ASUN Antibody

What is ASUN protein and why is it important to study?

ASUN (also known as INTS13) is a protein involved in multiple cellular processes including regulation of the mitotic cell cycle, centrosome localization, mitotic spindle organization, and protein localization to the nuclear envelope . It acts as a critical regulator of dynein localization during spermatogenesis and is essential for maintaining genomic stability and promoting proper gene transcription . The protein is located in both the cytoplasm and nuclear bodies, suggesting diverse functional roles across cellular compartments . Studying ASUN is particularly important for understanding fundamental cellular processes related to DNA damage repair, RNA processing, and cell division, with potential implications for developmental biology and cancer research .

What applications are validated for commercial ASUN antibodies?

Commercial ASUN antibodies have been validated for multiple research applications:

Most commercially available ASUN antibodies show reactivity specifically with human samples . Researchers should note that optimal dilutions may be sample-dependent and should be determined empirically for each experimental system .

What is the molecular weight of ASUN protein and how does this impact antibody selection?

The calculated molecular weight of ASUN protein is 80 kDa (based on its 706 amino acid sequence), while the observed molecular weight in experimental conditions typically ranges between 70-80 kDa . This discrepancy between calculated and observed molecular weights is important to consider when interpreting Western blot results. When selecting an ASUN antibody, researchers should verify that the antibody can detect proteins within this molecular weight range and should be prepared to observe some variability in the apparent molecular weight depending on experimental conditions, post-translational modifications, and the specific cell type being studied .

What are the common synonyms and identifiers for ASUN protein?

When searching literature or databases for information about ASUN protein and antibodies, researchers should be aware of the various synonyms and identifiers used:

• ASUN (primary name)

• INTS13 (Integrator Complex Subunit 13)

• C12orf11 (Chromosome 12 open reading frame 11)

• GCT1

• NET48

• Mat89Bb

• SPATA30

• FLJ10630

• Spermatogenesis regulator homolog (Drosophila)

• Cell cycle regulator Mat89Bb homolog

• Asunder

GenBank Accession Number: BC003081
Gene ID (NCBI): 55726
UniProt ID: Q9NVM9

Using these alternative names in literature searches will ensure comprehensive coverage of available research.

How do polyclonal and monoclonal ASUN antibodies differ in research applications?

Both polyclonal and monoclonal ASUN antibodies are available for research, each with distinct advantages depending on the application:

Polyclonal ASUN Antibodies:
Polyclonal antibodies like the rabbit polyclonal (NBP1-70427) are generated using synthetic peptides corresponding to specific regions of the ASUN protein . These antibodies recognize multiple epitopes on the ASUN protein, which can provide higher sensitivity for applications where protein abundance is low. The polyclonal nature makes them particularly useful for initial characterization studies and applications like Western blot where signal amplification is beneficial .

Monoclonal ASUN Antibodies:
Monoclonal antibodies like the ASUN/INTS13 Monoclonal Antibody (CAB22792) are generated using hybridoma technology and target specific epitopes of the ASUN protein . These antibodies provide higher specificity and lower background, making them ideal for more precise applications such as determining protein localization or studying protein-protein interactions. They are particularly valuable in immunofluorescence studies where specific subcellular localization patterns must be distinguished .

The choice between polyclonal and monoclonal antibodies should be guided by the specific research question, with consideration for sensitivity versus specificity requirements, and the particular application being undertaken.

What controls are essential when using ASUN antibodies in immunoprecipitation and ChIP experiments?

When using ASUN antibodies for immunoprecipitation (IP) or chromatin immunoprecipitation (ChIP) experiments, several controls are essential to ensure data reliability:

For Immunoprecipitation:

• Input Control: Analyze a small portion (5-10%) of the pre-immunoprecipitated lysate to confirm protein presence.

• Isotype Control: Use matched isotype IgG (rabbit IgG for polyclonal antibodies) processed identically to the experimental sample to identify non-specific binding.

• Negative Control Lysate: Use lysate from cells where ASUN is known to be absent or knockdown/knockout cells to confirm specificity.

• Reciprocal IP: If studying protein-protein interactions, perform reverse IP with antibodies against the suspected interacting protein.

For ChIP Experiments:

• Input DNA: Include non-immunoprecipitated chromatin sample (typically 5-10%) to normalize for differences in chromatin amounts.

• Isotype Control: Use matched isotype IgG to determine background signal levels.

• Positive Control Region: Include primers for a genomic region known to be bound by ASUN.

• Negative Control Region: Include primers for a genomic region not expected to be bound by ASUN.

The IP protocol for ASUN typically requires 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate, with MCF-7 cells being a validated positive sample . Both IP and ChIP applications with ASUN antibodies have been documented in peer-reviewed literature, confirming their utility for these advanced applications .

How can researchers troubleshoot inconsistent ASUN antibody results across different cell lines?

Inconsistent results when using ASUN antibodies across different cell lines can stem from multiple factors:

• Variable ASUN Expression Levels: ASUN expression may differ significantly between cell types. Validated positive samples for ASUN detection include HeLa, MCF-7, and Jurkat cells . When working with other cell lines, researchers should first verify ASUN expression at the mRNA level through RT-PCR or database mining.

• Post-translational Modifications: ASUN may undergo different post-translational modifications in different cell types, affecting antibody recognition. Using multiple antibodies targeting different epitopes can help address this issue.

• Protocol Optimization by Cell Type:

  • Lysis Conditions: Different cell types may require adjusted lysis buffers to efficiently extract ASUN.

  • Fixation Parameters: For IF/ICC, fixation conditions (paraformaldehyde vs. methanol, duration, temperature) may need cell-type-specific optimization.

  • Antigen Retrieval: For IHC, different antigen retrieval methods may be necessary (e.g., TE buffer pH 9.0 has been suggested for ASUN detection, with citrate buffer pH 6.0 as an alternative) .

• Antibody Validation Strategy:

  • Use siRNA knockdown or CRISPR knockout of ASUN as negative controls

  • Compare results across multiple antibodies targeting different epitopes

  • Consider using tagged ASUN overexpression systems for antibody validation

When inconsistent results are observed, systematic optimization of these parameters for each cell line is recommended, along with proper documentation of conditions that yield reproducible results.

What is known about ASUN's role in mitotic regulation and how can antibodies help elucidate its mechanism?

ASUN plays a critical role in mitotic regulation, particularly in centrosome localization, mitotic spindle organization, and protein localization to the nuclear envelope . Research has identified it as a conserved protein that acts as a critical regulator of dynein localization during cell division processes .

To elucidate ASUN's mitotic mechanisms, researchers can employ antibodies in the following strategic approaches:

• Temporal and Spatial Dynamics: Using ASUN antibodies for immunofluorescence microscopy during different stages of mitosis can reveal dynamic changes in ASUN localization. The recommended dilution for IF/ICC applications is 1:200-1:800, with MCF-7 cells serving as a validated positive control .

• Protein Complex Identification:

  • Immunoprecipitation using ASUN antibodies followed by mass spectrometry can identify mitosis-specific interaction partners

  • Proximity ligation assays (PLA) can confirm in vivo interactions with suspected binding partners

  • Co-immunoprecipitation during different cell cycle stages can reveal dynamic interaction networks

• Chromatin Association Studies:

  • ChIP-seq using ASUN antibodies can identify genomic regions directly regulated by ASUN

  • This approach has been validated in published studies

  • Results can be correlated with transcriptome analysis to establish functional consequences

• Functional Inhibition Studies:

  • Coupling ASUN antibody microinjection with live cell imaging to observe immediate functional consequences

  • Comparing results with siRNA or CRISPR-based depletion to distinguish acute versus adaptive effects

These approaches collectively can provide comprehensive insights into ASUN's mechanistic roles during mitotic progression, with antibodies serving as critical tools at each analytical stage.

What are the best practices for ASUN antibody validation before experimental use?

Thorough validation of ASUN antibodies is crucial for ensuring experimental reliability. Recommended validation approaches include:

• Western Blot Validation:

  • Verify detection of a band at the expected molecular weight (70-80 kDa)

  • Test across multiple positive control cell lines (HeLa, MCF-7, Jurkat)

  • Include negative controls (knockdown/knockout samples)

  • Test different antibody dilutions to determine optimal working concentration (1:5000-1:50000 recommended for WB)

• Orthogonal Method Validation:

  • Correlate protein detection with mRNA expression (RT-PCR, RNA-seq)

  • Compare results using multiple antibodies targeting different epitopes

  • Confirm specificity using tagged recombinant ASUN expression

• Application-Specific Validation:

  • For IF/ICC: Verify expected subcellular localization pattern (cytoplasmic and nuclear body staining)

  • For IHC: Include positive control tissues (human cervical cancer tissue, human testis tissue)

  • For IP/ChIP: Confirm enrichment of known interacting proteins or genomic regions

• Independent Method Validation:

  • Mass spectrometry confirmation of immunoprecipitated proteins

  • Genetic approaches (siRNA, CRISPR) to confirm antibody specificity

Proper validation should be performed for each new lot of antibody and for each specific application and cell type/tissue. Documentation of validation results is essential for reproducibility and troubleshooting.

How should researchers optimize sample preparation for ASUN antibody-based detection methods?

Optimal sample preparation is crucial for successful ASUN detection across different experimental platforms:

For Western Blot:

• Lysis Buffer Selection: Use RIPA buffer supplemented with protease inhibitors for general applications; consider NP-40 buffer for preserving protein-protein interactions.

• Sample Handling: Maintain samples at 4°C throughout processing to prevent degradation.

• Protein Quantification: Ensure equal loading using Bradford or BCA assays.

• Denaturation Conditions: Standard conditions (95°C for 5 minutes in Laemmli buffer with β-mercaptoethanol) are generally suitable.

• Recommended Positive Controls: Include lysates from HeLa, MCF-7, or Jurkat cells .

For Immunofluorescence/Immunocytochemistry:

• Fixation Method: 4% paraformaldehyde for 15 minutes at room temperature preserves most epitopes.

• Permeabilization: 0.1-0.5% Triton X-100 for cytoplasmic and nuclear epitopes.

• Blocking: 5% normal serum (matching secondary antibody host) for 1 hour at room temperature.

• Antibody Dilution: 1:200-1:800 in blocking buffer .

• Validated Cell Line: MCF-7 cells serve as a positive control .

For Immunohistochemistry:

• Fixation: Formalin-fixed, paraffin-embedded tissues are standard.

• Antigen Retrieval: TE buffer pH 9.0 is recommended; citrate buffer pH 6.0 is an alternative .

• Endogenous Peroxidase Blocking: 3% hydrogen peroxide for 10 minutes.

• Antibody Dilution: 1:20-1:200 .

• Positive Control Tissues: Human cervical cancer tissue and human testis tissue .

For Immunoprecipitation:

• Lysis Conditions: Use NP-40 or CHAPS-based buffers to preserve protein-protein interactions.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Antibody Amount: Use 0.5-4.0 μg antibody per 1.0-3.0 mg total protein lysate .

• Incubation: Overnight at 4°C with gentle rotation.

• Washing: Multiple stringent washes to reduce background.

Optimization of these parameters for each specific experimental system is recommended to achieve optimal results.

What experimental design considerations are important when studying ASUN in different cellular contexts?

When designing experiments to study ASUN across different cellular contexts, researchers should consider:

• Cell Type Selection:

  • Include validated positive cell lines (HeLa, MCF-7, Jurkat) as controls

  • Consider developmental or tissue context relevant to research question (e.g., testis tissue for spermatogenesis studies)

  • When expanding to new cell lines, verify ASUN expression levels first

• Experimental Controls:

  • Positive Controls: Include conditions where ASUN function is well-characterized

  • Negative Controls: Generate ASUN knockdown/knockout models for antibody validation

  • Complementation Controls: Rescue experiments with wild-type and mutant ASUN to confirm specificity

• Cell Cycle Considerations:

  • Given ASUN's role in mitotic regulation, synchronize cells at specific cell cycle stages

  • Document cell confluence and passage number, as these can affect ASUN expression

  • Consider time-course experiments to capture dynamic changes in ASUN localization and interactions

• Signal Verification Approach:

  • Use multiple detection methods (e.g., IF/ICC and WB) to cross-validate findings

  • Apply quantitative approaches (fluorescence intensity measurement, Western blot densitometry)

  • Employ super-resolution microscopy techniques for detailed localization studies

• Functional Assessment Strategy:

  • Combine ASUN antibody-based detection with functional readouts

  • Consider correlation with phenotypic assays (e.g., mitotic index, spindle morphology)

  • Design experiments to distinguish direct from indirect effects

By systematically addressing these considerations, researchers can generate more robust and interpretable data about ASUN function across different cellular contexts.

How can researchers effectively use ASUN antibodies in multiplexed imaging applications?

Multiplexed imaging with ASUN antibodies allows simultaneous visualization of ASUN along with other proteins, enabling studies of co-localization and contextual function. Effective implementation requires careful consideration of several factors:

• Antibody Compatibility Planning:

  • Select ASUN antibodies from different host species than other target antibodies

  • If using multiple rabbit antibodies, consider sequential staining with direct labeling or use tyramide signal amplification

  • Verify that secondary antibodies do not cross-react with primaries from other species

• Optimization for Specific Applications:

  • For standard fluorescence microscopy: Use fluorophores with minimal spectral overlap

  • For confocal microscopy: Optimize sequential scanning to minimize bleed-through

  • For super-resolution techniques: Validate ASUN antibody performance under specific fixation conditions required by the technique

• Controls for Multiplexed Applications:

  • Single antibody controls to verify signal specificity

  • Secondary-only controls to assess background fluorescence

  • Absorption controls (pre-incubating antibody with immunizing peptide) to confirm specificity

• Recommendations for ASUN Visualization:

  • For co-localization with centrosomal markers: Use ASUN antibody at 1:500 dilution with γ-tubulin antibody

  • For nuclear envelope studies: Combine with lamin antibodies

  • For mitotic spindle analyses: Pair with α-tubulin antibodies

• Image Acquisition and Analysis Considerations:

  • Use appropriate exposure settings to avoid signal saturation

  • Apply consistent thresholding methods across experiments

  • Employ quantitative co-localization analyses (Pearson's correlation, Manders' overlap)

Following these recommendations ensures that multiplexed imaging experiments with ASUN antibodies yield interpretable and reproducible results that accurately represent the biological reality of ASUN's interactions and functions.

How should researchers interpret discrepancies between ASUN antibody results and genetic knockdown experiments?

Discrepancies between antibody-based detection and genetic manipulation experiments are not uncommon and require careful interpretation:

• Possible Causes of Discrepancies:

  • Antibody Specificity Issues: The antibody may recognize proteins other than ASUN

  • Knockdown Efficiency: Incomplete siRNA-mediated knockdown might leave sufficient protein for detection

  • Protein Stability: ASUN protein may have a long half-life, persisting after mRNA reduction

  • Epitope Accessibility: Protein interactions or conformational changes may mask antibody epitopes

  • Compensatory Mechanisms: Cells may upregulate related proteins following ASUN knockdown

• Systematic Resolution Approach:

  • Quantify knockdown efficiency at both mRNA (qRT-PCR) and protein (Western blot) levels

  • Test multiple ASUN antibodies targeting different epitopes

  • Perform time-course experiments following knockdown to assess protein turnover

  • Use complete knockout systems (CRISPR-Cas9) rather than knockdown where possible

• Complementary Validation Strategies:

  • Rescue experiments with exogenous ASUN expression resistant to knockdown

  • Mass spectrometry validation of antibody-detected bands

  • Use of tagged ASUN constructs to track expression independent of antibody detection

• Interpretation Framework:

  • Consider that both approaches may reveal complementary aspects of biology

  • Document conditions where concordance is observed versus where discrepancies occur

  • Consider that discrepancies might reflect biologically meaningful regulatory mechanisms

Careful documentation and transparent reporting of discrepancies help advance understanding of both the biology of ASUN and the technical limitations of different experimental approaches.

What are common pitfalls in ASUN antibody-based experiments and how can they be avoided?

Researchers working with ASUN antibodies should be aware of common pitfalls and employ appropriate strategies to avoid them:

• Non-specific Binding:

  • Pitfall: False positive signals due to antibody cross-reactivity

  • Solution: Validate specificity with ASUN knockdown/knockout samples; use recommended antibody dilutions (1:5000-1:50000 for WB; 1:20-1:200 for IHC; 1:200-1:800 for IF/ICC)

• Inconsistent Results Between Experiments:

  • Pitfall: Variability due to differences in sample preparation or experimental conditions

  • Solution: Standardize protocols; document lot numbers; include positive control samples (HeLa, MCF-7, Jurkat cells)

• Poor Signal-to-Noise Ratio:

  • Pitfall: High background obscuring specific ASUN signal

  • Solution: Optimize blocking conditions; increase washing stringency; adjust antibody concentration; consider signal amplification systems for low-abundance detection

• Epitope Masking:

  • Pitfall: Inability to detect ASUN due to protein interactions or conformational changes

  • Solution: Test multiple antibodies targeting different ASUN epitopes; optimize sample preparation to preserve epitope accessibility

• Fixation Artifacts in Microscopy:

  • Pitfall: Altered ASUN localization due to fixation method

  • Solution: Compare multiple fixation protocols; validate with live-cell imaging of tagged ASUN where possible

• Misinterpretation of Molecular Weight:

  • Pitfall: Confusion between calculated (80 kDa) and observed (70-80 kDa) molecular weights

  • Solution: Include molecular weight markers; verify with positive control lysates; consider post-translational modifications

• Overlooking Cell Cycle Dependence:

  • Pitfall: Failing to account for ASUN's cell cycle-dependent regulation

  • Solution: Synchronize cells; perform time-course experiments; correlate with cell cycle markers

By anticipating these common pitfalls and implementing preventative strategies, researchers can improve the reliability and reproducibility of their ASUN antibody-based experiments.

How can researchers apply ASUN antibodies to study its role in disease models?

ASUN's involvement in fundamental cellular processes like mitotic regulation, nuclear envelope dynamics, and genomic stability suggests potential roles in various diseases. Researchers can leverage ASUN antibodies to investigate these connections:

• Cancer Research Applications:

  • Use ASUN antibodies for immunohistochemical analysis of tumor tissue microarrays to correlate expression with clinical outcomes

  • Investigate ASUN localization changes in cancer cells with chromosomal instability

  • Apply validated dilutions (1:20-1:200) for IHC on human cancer tissues

  • Human cervical cancer tissue has been validated as a positive control

• Reproductive Biology and Infertility:

  • Utilize ASUN antibodies to study its role in spermatogenesis, given its documented function in Drosophila spermatogenesis

  • Human testis tissue has been validated for ASUN antibody detection

  • Correlate ASUN expression and localization with male fertility parameters

• Neurodevelopmental Disorders:

  • Investigate ASUN in neural progenitor cell division using immunofluorescence approaches

  • Study potential dysregulation in neurodevelopmental disease models

  • Apply recommended IF/ICC dilutions (1:200-1:800)

• Experimental Design Considerations:

  • Include appropriate disease and control samples

  • Quantify both expression levels and subcellular localization patterns

  • Correlate antibody findings with functional assays specific to each disease context

  • Consider complementary genetic approaches (patient-derived mutations, CRISPR models)

• Translational Research Potential:

  • Development of ASUN as a potential biomarker for specific disease states

  • Correlation of ASUN dysregulation with treatment response

  • Identification of ASUN interaction partners as potential therapeutic targets

These research directions leverage the specificity and versatility of ASUN antibodies to expand understanding of its roles in pathological states, potentially opening new diagnostic or therapeutic avenues.

What emerging technologies can enhance ASUN antibody-based research?

Several cutting-edge technologies can significantly advance ASUN antibody-based research:

• Proximity Labeling Techniques:

  • BioID or APEX2 fusion with ASUN to identify proximal proteins in living cells

  • Combination with mass spectrometry to map comprehensive interaction networks

  • Validation of identified interactions using traditional co-immunoprecipitation with ASUN antibodies (0.5-4.0 μg per 1.0-3.0 mg lysate)

• Advanced Imaging Technologies:

  • Super-resolution microscopy (STORM, PALM, SIM) to precisely map ASUN localization

  • Live-cell imaging with ASUN antibody fragments to track dynamics

  • Lattice light-sheet microscopy for long-term visualization with minimal phototoxicity

  • Correlative light and electron microscopy (CLEM) to place ASUN in ultrastructural context

• Single-Cell Technologies:

  • Single-cell Western blotting to examine ASUN expression heterogeneity

  • Mass cytometry (CyTOF) with ASUN antibodies for high-dimensional analysis

  • Integration with single-cell transcriptomics to correlate protein and mRNA levels

• Spatial Transcriptomics Integration:

  • Combining ASUN immunohistochemistry with spatial transcriptomics

  • Correlating protein localization with local gene expression profiles

  • Application to tissue sections using validated IHC conditions (1:20-1:200 dilution)

• Functional Genomics Approaches:

  • CRISPR screens combined with ASUN antibody-based readouts

  • Synthetic lethality studies to identify context-dependent functions

  • CRISPR base editing to introduce specific mutations and assess effects on ASUN localization

• Computational Approaches:

  • Machine learning for automated analysis of ASUN localization patterns

  • Integrative multi-omics approaches incorporating antibody-based data

  • Predictive modeling of ASUN interaction networks

By integrating these emerging technologies with established ASUN antibody applications, researchers can gain unprecedented insights into ASUN biology across different cellular contexts and disease states.