SMYD4 Monoclonal Antibody

What is SMYD4 and why is it significant in research?

SMYD4 is a protein coding gene belonging to the SET and MYND domain-containing protein family. It plays a critical role in cardiac development and functions as a key epigenetic regulator of gene expression . The significance of SMYD4 in research stems from:

• Its dual functions as a methyltransferase and as a negative regulator of HDAC1

• Its association with diseases including Congenital Contractures Of The Limbs And Face, Hypotonia, And Developmental Delay

• Its involvement in cardiac development pathways, with rare variants linked to congenital heart defects (CHDs)

• Its potential as a model for understanding epigenetic regulation mechanisms

Investigating SMYD4 can provide crucial insights into developmental biology, particularly cardiac development, and may lead to therapeutic strategies for associated disorders.

What are the structural characteristics and functional domains of SMYD4?

SMYD4 contains four key functional domains that contribute to its biological activity:

• Two TPR (Tetratricopeptide Repeat) domains located at the N- and C-termini

• A MYND domain that mediates protein-protein interactions, particularly with HDAC1

• A SET domain that functions as a methyltransferase

The MYND domain has been specifically identified as responsible for the interaction between SMYD4 and HDAC1 through deletion mutant studies . This domain architecture enables SMYD4 to function both as a methyltransferase and as a regulator of histone deacetylation, making it a versatile epigenetic regulator during development.

Structurally, SMYD4 localizes to both the nucleus and cytoplasm, consistent with its roles in both nuclear (gene regulation) and cytoplasmic functions .

What are the key differences between SMYD4 monoclonal and polyclonal antibodies?

Researchers should choose the appropriate antibody type based on their experimental needs, with monoclonal antibodies being preferable for highly specific detection and polyclonal antibodies advantageous for applications requiring higher sensitivity.

How can SMYD4 monoclonal antibodies be used to investigate protein-protein interactions?

SMYD4 monoclonal antibodies can be effectively utilized to investigate protein-protein interactions, particularly the critical interaction between SMYD4 and HDAC1, through several methodological approaches:

• Co-immunoprecipitation (Co-IP): Using anti-SMYD4 monoclonal antibodies to immunoprecipitate SMYD4 protein complexes followed by western blotting to detect interacting partners. Research has demonstrated this approach successfully identified HDAC1 as a major interaction partner of SMYD4 .

• Immunoprecipitation followed by mass spectrometry: This approach was used to identify HDAC1 as one of the major proteins interacting with SMYD4. The methodology involves:

  • Overexpression of flag-tagged SMYD4 in cardiac cell lines

  • Immunoprecipitation using anti-Flag affinity gel

  • SDS-PAGE resolution of eluates

  • Mass spectrometry analysis of the resulting protein bands

• Domain-specific interaction studies: SMYD4 monoclonal antibodies can be used alongside deletion mutants to map specific interaction domains. Research has demonstrated that the MYND domain of SMYD4 is responsible for its interaction with HDAC1 using this approach .

This methodological framework enables detailed investigation of how SMYD4 participates in multiprotein complexes that regulate epigenetic modifications and gene expression.

What approaches can be used to study SMYD4 variants associated with congenital heart defects?

Researchers can employ several sophisticated approaches to study disease-associated SMYD4 variants (such as G345D and R579Q) using monoclonal antibodies:

• Structural-functional correlation studies: Computational structure prediction can be combined with biochemical studies to understand how mutations affect protein structure. Research shows both G345D and R579Q mutations lead to significant changes in SMYD4 protein structure compared to wild-type .

• Interaction profile analysis: Co-IP/western blotting assays have demonstrated that the SMYD4(G345D) variant has dramatically reduced ability to interact with HDAC1 compared to wild-type SMYD4 . Similar approaches can be used to:

  • Quantify binding affinity differences between wild-type and mutant SMYD4 with various interaction partners

  • Identify altered protein-protein interaction networks

• Cellular localization studies: Immunofluorescence using monoclonal antibodies can determine if disease-associated variants show altered subcellular localization compared to wild-type SMYD4.

• Functional enzymatic assays: Methyltransferase activity assays can assess how mutations affect the enzymatic activity of SMYD4.

These approaches provide mechanistic insights into how SMYD4 variants contribute to pathological phenotypes in congenital heart defects.

What are the optimal conditions for using SMYD4 monoclonal antibody in Western blot analysis?

For optimal Western blot results with SMYD4 monoclonal antibody, researchers should consider the following methodological guidelines:

• Antibody dilution: The recommended dilution range for SMYD4 monoclonal antibody (e.g., MAB8729) in Western blot applications is 1:100-1:2000 . Validation studies have shown effective detection at 1:2000 dilution for recombinant SMYD4 protein with a GST tag .

• Expected band size: The predicted molecular weight of SMYD4 is approximately 60 kDa, and this has been confirmed in experimental validation with recombinant SMYD4 protein .

• Sample preparation:

  • Use RIPA buffer containing 1 mM PMSF and complete protease inhibitors for cell lysis

  • Include phosphatase inhibitors if studying phosphorylation states

  • Denature samples at 95°C for 5 minutes in loading buffer containing SDS and reducing agent

• Blocking conditions: 5% non-fat dry milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature.

• Antibody incubation: Overnight incubation at 4°C with primary antibody diluted in blocking buffer is typically optimal for specific detection.

• Detection method: HRP-conjugated secondary antibodies with enhanced chemiluminescence (ECL) detection systems provide suitable sensitivity for SMYD4 detection.

Optimization of these parameters should be performed for each experimental system to ensure reliable and reproducible results.

How can researchers design co-immunoprecipitation experiments to study SMYD4-HDAC1 interactions?

A robust co-immunoprecipitation (Co-IP) protocol to study SMYD4-HDAC1 interactions should include:

• Experimental design:

  • Expression system: Either endogenous protein detection or overexpression of tagged proteins (Flag-tagged SMYD4 and HA-tagged HDAC1 have been successfully used)

  • Controls: IgG control, input control, and single-transfection controls

  • Cell model: Cardiac cell lines like HL-1 or HEK293T cells have been successfully used for SMYD4-HDAC1 interaction studies

• Cell lysis protocol:

  • Lyse cells in RIPA buffer containing 1 mM PMSF and complete protease inhibitors

  • Gentle lysis conditions to preserve protein-protein interactions

  • Clear lysates by centrifugation at 12,000 × g for 15 minutes at 4°C

• Immunoprecipitation:

  • For tagged proteins: Use anti-Flag affinity gel for immunoprecipitation

  • For endogenous proteins: Use protein G-conjugated beads with anti-SMYD4 monoclonal antibody

  • Incubate lysates with antibody-conjugated beads for 4 hours to overnight at 4°C with gentle rotation

• Washing and elution:

  • Wash beads 4-5 times with lysis buffer

  • Elute proteins with SDS sample buffer by boiling for 5 minutes

• Detection:

  • Resolve proteins by SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane

  • Probe with appropriate antibodies against SMYD4 and HDAC1

  • Use appropriate secondary antibodies and detection methods

This methodology has successfully demonstrated that HDAC1 is a major interaction partner of SMYD4 and that the MYND domain is responsible for this interaction .

What are common issues when using SMYD4 monoclonal antibody and how can they be resolved?

How can researchers validate the specificity of SMYD4 monoclonal antibody?

To validate SMYD4 monoclonal antibody specificity, researchers should implement the following methodological approaches:

• Positive control testing: Use recombinant SMYD4 protein with appropriate tags (e.g., GST tag) to confirm antibody detection at the predicted molecular weight (60 kDa) .

• Epitope mapping: Determine the specific region of SMYD4 recognized by the antibody to ensure it won't cross-react with related proteins like SMYD2 (an important paralog of SMYD4) .

• Cross-reactivity assessment:

  • Test the antibody against related SMYD family proteins

  • Perform Western blot analysis in multiple cell lines with known SMYD4 expression

  • Include negative control samples (cells with SMYD4 knockdown)

• Validation across applications: If using the antibody for multiple applications (Western blot, immunoprecipitation, immunofluorescence), validate specificity in each context separately.

• Antibody characterization data review: Review supplier validation data showing predicted vs. observed band size (e.g., 60 kDa for SMYD4) and dilution optimization experiments.

Thorough validation ensures experimental reliability and reproducibility when studying SMYD4 in diverse research contexts.

How can SMYD4 monoclonal antibodies contribute to understanding epigenetic regulation mechanisms?

SMYD4 monoclonal antibodies can be leveraged in several sophisticated experimental approaches to elucidate epigenetic regulation mechanisms:

• Chromatin Immunoprecipitation (ChIP) assays: Using SMYD4 monoclonal antibodies to:

  • Identify genomic regions bound by SMYD4

  • Map the distribution of SMYD4 across the genome

  • Correlate SMYD4 binding with specific histone modifications and gene expression patterns

• Proximity ligation assays (PLA): To visualize and quantify SMYD4 interactions with HDAC1 and other epigenetic regulators in situ, providing spatial context to protein-protein interactions within the nuclear and cytoplasmic compartments.

• Sequential ChIP (Re-ChIP): To identify genomic regions co-occupied by SMYD4 and HDAC1, revealing loci under coordinated regulation by both factors.

• Proteomics approaches: Combining immunoprecipitation with mass spectrometry to:

  • Identify the complete SMYD4 interactome

  • Characterize how this interactome changes in different developmental or disease contexts

  • Map post-translational modifications of SMYD4 itself

• Functional genomics integration: Correlating SMYD4 binding sites with:

  • Histone modification patterns

  • Chromatin accessibility data

  • Gene expression datasets

  • Disease-associated genetic variants

These approaches can collectively provide mechanistic insights into how SMYD4 contributes to epigenetic regulation during cardiac development and how dysregulation contributes to congenital heart defects .

What research directions might emerge from studying SMYD4 variants in disease models?

Investigation of SMYD4 variants associated with congenital heart defects (such as G345D and R579Q) opens several promising research directions:

• Structure-function studies: Detailed molecular analysis of how these mutations affect:

  • Protein folding and stability

  • Enzymatic activity (methyltransferase function)

  • Protein-protein interactions, particularly with HDAC1

  • Subcellular localization

• Animal models: Development of knock-in mouse models harboring SMYD4 mutations (G345D or R579Q) to:

  • Recapitulate cardiac developmental defects

  • Study the temporal and spatial requirements for SMYD4 function

  • Test potential therapeutic interventions

• Patient-derived models: Generation of induced pluripotent stem cells (iPSCs) from patients with SMYD4 mutations to:

  • Create cardiac organoids for disease modeling

  • Perform drug screening

  • Test gene therapy approaches

• Epigenomic profiling: Compare epigenetic landscapes in wildtype versus mutant SMYD4 models to:

  • Identify dysregulated target genes

  • Map altered histone modification patterns

  • Discover compensatory epigenetic mechanisms

• Therapeutic development: Design of small molecules or peptides that could:

  • Restore SMYD4-HDAC1 interaction in G345D mutants

  • Modulate HDAC1 activity to compensate for SMYD4 dysfunction

  • Target downstream effectors in the SMYD4 pathway

These approaches could significantly advance our understanding of how SMYD4 variants contribute to congenital heart defects and potentially lead to novel therapeutic strategies for affected patients .