ASMT Antibody

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

ASMT (acetylserotonin O-methyltransferase) is a key enzyme in the melatonin biosynthesis pathway that catalyzes the transfer of a methyl group onto N-acetylserotonin, producing melatonin (N-acetyl-5-methoxytryptamine). In humans, the canonical protein has 345 amino acid residues with a molecular mass of 38.5 kDa . It's primarily expressed in the pineal gland and belongs to the cation-independent O-methyltransferase protein family . The significance of ASMT extends beyond melatonin synthesis, as recent research has revealed its potential role in circadian rhythm regulation and cancer progression, particularly in breast cancer metastasis . Alternative splicing yields three different isoforms, which may have tissue-specific functions that are still being elucidated. ASMT is also known by several synonyms including HIOMT, HIOMTY, acetylserotonin N-methyltransferase, acetylserotonin methyltransferase (Y chromosome), hydroxyindole O-methyltransferase, and ASMTY .

What types of ASMT antibodies are available for research, and how should I select the appropriate one?

Multiple types of ASMT antibodies are commercially available with varying specifications to suit different experimental designs. The selection process should involve careful consideration of several technical parameters:

Table 1: Key Selection Criteria for ASMT Antibodies

When selecting an ASMT antibody, evaluate your specific experimental needs and the protein characteristics you're investigating. For instance, if studying specific isoforms, select antibodies targeting unique regions of your isoform of interest. Current commercial offerings include over 150 ASMT antibodies across 19 suppliers with various applications including Western blotting, immunohistochemistry, flow cytometry, and fluorescence-linked immunosorbent assays .

How can I optimize ASMT antibody use in Western blotting experiments?

Optimization of ASMT antibody use in Western blotting requires consideration of several technical parameters:

• Sample Preparation:

  • For cellular samples, use RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if studying post-translational modifications

  • Optimal protein loading: 20-40 μg of total protein per lane

• Electrophoresis and Transfer:

  • Use 10-12% SDS-PAGE gels for optimal separation of the 38.5 kDa ASMT protein

  • Transfer to PVDF membranes (0.45 μm pore size) at 100V for 60-90 minutes in cold transfer buffer

• Blocking and Antibody Incubation:

  • Block with 5% non-fat dry milk in TBST (preferred over BSA for ASMT detection)

  • Primary antibody dilution: Typically 1:500-1:2000 (optimize based on antibody specificity)

  • Incubation time: Overnight at 4°C with gentle rocking

  • Include appropriate controls, especially when studying ASMT knockdown effects as demonstrated in breast cancer cell line studies

• Validation Controls:

  • Use ASMT siRNA-treated samples as negative controls

  • Include loading controls such as GAPDH or tubulin as demonstrated in research on ASMT inhibition

When analyzing Western blot results, quantify ASMT protein levels relative to housekeeping proteins like GAPDH or tubulin, as shown in studies examining ASMT inhibition in breast cancer cell lines . This approach enables accurate comparative analysis across experimental conditions.

What are the critical considerations for immunohistochemical detection of ASMT in tissue samples?

Successful immunohistochemical detection of ASMT requires addressing several technical challenges:

• Tissue Preparation:

  • For FFPE samples: Use consistent fixation protocols (10% neutral buffered formalin for 24-48 hours)

  • For frozen sections: Optimal cutting temperature (OCT) embedding followed by 8-10 μm sections

  • Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically most effective

• Antibody Optimization:

  • Titrate antibody concentrations (typically 1:50-1:500 dilutions)

  • Include both positive controls (pineal gland tissue) and negative controls (antibody diluent only)

  • Consider dual staining with circadian clock proteins (CLOCK, BMAL1, PER1) when studying relationships between these systems

• Signal Detection and Quantification:

  • Use automated image analysis software for objective quantification

  • Establish consistent scoring criteria for ASMT expression levels

  • For correlation studies, use statistical approaches like those applied in breast cancer tissue analysis where significant correlations were found between ASMT and circadian clock proteins

• Interpretation Guidelines:

  • Account for subcellular localization patterns (cytoplasmic vs. nuclear staining)

  • Consider semi-quantitative scoring systems (H-score, Allred score) for comparing expression across samples

  • Correlate with clinicopathological parameters as demonstrated in breast cancer studies

Tissue-specific considerations are important as ASMT expression patterns vary between normal and pathological states, as observed in comparative studies between triple-negative breast cancer tissues and para-carcinoma tissues .

How can ASMT antibodies be effectively employed in circadian rhythm research?

ASMT antibodies provide valuable tools for investigating the crosstalk between melatonin synthesis and circadian regulation:

• Temporal Expression Profiling:

  • Collect samples at multiple time points across 24-hour cycles

  • Use synchronized cell cultures (serum shock or dexamethasone pulse methods)

  • Employ Western blotting with ASMT antibodies alongside core clock proteins (CLOCK, BMAL1, PER1)

  • Quantify relative expression changes across the circadian cycle

• Co-localization Studies:

  • Perform dual immunofluorescence with ASMT antibodies and clock protein antibodies

  • Analyze subcellular localization patterns using confocal microscopy

  • Quantify co-localization using Pearson's or Mander's coefficients

• Functional Interrogation:

  • Design siRNA knockdown experiments targeting ASMT to examine effects on clock protein expression

  • Implement real-time monitoring of circadian rhythms following ASMT manipulation

  • Analyze phase shifts, amplitude changes, and period alterations in circadian gene expression

• Experimental Design Considerations:

  • Include both positive controls (known circadian regulators) and negative controls

  • Account for cell-type specific differences in ASMT expression

  • Consider the impact of experimental conditions (serum composition, light exposure) on melatonin synthesis

Research has demonstrated significant correlations between ASMT expression and circadian clock proteins in both triple-positive and triple-negative breast cancer tissues, suggesting regulatory relationships between these systems . When designing experiments, consider the bidirectional relationship - ASMT inhibition reduces circadian clock protein levels in breast cancer cell lines, indicating ASMT functions upstream of clock regulation in certain contexts .

What are the technical challenges and solutions when using ASMT antibodies for cancer research?

Cancer research applications of ASMT antibodies present specific technical challenges requiring methodological solutions:

• Heterogeneous Expression Patterns:

  • Challenge: ASMT expression varies across cancer subtypes and even within the same tumor

  • Solution: Implement tissue microarray analysis with multiple cores per tumor

  • Approach: Use digital pathology quantification methods to account for intratumoral heterogeneity

• Correlation with Clinical Parameters:

  • Challenge: Establishing meaningful correlations with disease progression

  • Solution: Comprehensive clinicopathological annotation of samples

  • Methodology: Apply multivariate statistical analysis as demonstrated in studies correlating ASMT expression with lymphatic invasion in TNBC

• Functional Validation:

  • Challenge: Determining causality versus correlation

  • Solution: Implement knockdown/overexpression studies with appropriate controls

  • Example: ASMT siRNA treatment in MDA-MB-231 cells demonstrated reduced migration and invasion capacities, validating the functional significance of ASMT in TNBC

• Reproducibility Issues:

  • Challenge: Variability between antibody lots and experimental conditions

  • Solution: Standardized protocols with extensive validation

  • Approach: Include multiple positive and negative controls, and verify with alternative detection methods

When designing cancer research experiments using ASMT antibodies, researchers should consider the emerging evidence that ASMT expression correlates with lymphatic invasion in triple-negative breast cancer but not in triple-positive breast cancer, suggesting context-specific roles . This differential association highlights the importance of cancer subtype stratification in experimental design.

How does ASMT interact with circadian clock proteins, and how can these interactions be studied?

The interaction between ASMT and circadian clock proteins represents a complex molecular relationship that can be investigated through multiple methodological approaches:

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation using ASMT antibodies followed by Western blotting for clock proteins

  • Proximity ligation assays to visualize and quantify direct interactions in situ

  • FRET/BRET assays for real-time monitoring of dynamic interactions

• Transcriptional Regulation Analysis:

  • ChIP assays using CLOCK/BMAL1 antibodies to assess binding to ASMT promoter regions

  • Luciferase reporter assays with ASMT promoter constructs to evaluate clock-mediated regulation

  • RT-qPCR time-course experiments to correlate ASMT mRNA levels with circadian gene expression

• Signaling Pathway Dissection:

  • Pharmacological inhibition of key signaling molecules followed by ASMT/clock protein expression analysis

  • Phosphorylation-specific antibodies to track post-translational modifications in response to circadian cues

  • Pathway interference studies using small molecule inhibitors or genetic approaches

Research has established that inhibition of ASMT through siRNA treatment leads to significant reductions in CLOCK, BMAL1, and PER1 protein levels in both BT-474 (triple-positive) and MDA-MB-231 (triple-negative) breast cancer cells . This indicates ASMT functions upstream of these clock proteins in certain cellular contexts. The relationship appears bidirectional, as CLOCK overexpression partially reverses the phenotypic effects of ASMT inhibition , suggesting a feedback regulatory mechanism between ASMT and the circadian system.

What are the methodological approaches for studying ASMT's role in cancer cell migration and invasion?

Investigating ASMT's influence on cancer cell migration and invasion requires robust methodological approaches:

• In Vitro Migration Assays:

  • Wound healing (scratch) assays following ASMT manipulation

  • Transwell migration assays with quantification of cells traversing membrane

  • Time-lapse microscopy with single-cell tracking for detailed migration dynamics

  • Experimental approach: Compare migration rates between ASMT-inhibited cells and controls

• Invasion Assessment Techniques:

  • Matrigel-coated transwell assays to evaluate invasive potential

  • 3D spheroid invasion assays in extracellular matrix

  • Quantification method: Crystal violet staining of invaded cells with microscopic counting

  • Data analysis: Statistical comparison of invasion indices between experimental groups

• Molecular Mechanism Elucidation:

  • Analysis of epithelial-mesenchymal transition markers following ASMT manipulation

  • Matrix metalloproteinase activity assays to assess extracellular matrix degradation

  • Focal adhesion dynamics monitoring through live-cell imaging

  • Intervention studies: Test if CLOCK overexpression rescues migration/invasion phenotypes in ASMT-inhibited cells

• Experimental Controls and Validation:

  • Multiple siRNA sequences to confirm specificity of ASMT knockdown effects

  • Rescue experiments through ASMT re-expression

  • Positive controls using known modulators of migration/invasion

  • Validation through orthogonal approaches (e.g., confirming transwell results with 3D invasion models)

Research has demonstrated that ASMT inhibition significantly reduces both migration and invasion in MDA-MB-231 triple-negative breast cancer cells, while overexpression of CLOCK partially reverses these effects . These findings suggest ASMT promotes invasiveness in TNBC partially through clock-dependent mechanisms, revealing potential therapeutic targets for intervention.

How can ASMT antibodies be used for developing potential cancer therapeutics?

The emerging role of ASMT in cancer progression suggests several approaches for therapeutic development using ASMT antibodies:

• Target Validation Strategies:

  • Implement systematic ASMT knockdown studies across diverse cancer cell lines

  • Evaluate phenotypic consequences on proliferation, migration, and invasion

  • Correlate ASMT expression with patient outcomes using tissue microarrays

  • Research finding: ASMT inhibition reduces invasiveness in TNBC cells, suggesting therapeutic potential

• Antibody-Drug Conjugate (ADC) Development:

  • Screen ASMT antibodies for internalization efficiency in cancer cells

  • Optimize linker chemistry and cytotoxic payload selection

  • Evaluate specificity using comparative binding studies in normal versus cancer cells

  • Considerations: Select antibodies targeting epitopes with cancer-specific accessibility

• Companion Diagnostic Applications:

  • Develop immunohistochemical protocols for patient stratification

  • Establish scoring systems correlating with therapeutic response

  • Create standardized testing methodologies

  • Clinical relevance: ASMT expression correlates with lymphatic invasion in TNBC, suggesting potential as a prognostic biomarker

• Combination Therapy Approaches:

  • Investigate synergistic effects of ASMT inhibition with clock-targeting compounds

  • Design rational drug combinations based on pathway analysis

  • Evaluate temporal dosing schedules considering circadian influences

  • Experimental design: Test if timed administration enhances therapeutic efficacy

Research indicates that ASMT may represent a particularly valuable drug target for triple-negative breast cancer, as its expression correlates specifically with lymphatic invasion in this aggressive cancer subtype but not in triple-positive breast cancer . This subtype specificity suggests the potential for precision medicine approaches targeting ASMT in appropriately selected patient populations.

What are the technical considerations for developing dual-targeting approaches using ASMT antibodies in bispecific formats?

Developing bispecific antibodies incorporating ASMT binding domains presents unique technical challenges and opportunities:

• Format Selection Considerations:

  • Evaluate symmetric versus asymmetric configurations based on target biology

  • Consider molecular architecture effects on expression yield and stability

  • Assess structural configurations for optimal dual antigen engagement

  • Technical insight: Molecular format significantly affects bispecific antibody functionality and developability

• Domain Engineering Approaches:

  • Fusion of single-domain antibodies (sdAbs) onto IgG scaffolds

  • CDR grafting techniques to humanize murine-derived ASMT-binding domains

  • Stability engineering to address potential liabilities in non-natural antibody formats

  • Design consideration: Structural configuration impacts expression yield and biophysical stability

• Functional Characterization Requirements:

  • Develop binding assays capable of measuring simultaneous target engagement

  • Implement flow-induced dispersion analysis (FIDA) for in-solution binding characterization

  • Assess effector functions including FcγR binding and complement activation

  • Methodological advantage: FIDA enables quantitative analysis of complex dual-binding events

• Manufacturing and Quality Considerations:

  • Evaluate chain mispairing potential in asymmetric formats

  • Implement purification strategies for removing product-related impurities

  • Assess aggregation propensity across different bispecific formats

  • Technical challenge: Asymmetric bispecifics with >2 polypeptide chains are prone to forming product-related impurities through chain mispairing

Table 2: Bispecific Antibody Format Comparison for ASMT Targeting Applications

When developing bispecific antibodies targeting ASMT alongside another target (e.g., a circadian clock protein), researchers must carefully select molecular formats that are fit-for-purpose based on the intended mechanism of action . This selection process should account for both functional requirements and developability considerations to ensure successful translation to clinical applications.

What are common issues encountered with ASMT antibodies and how can they be addressed?

Researchers frequently encounter technical challenges when working with ASMT antibodies that require systematic troubleshooting approaches:

• Nonspecific Binding Issues:

  • Problem: High background or multiple bands in Western blots

  • Diagnosis: Compare multiple ASMT antibodies targeting different epitopes

  • Solution: Optimize blocking conditions (5% milk often superior to BSA for ASMT detection)

  • Validation approach: Include ASMT knockdown controls to confirm specificity

• Variable Signal Intensity:

  • Problem: Inconsistent staining or detection across experiments

  • Diagnosis: Evaluate antibody lot-to-lot variability and storage conditions

  • Solution: Prepare single-use aliquots and avoid freeze-thaw cycles

  • Quality control: Include internal reference samples across experimental batches

• Tissue-Specific Detection Challenges:

  • Problem: Different optimal conditions for various tissue types

  • Diagnosis: Systematic comparison of fixation and antigen retrieval methods

  • Solution: Customize protocols for specific tissue types or cell lines

  • Approach: Develop tissue-specific positive controls (e.g., pineal gland for high ASMT expression)

• Quantification Reliability:

  • Problem: Variability in quantitative measurements

  • Diagnosis: Assess linearity of signal across protein concentration range

  • Solution: Establish standard curves with recombinant ASMT protein

  • Methodological improvement: Use digital image analysis with appropriate normalization

How can I validate the specificity of ASMT antibodies for research applications?

Rigorous validation of ASMT antibody specificity is essential for ensuring experimental reproducibility and accurate data interpretation:

• Genetic Manipulation Controls:

  • Implement ASMT knockdown using multiple siRNA sequences or shRNA

  • Generate ASMT knockout cell lines using CRISPR-Cas9

  • Perform ASMT overexpression with tagged constructs

  • Validation criterion: Signal reduction in knockdown/knockout samples and enhancement in overexpression samples

• Peptide Competition Assays:

  • Pre-incubate antibody with blocking peptide corresponding to the target epitope

  • Include graduated concentrations of blocking peptide

  • Compare signal with and without peptide competition

  • Interpretation: Specific signal should be competitively reduced by the peptide

• Cross-Reactivity Assessment:

  • Test antibodies against recombinant proteins with similar domains

  • Evaluate detection in species with known sequence divergence

  • Analyze potential cross-reactivity with ASMT isoforms

  • Strategy: Use multiple antibodies targeting different epitopes to confirm findings

• Orthogonal Validation Approaches:

  • Correlate protein detection with mRNA expression

  • Compare results across different detection methodologies

  • Implement mass spectrometry for antibody-independent validation

  • Technical consideration: Combine immunodetection with functional assays to confirm biological relevance

In published research on ASMT's role in breast cancer, antibody specificity was validated through siRNA-mediated knockdown of ASMT, with Western blotting demonstrating significant reduction in ASMT protein levels following siRNA treatment . This approach provides convincing evidence of antibody specificity while simultaneously establishing experimental systems for functional studies.