAD2 bands from non-specific signals requires careful experimental design and controls. Phospho-SMAD2 should appear at approximately 60-68 kDa on Western blots . To confirm band specificity:

• Treatment controls: Compare untreated cells (-) with TGF-β-treated cells (+) to identify inducible bands. Specific phospho-SMAD2 signals should increase substantially after TGF-β treatment (typically 10 ng/mL for 30 minutes)

• Molecular weight markers: Verify that the detected band corresponds to the expected molecular weight of phospho-SMAD2 (approximately 60-68 kDa)

• Phosphatase treatment: Treat some lysate samples with lambda phosphatase to remove phosphorylation; the phospho-SMAD2 band should disappear in these samples

• Blocking peptide: If available, pre-incubate the antibody with a phospho-peptide containing the S465/S467 phosphorylation sites to block specific binding

• Knockdown validation: Use SMAD2 siRNA or CRISPR knockout cells as negative controls to confirm antibody specificity

Non-specific bands that appear consistently across all lanes, including negative controls, should be documented but excluded from analysis. If multiple specific bands appear, these could represent different isoforms or post-translationally modified forms of SMAD2. For maximum specificity, researchers might consider using monoclonal antibodies like the 138D4 clone, which is reported to detect SMAD2 only when dually phosphorylated at serines 465 and 467 .

What are common pitfalls in phospho-SMAD2 detection and how can they be addressed?

Researchers frequently encounter challenges when detecting phospho-SMAD2. Common issues and their solutions include:

• Weak or absent signals:

  • Insufficient TGF-β stimulation: Verify TGF-β activity and increase concentration (10 ng/mL is typically effective)

  • Rapid dephosphorylation: Include phosphatase inhibitors in all buffers

  • Suboptimal antibody concentration: Titrate antibody (1:1000 dilution or 2 μg/mL are common starting points)

• High background:

  • Non-specific antibody binding: Optimize blocking conditions and antibody dilution

  • Insufficient washing: Increase wash duration and volume

  • Secondary antibody cross-reactivity: Use highly cross-adsorbed secondary antibodies

• Inconsistent results:

  • Variable TGF-β treatment timing: Standardize the stimulation protocol (30 minutes is commonly used)

  • Cell density variations: Maintain consistent cell confluence between experiments

  • Antibody lot-to-lot variations: Test new lots against reference samples

• Multiple bands or unexpected band size:

  • Protein degradation: Use fresh protease inhibitors and keep samples cold

  • Post-translational modifications: Characterize bands using additional techniques (e.g., mass spectrometry)

  • Cross-reactivity with SMAD3: Some antibodies detect both phospho-SMAD2 and phospho-SMAD3; use SMAD2-specific antibodies if differentiation is required

• Poor nuclear localization in immunofluorescence:

  • Inadequate fixation: Optimize fixation protocol to preserve phospho-epitopes

  • Insufficient permeabilization: Adjust detergent concentration to allow nuclear antibody access

  • Timing of fixation: Fix cells at peak nuclear accumulation (typically 30-60 minutes after TGF-β stimulation)

Creating a detailed standardized protocol with precise timing, buffer compositions, and cell handling procedures can significantly improve reproducibility in phospho-SMAD2 detection experiments.

How can phospho-SMAD2 antibodies be used to investigate cross-talk between TGF-β and other signaling pathways?

Investigating signaling cross-talk using phospho-SMAD2 antibodies requires thoughtful experimental design. TGF-β signaling intersects with numerous other pathways, including MAPK, PI3K/Akt, Wnt, and Hippo signaling. To study these interactions:

• Combined pathway stimulation/inhibition:

  • Treat cells with TGF-β (10 ng/mL) alone or in combination with activators/inhibitors of other pathways

  • Monitor phospho-SMAD2 levels by Western blot (recommended dilution 1:1000)

  • Assess nuclear translocation by immunofluorescence using antibody dilutions of 1:50-1:100

• Time-course experiments:

  • Collect samples at multiple time points after stimulation

  • Analyze both immediate (30 minutes) and delayed (24 hours) effects on SMAD2 phosphorylation

• Additional phosphorylation sites:

  • Beyond the C-terminal S465/467 sites, examine linker region phosphorylation (e.g., Ser245/250/255) which can be regulated by MAPKs

  • Use site-specific antibodies to distinguish different phosphorylation patterns

• Protein-protein interactions:

  • Combine phospho-SMAD2 detection with co-immunoprecipitation to identify novel interacting partners

  • Use proximity ligation assays to visualize interactions in situ

Research has demonstrated that PMA treatment can affect SMAD2/3 phosphorylation , highlighting cross-talk with PKC signaling. The publication by Leung et al. cited in search result investigated interactions between SMAD4 and KRAS signaling in pancreatic cancer, demonstrating how phospho-SMAD2 antibodies can be used to dissect complex signaling networks. When designing these experiments, include appropriate positive controls (TGF-β only) and negative controls (unstimulated cells) to accurately interpret pathway interactions.

What methodological approaches can be used to study the dynamics of SMAD2 phosphorylation in living cells?

Studying SMAD2 phosphorylation dynamics in living cells requires approaches that extend beyond traditional fixed cell immunodetection methods. Advanced methodological approaches include:

• Fluorescent biosensors:

  • FRET-based sensors that change conformation upon SMAD2 phosphorylation

  • Split fluorescent protein systems that reassemble when SMAD2 is phosphorylated and complexes with SMAD4

• Live-cell imaging combinations:

  • Express fluorescently-tagged SMAD2 to track nuclear translocation in real-time

  • Correlate translocation dynamics with phosphorylation status by fixing cells at specific timepoints for phospho-SMAD2 immunofluorescence analysis

  • Dilute antibodies to 1:50-1:100 for optimal detection in fixed samples

• Mass spectrometry approaches:

  • Parallel reaction monitoring (PRM) to quantify specific phosphopeptides

  • Pulse-chase SILAC to measure phosphorylation/dephosphorylation rates

  • Analyze samples from cells treated with 10 ng/mL TGF-β for various durations

• Optogenetic tools:

  • Light-inducible TGF-β receptor activation to precisely control signaling initiation

  • Combine with live-cell imaging of SMAD2 translocation and subsequent validation of phosphorylation status

• Single-cell analysis:

  • Correlate phospho-SMAD2 levels with cellular behaviors in heterogeneous populations

  • Use flow cytometry or mass cytometry with phospho-SMAD2 antibodies to quantify single-cell responses

When validating these dynamic approaches, researchers should establish baseline parameters using standard Western blot analysis to detect phospho-SMAD2 levels (approximately 60-68 kDa) in TGF-β-treated versus untreated cells . These baseline measurements can then serve as reference points for calibrating more sophisticated dynamic measurements. Creating standardized protocols for each technique is essential, as the dynamic nature of phosphorylation events makes them particularly sensitive to experimental variations.

How can phospho-SMAD2 antibodies be applied to cancer research models?

Phospho-SMAD2 antibodies have significant applications in cancer research, where TGF-β signaling often exhibits context-dependent tumor-suppressive or tumor-promoting effects. When applying these antibodies to cancer research:

• Cell line model applications:

  • Compare phospho-SMAD2 levels between normal and cancer cell lines using Western blot

  • HepG2 human hepatocellular carcinoma cells show robust phospho-SMAD2 response to TGF-β treatment (10 ng/mL for 30 minutes)

  • Evaluate nuclear translocation patterns using immunofluorescence at antibody dilutions of 1:50-1:100

• Tumor tissue analysis:

  • Optimize antigen retrieval methods for FFPE tissue sections

  • Use phospho-SMAD2 antibodies to assess pathway activation in tumor versus adjacent normal tissue

  • Correlate phospho-SMAD2 levels with patient outcomes or treatment responses

• Genetic manipulation studies:

  • Monitor phospho-SMAD2 levels after SMAD4 loss, as demonstrated in pancreatic cancer models

  • Study how oncogene activation (e.g., KRAS) affects TGF-β-induced SMAD2 phosphorylation

• Drug response assessment:

  • Use phospho-SMAD2 as a pharmacodynamic marker for TGF-β pathway inhibitors

  • Combine with other pathway markers to understand signaling network adaptations

Researchers studying pancreatic cancer models can reference the Leung et al. publication cited in search result , which demonstrated how loss of canonical Smad4 signaling impacts KRAS-driven malignant transformation. This study utilized phospho-SMAD2/3 antibodies to monitor pathway activation in pancreatic duct epithelial cells. Including appropriate positive controls (TGF-β-treated cells) is essential for accurate interpretation of phospho-SMAD2 levels in cancer models, where pathway components may be dysregulated.

What considerations are important when analyzing phospho-SMAD2 in clinical samples?

Analyzing phospho-SMAD2 in clinical samples presents unique challenges that require careful methodological considerations:

• Sample preservation:

  • Phosphorylation states degrade rapidly; minimize time between sample collection and processing

  • For surgical specimens, rapid freezing or immediate fixation is critical

  • Document cold ischemia time (time between removal from patient and fixation/freezing)

• Fixation optimization:

  • Standard formalin fixation can mask phospho-epitopes

  • Test multiple fixation protocols with control samples

  • Consider phospho-specific fixatives that better preserve phosphorylation status

• Controls and standardization:

  • Include cell line controls (TGF-β-treated and untreated) on the same blot/slide as clinical samples

  • Use tissue microarrays with known phospho-SMAD2 status to validate antibody performance across multiple samples

  • Implement digital pathology quantification for consistent scoring

• Signal amplification methods:

  • For IHC/IF in tissue sections, consider tyramide signal amplification

  • For Western blot of limiting samples, use high-sensitivity detection systems

• Technical validation:

  • Confirm phospho-SMAD2 antibody specificity in the specific clinical sample type

  • Validate antibody at the recommended dilution (1:1000 for Western blot or 1:50-1:100 for IHC/IF )

  • Consider parallel assessment with multiple antibody clones

• Biological context:

  • Correlate phospho-SMAD2 staining with total SMAD2 levels

  • Evaluate other TGF-β pathway components (receptors, SMAD4)

  • Interpret results in the context of disease stage and patient history

The expected molecular weight for phospho-SMAD2 is approximately 60-68 kDa on Western blots . When analyzing immunohistochemistry, positive staining should show nuclear localization in cells responsive to TGF-β. Pre-analytical variables such as time to fixation and fixation duration should be carefully documented for meaningful comparison across clinical samples. Methodology papers validating phospho-SMAD2 antibodies in specific clinical contexts should be consulted when establishing new clinical applications.

How does Simple Western technology compare to traditional Western blotting for phospho-SMAD2 detection?

Simple Western (also known as automated capillary Western) offers several advantages over traditional Western blotting for phospho-SMAD2 detection, particularly for quantitative applications. Based on search result :

Comparison Table: Simple Western vs. Traditional Western for Phospho-SMAD2 Detection

For phospho-SMAD2 detection, Simple Western technology demonstrates particular advantages when:

The search results indicate that Simple Western successfully detected phospho-SMAD2/3 in HEK293T cells treated with PMA, showing a specific band at approximately 68 kDa using antibody dilutions between 1:50-1:100 . This technology maintains the specificity advantages of traditional Western blotting while reducing technical variability and improving reproducibility, making it particularly valuable for phosphorylation studies where small changes in signal intensity may have biological significance.

What emerging techniques are advancing our ability to study context-specific SMAD2 phosphorylation?

Emerging technologies are revolutionizing our ability to study context-specific SMAD2 phosphorylation with increased sensitivity, resolution, and throughput:

• Mass spectrometry-based phosphoproteomics:

  • Targeted MS approaches can quantify multiple phosphorylation sites simultaneously

  • Phospho-site occupancy measurement provides absolute quantification

  • Single-cell phosphoproteomics is emerging for heterogeneity analysis

• Spatial proteomics approaches:

  • Imaging mass cytometry can map phospho-SMAD2 distribution in tissue sections

  • Highly multiplexed immunofluorescence allows correlation with multiple pathway components

  • Spatial transcriptomics combined with phospho-protein detection links signaling to gene expression

• Advanced microscopy techniques:

  • Super-resolution microscopy reveals nanoscale organization of phospho-SMAD2

  • Light-sheet microscopy enables 3D imaging of phospho-SMAD2 in organoids and tissue samples

  • Correlative light and electron microscopy connects phospho-SMAD2 to ultrastructural features

• Miniaturized assay formats:

  • Microfluidic Western blotting reduces sample requirements

  • Antibody-based microarrays enable high-throughput phosphorylation profiling

  • Droplet-based single-cell Western blotting for heterogeneity analysis

• CRISPR-based approaches:

  • Base editing to mutate endogenous SMAD2 phosphorylation sites

  • Selective tagging of endogenous SMAD2 for live imaging

  • CUT&Tag approaches to map phospho-SMAD2 genomic binding sites

When implementing these advanced approaches, researchers should validate findings against established methods, such as Western blotting with well-characterized antibodies diluted appropriately (1:1000 for traditional Western blot or 1:50-1:100 for more specialized applications ). The expected molecular weight for phospho-SMAD2 (approximately 60-68 kDa) serves as an important reference point across different detection platforms. These emerging technologies are particularly valuable for understanding the complex regulation of SMAD2 phosphorylation in developmental processes, disease progression, and therapeutic responses.