smad5 Antibody

What is SMAD5 and why is it significant in biological research?

SMAD5 (SMAD family member 5) is a receptor-regulated SMAD (R-SMAD) that functions as a transcriptional modulator in the bone morphogenetic protein (BMP) signaling pathway. It plays critical roles in embryonic development, cell differentiation, angiogenesis, and tissue homeostasis . Upon BMP ligand binding to cell surface receptors, SMAD5 is phosphorylated by type I BMP receptors (BMPRIs) at specific serine residues (S463/465) and forms a complex with SMAD4, which translocates to the nucleus to regulate gene expression . Beyond its canonical role in BMP signaling, SMAD5 also has a cytoplasmic function in energy metabolism by promoting mitochondrial respiration and glycolysis through interaction with hexokinase 1 (HK1) . This dual functionality makes SMAD5 an important target for research in developmental biology, cancer research, and metabolic studies.

What types of SMAD5 antibodies are available for research applications?

Based on current research resources, SMAD5 antibodies are available in multiple formats:

The choice depends on the specific research application, with recombinant antibodies offering superior lot-to-lot consistency for longitudinal studies .

How should I select the appropriate SMAD5 antibody for my specific experimental application?

Selection should be based on multiple factors aligned with your experimental goals:

• Application compatibility: Verify the antibody has been validated for your intended application (WB, IHC, IF, etc.). For example, antibody ABIN3043980 is specifically validated for WB and IHC-P in human, mouse, and rat samples .

• Target region specificity: Consider which domain of SMAD5 is relevant to your research:

  • For total SMAD5 detection, antibodies targeting conserved regions work well

  • For phosphorylation studies, use phospho-specific antibodies (e.g., pS463/465)

  • For distinguishing between nuclear and cytoplasmic SMAD5, antibodies targeting the MH1 domain may be preferred

• Species reactivity: Confirm cross-reactivity with your experimental model organism. Most commercial antibodies show reactivity with human, mouse, and rat SMAD5 .

• Validation evidence: Review validation data and publications citing the antibody. Antibodies with extensive literature citations like Cell Signaling #9517 have demonstrated reliability across multiple studies .

• Format considerations: For multi-color imaging, select conjugated antibodies; for sensitive detection in low-expressing samples, consider high-affinity monoclonal antibodies.

What are the recommended protocols for detecting SMAD5 in Western blotting experiments?

For optimal SMAD5 detection by Western blotting:

Sample Preparation:

• Lyse cells in buffer containing phosphatase inhibitors to preserve phosphorylation status

• Use gentle lysis conditions as harsh detergents may affect epitope recognition

• For phospho-SMAD5 detection, stimulate cells with BMP ligands (e.g., BMP4) for 30-60 minutes before lysis

Electrophoresis and Transfer:

• Load 20-40 μg of total protein per lane

• Use 8-10% SDS-PAGE gels for optimal resolution of the 52-60 kDa SMAD5 protein

• Transfer to PVDF membranes at 100V for 60-90 minutes or 30V overnight at 4°C

Antibody Incubation:

• Block membranes with 5% BSA in TBST (preferred over milk for phospho-antibodies)

• Dilute primary antibodies according to manufacturer recommendations:

  • Cell Signaling SMAD5 antibody: 1:1000

  • Proteintech SMAD5 antibody: 1:2000-1:16000

  • Boster SMAD5 antibody: 1:1000-1:6000

• Incubate overnight at 4°C for optimal results

• For detection, use HRP-conjugated secondary antibodies with enhanced chemiluminescence

Expected Results:

• Total SMAD5 appears as a band at approximately 52-60 kDa

• Phosphorylated SMAD5 may show slightly higher apparent molecular weight

How can I design experiments to study SMAD5 nucleocytoplasmic shuttling in response to pH changes?

Based on research by Alborzinia et al. , SMAD5 exhibits pH-dependent nucleocytoplasmic shuttling that is independent of BMP signaling. To study this phenomenon:

Experimental Setup:

• Cell system selection: Use cells with endogenous SMAD5 expression or transfect with fluorescently tagged SMAD5 (e.g., GFP-SMAD5)

• pH manipulation methods:

  • Extracellular pH (pHe) adjustment: Prepare culture media at different pH values (6.0-8.0)

  • Intracellular pH (pHi) measurement: Use pHluorin or other pH-sensitive fluorescent proteins

  • Permeabilization approach: Treat cells with digitonin (160 μg/ml) to allow direct control of cytoplasmic pH

Imaging Protocol:

• Perform live-cell imaging using confocal microscopy at 37°C with CO₂ control

• Capture baseline SMAD5 distribution before pH manipulation

• Monitor localization changes in real-time after pH alteration

• Quantify nuclear/cytoplasmic signal ratio over time

Controls and Validation:

• Include SMAD5 mutants lacking nuclear export signals (NESs) as controls

• Compare with BMP pathway inhibitors (e.g., LDN193189) to confirm BMP independence

• Use SMAD5 phosphorylation-deficient mutants (e.g., SSVS→AAVA) to verify signaling independence

Analysis Considerations:

• Calculate nuclear/cytoplasmic ratios across multiple cells

• Correlate SMAD5 localization with measured intracellular pH

• Determine kinetics of translocation in response to pH changes

This approach allows for precise characterization of how SMAD5 responds to physiological pH fluctuations independent of its canonical signaling role .

What controls should be included when using SMAD5 antibodies for immunohistochemistry?

For rigorous IHC experiments with SMAD5 antibodies, include these controls:

Positive Controls:

• Tissues with known SMAD5 expression:

  • Human colon epithelial cells show specific cytoplasmic SMAD5 staining

  • Embryonic tissues with active BMP signaling

  • Cell lines with confirmed SMAD5 expression (e.g., HeLa, K-562, NIH/3T3)

Negative Controls:

• Primary antibody omission: Replace primary antibody with antibody diluent

• Isotype control: Use matched isotype IgG at the same concentration

• Blocking peptide competition: Pre-incubate antibody with immunizing peptide

• SMAD5 knockdown/knockout tissues: If available, use genetically modified samples

Technical Controls:

• Antibody titration: Test multiple dilutions (e.g., 1:150-1:600 for Proteintech antibody)

• Antigen retrieval comparison: Test both citrate buffer (pH 6.0) and TE buffer (pH 9.0)

• Detection system control: Include a sample processed without the detection reagent

Tissue Processing Considerations:

• Use identical fixation conditions across all samples

• Monitor staining development time carefully

• For dual staining with phospho-SMAD5, use sequential protocols with appropriate blocking steps

Proper controls ensure that observed staining truly represents SMAD5 and is not due to non-specific binding or technical artifacts.

How can I troubleshoot weak or absent SMAD5 signal in Western blotting?

Weak or absent SMAD5 signal can result from multiple factors. Address these systematically:

Sample Preparation Issues:

• Protein degradation: Add protease inhibitors to lysis buffer; keep samples cold and process quickly

• Low expression levels: Increase protein loading (40-60 μg); concentrate samples if necessary

• Improper lysis: Ensure complete cell lysis with appropriate buffer; for nuclear SMAD5, use nuclear extraction protocols

Technical Problems:

• Inefficient transfer: Optimize transfer conditions; verify with reversible staining (Ponceau S)

• Insufficient blocking: Increase blocking time or BSA concentration (5-10%)

• Antibody dilution: Try more concentrated primary antibody; titrate from 1:500 to 1:2000

• Exposure time: For weak signals, use longer exposure or more sensitive detection systems

Signal Specificity Concerns:

• Wrong molecular weight: SMAD5 appears at 52-60 kDa; verify with positive control lysate

• Non-specific bands: Try different antibody clones or more stringent washing

• Phosphorylation states: For phospho-SMAD5, ensure activation of signaling pathway

Methodological Solutions:

• Enrichment approaches: Use immunoprecipitation to concentrate SMAD5 before WB

• Signal amplification: Apply HRP-polymer detection systems or TSA amplification

• Alternative antibodies: Test different epitope antibodies (e.g., N-terminal vs. middle region)

By systematically addressing these potential issues, researchers can improve SMAD5 detection sensitivity and specificity.

How should I interpret differences between phosphorylated and total SMAD5 staining patterns?

Interpreting phospho-SMAD5 versus total SMAD5 requires careful analysis:

Expected Patterns:

• Total SMAD5: Generally shows both cytoplasmic and nuclear localization depending on cell type and conditions; often more cytoplasmic in unstimulated cells

• Phospho-SMAD5 (pS463/465): Primarily nuclear localization in BMP-stimulated cells; represents active signaling

Interpretation Guidelines:

• Ratio analysis: Calculate the phospho-SMAD5/total SMAD5 ratio to normalize for expression differences

• Subcellular localization: Nuclear phospho-SMAD5 with cytoplasmic total SMAD5 indicates active BMP signaling

• Temporal dynamics: Sequential sampling shows activation kinetics (phosphorylation peaks 30-60 min after stimulation)

• Cell-type heterogeneity: Note differences across cell types in the same tissue

Complicating Factors:

• Antibody cross-reactivity: Phospho-antibodies may detect multiple phosphorylated SMADs (e.g., pSMAD1/5/8)

• pH sensitivity: Changes in intracellular pH can alter SMAD5 localization independent of phosphorylation

• Baseline phosphorylation: Some tissues show constitutive SMAD5 phosphorylation

Methodological Considerations:

• Use consecutive tissue sections for phospho and total staining comparison

• For dual staining, use spectrally distinct fluorophores and carefully control for antibody cross-reactivity

• Include pathway activation controls (BMP treatment) and inhibition controls (LDN193189)

This comparative approach provides insights into both SMAD5 expression levels and activation status within the same experimental system.

How can I distinguish between canonical BMP-dependent and non-canonical pH-dependent functions of SMAD5?

Differentiation between these pathways requires specific experimental designs:

Genetic Approaches:

• Mutant constructs:

  • Use SMAD5 SSVS→AAVA (3A) or SSVS→DDVD (3D) mutations to block phosphorylation-dependent functions

  • Create SMAD5 NES mutants (all three NESs mutated) to block pH-dependent nucleocytoplasmic shuttling

  • Employ deletion mutant (GFP-SMAD5 Δ11) lacking C-terminal phosphorylation sites

Pharmacological Intervention:

• Pathway inhibition:

  • Use BMP pathway inhibitors (LDN193189) to block canonical signaling

  • Apply pH modifiers (e.g., NH₄Cl or acetate) to alter intracellular pH

  • Measure effects under each condition separately and in combination

Protein Interaction Analysis:

• Co-immunoprecipitation studies:

  • For canonical function: Detect SMAD5-SMAD4 complexes

  • For pH-dependent function: Examine SMAD5-HK1 interaction in cytoplasm

  • Use size exclusion chromatography to separate different complex types

Functional Readouts:

• Pathway-specific targets:

  • BMP-dependent: Measure ID1, ID2 gene expression by qPCR

  • pH-dependent: Assess glycolysis rate using extracellular flux analysis

  • Compare responses under pathway-specific conditions

Localization Studies:

• High-resolution imaging:

  • Use fractionation followed by Western blotting to quantify distribution

  • Perform time-lapse imaging with fluorescently tagged SMAD5 variants

  • Correlate localization with functional outcomes

This multi-faceted approach allows researchers to disambiguate canonical BMP-regulated nuclear functions from non-canonical pH-responsive metabolic functions of SMAD5 .

What are the considerations for studying SMAD5 in conjunction with other SMAD family members?

SMAD family proteins share structural similarities and overlapping functions, requiring careful experimental planning:

Antibody Selection Considerations:

• Cross-reactivity assessment:

  • Some antibodies recognize multiple SMADs due to sequence homology

  • Phospho-antibodies may detect pSMAD1/5/8 due to conserved phosphorylation motifs

  • Verify specificity using recombinant proteins or knockout controls

Expression Pattern Analysis:

• Co-expression studies:

  • Use multiplexed immunofluorescence with validated antibodies

  • Employ RNA-scope for mRNA-level co-expression analysis

  • Compare expression across developmental stages and tissue types

Functional Redundancy Evaluation:

• Knockdown strategies:

  • Design single and combinatorial knockdowns (e.g., SMAD1/5)

  • Use rescue experiments with specific SMAD isoforms

  • Apply inducible systems for temporal control

Pathway-Specific Activation:

• Selective stimulation:

  • BMP ligands preferentially activate SMAD1/5/8

  • TGF-β ligands primarily activate SMAD2/3

  • Use receptor-selective compounds for pathway distinction

Data Analysis Framework:

• Quantitative approaches:

  • Apply computational modeling for SMAD network interactions

  • Use phosphorylation kinetics to distinguish primary vs. secondary effects

  • Correlate SMAD ratios with biological outcomes

Practical Implementation Table:

This integrated approach enables researchers to decipher the specific contributions of SMAD5 within the broader context of SMAD-mediated signaling networks.

What methods can be used to validate SMAD5 antibody specificity?

Ensuring antibody specificity is critical for meaningful results. Implement these validation approaches:

Genetic Validation:

• Knockout/knockdown controls: Test antibody on SMAD5-knockout or CRISPR-edited cell lines

• Overexpression systems: Compare signal in wild-type vs. SMAD5-overexpressing samples

• Epitope mutation: Introduce mutations in the antibody recognition site to confirm specificity

Biochemical Validation:

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Immunoprecipitation-mass spectrometry: Confirm pulled-down protein identity

• Multiple antibody comparison: Test antibodies targeting different SMAD5 epitopes

Application-Specific Validation:

• Orthogonal methods: Correlate protein detection with mRNA levels (RT-qPCR)

• Expected biology verification: Confirm nuclear translocation after BMP stimulation

• Signal modulation: Verify signal increases/decreases with known treatments

Documentation Requirements:

• Record lot numbers and validation results for reproducibility

• Include validation controls in publications

• Document optimization parameters for each experimental system

These validation approaches significantly enhance data reliability and interpretation confidence.

How can I quantitatively analyze SMAD5 expression and activation in complex tissue samples?

Quantitative analysis of SMAD5 in tissues requires systematic approaches:

Image Analysis Methods:

• Digital pathology tools:

  • Use automated cell counting with nuclear/cytoplasmic segmentation

  • Apply machine learning algorithms for pattern recognition

  • Quantify staining intensity using standardized scales (H-score, Allred)

Multi-parameter Analysis:

• Multiplexed detection:

  • Combine SMAD5 with lineage markers for cell-type specific analysis

  • Co-stain for phospho-SMAD5 and total SMAD5 to calculate activation ratio

  • Include proliferation or differentiation markers for functional correlation

Tissue Heterogeneity Considerations:

• Spatial analysis:

  • Use tissue microarrays for standardized comparison

  • Apply spatial statistics to identify expression patterns

  • Consider region-specific normalization methods

Standardization Approaches:

• Reference standards:

  • Include calibration samples with known SMAD5 expression

  • Use digital image standards for inter-laboratory comparison

  • Apply spectral unmixing for accurate multiplexed quantification

Statistical Methods:

• Advanced analytics:

  • Use hierarchical clustering to identify expression patterns

  • Apply principal component analysis for multi-parameter reduction

  • Consider mixed effects models for longitudinal studies

This quantitative framework enables robust comparative analysis of SMAD5 across different experimental conditions, tissue types, and disease states.

What emerging technologies are enhancing SMAD5 antibody-based research?

Recent technological advances are expanding the capabilities of SMAD5 antibody applications:

Next-Generation Antibody Technologies:

• Recombinant antibody production: Offers superior lot-to-lot consistency for longitudinal studies

• Single-domain antibodies: Provide access to previously inaccessible epitopes

• Intrabodies: Allow real-time tracking of SMAD5 in living cells

Advanced Imaging Methods:

• Super-resolution microscopy: Reveals nanoscale SMAD5 complex organization

• Live-cell imaging with split fluorescent proteins: Monitors dynamic SMAD5 interactions

• Lattice light-sheet microscopy: Captures rapid SMAD5 translocation events with reduced phototoxicity

Omics Integration:

• Spatial transcriptomics: Correlates SMAD5 protein localization with transcriptional changes

• Single-cell proteomics: Detects cell-to-cell variation in SMAD5 signaling

• Proximity labeling (BioID/TurboID): Maps SMAD5 protein interaction networks in situ

Emerging Applications:

• CUT&Tag/CUT&RUN: Maps SMAD5 genomic binding sites with improved resolution

• Optogenetic SMAD5 control: Enables precise spatiotemporal pathway manipulation

• Nanobody-based biosensors: Detects SMAD5 conformational changes in real time

These technologies are expanding our understanding of SMAD5 biology and creating new experimental possibilities for researchers in this field.

How can researchers effectively compare results obtained using different SMAD5 antibodies in the literature?

Cross-study comparison requires careful consideration of methodological differences:

Documentation Assessment:

• Antibody identification: Check catalog numbers, clones, and lot numbers

• Protocol differences: Compare fixation methods, antigen retrieval, and detection systems

• Validation approaches: Evaluate controls used to verify specificity

Normalization Strategies:

• Internal controls: Look for housekeeping proteins or loading controls

• Relative quantification: Convert absolute values to fold changes when possible

• Reference standards: Identify studies using common reference samples

Meta-analysis Framework:

• Systematic review approach: Apply PRISMA guidelines for literature comparison

• Effect size calculation: Use standardized mean difference for quantitative comparison

• Heterogeneity assessment: Apply I² statistics to determine consistency across studies

Practical Comparison Worksheet: