DNASE1L1 Antibody

What is DNASE1L1 and why is it important in scientific research?

DNASE1L1 (Deoxyribonuclease 1 Like 1) is a protein-coding gene that encodes a deoxyribonuclease protein showing high sequence similarity to DNase I. The encoded protein is localized to the endoplasmic reticulum and undergoes N-linked glycosylation modification. DNASE1L1 plays a crucial role in DNA degradation, particularly during apoptosis, by mediating internucleosomal DNA fragmentation through its catalytic activity . This function is vital for maintaining cellular homeostasis and preventing damaged DNA accumulation, which can lead to various diseases including cancer . DNASE1L1 is predominantly expressed at high levels in cardiac and skeletal muscle tissues, suggesting an important role in muscle physiology and development . Research on DNASE1L1 contributes to our understanding of DNA degradation pathways, apoptotic mechanisms, and potentially to insights regarding diseases associated with this gene, such as Barth Syndrome and Human Monocytic Ehrlichiosis .

What are the key characteristics of commercially available DNASE1L1 antibodies?

Available DNASE1L1 antibodies come in multiple formats with distinct characteristics. Polyclonal antibodies like the rabbit-derived 13653-1-AP detect the full DNASE1L1 protein (calculated molecular weight of 34 kDa, 302 amino acids) in human, mouse, and rat samples . This antibody has been validated for multiple applications including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence/Immunocytochemistry (IF/ICC), Immunoprecipitation (IP), and ELISA . Monoclonal antibodies such as KK-M3 (mouse IgG1 kappa light chain) are also available, specifically detecting human DNASE1L1 via WB, IP, and ELISA applications . The choice between polyclonal and monoclonal antibodies depends on the specific research requirements - polyclonals offer broader epitope recognition but potentially lower specificity, while monoclonals provide higher specificity for a single epitope but may be more sensitive to conformational changes in the target protein.

How does DNASE1L1 differ from other DNASE family members, and what implications does this have for antibody selection?

DNASE1L1 shows high sequence similarity to DNase I but has distinct characteristics and functions. Unlike DNASE1, which is secreted into body fluids by various exocrine and endocrine organs, DNASE1L1 is predominantly localized to the endoplasmic reticulum . An important paralog of DNASE1L1 is DNASE1L3, with both potentially having complementary functions in certain pathways . While DNASE1 and DNASE1L3 together play key roles in degrading neutrophil extracellular traps (NETs), DNASE1L1's role appears more focused on intracellular DNA degradation during processes like apoptosis . When selecting antibodies, researchers should carefully verify specificity to DNASE1L1 versus other family members by examining the immunogen sequences and cross-reactivity testing data. Antibodies raised against unique regions of DNASE1L1 not conserved in other family members will provide greater specificity. Validation through knockout/knockdown experiments is highly recommended to confirm antibody specificity, especially when studying multiple DNASE family members simultaneously.

What are the optimal conditions for Western blotting with DNASE1L1 antibodies?

For optimal Western blotting with DNASE1L1 antibodies, researchers should follow these methodological considerations:

Always titrate the antibody in each testing system to obtain optimal results, as the performance can be sample-dependent .

What considerations should be taken into account when designing immunohistochemistry experiments with DNASE1L1 antibodies?

When designing immunohistochemistry (IHC) experiments with DNASE1L1 antibodies, researchers should consider several methodological aspects:

• Tissue selection: Human skeletal muscle tissue has been confirmed as positive for DNASE1L1 detection and serves as an excellent positive control . Given DNASE1L1's high expression in cardiac and skeletal muscle, these tissues are ideal for initial protocol optimization.

• Antigen retrieval: For optimal results with DNASE1L1 antibodies, suggested antigen retrieval should be performed with TE buffer pH 9.0. Alternatively, citrate buffer pH 6.0 may also be effective . Comparative testing of both methods is recommended for new tissue types.

• Antibody dilution: For antibodies like 13653-1-AP, the recommended dilution range for IHC is 1:20-1:200 . Starting with a mid-range dilution (e.g., 1:100) and adjusting based on signal intensity and background levels is advisable.

• Detection system: Choose a detection system compatible with the host species of the primary antibody (rabbit for 13653-1-AP). Consider signal amplification systems for tissues with lower DNASE1L1 expression.

• Controls: Include both positive (muscle tissue) and negative controls (primary antibody omission, isotype control, or tissues known to lack DNASE1L1 expression). For definitive validation, DNASE1L1 knockdown/knockout tissues provide the most rigorous specificity control.

Optimize incubation times and temperatures for both primary and secondary antibodies to achieve the best signal-to-noise ratio for your specific experimental conditions.

How can immunofluorescence experiments with DNASE1L1 antibodies be optimized for subcellular localization studies?

Optimizing immunofluorescence (IF) experiments with DNASE1L1 antibodies for subcellular localization studies requires attention to several critical factors:

• Cell line selection: MCF-7 cells have been validated for positive detection of DNASE1L1 by IF/ICC with antibodies like 13653-1-AP . Consider using these cells as positive controls when establishing protocols.

• Fixation method: Since DNASE1L1 is predominantly localized to the endoplasmic reticulum , compare different fixation methods (e.g., paraformaldehyde, methanol, or acetone) to determine which best preserves both antigenicity and subcellular structures.

• Permeabilization optimization: Test different permeabilization agents (Triton X-100, saponin, digitonin) and concentrations to ensure adequate access to endoplasmic reticulum-associated DNASE1L1 while maintaining cellular architecture.

• Antibody dilution: For IF/ICC applications with antibodies like 13653-1-AP, the recommended dilution range is 1:10-1:100 . Begin with a 1:50 dilution and adjust as needed based on signal strength and background.

• Co-localization markers: Include established endoplasmic reticulum markers (e.g., calnexin, PDI, or KDEL-tagged proteins) for co-localization studies to confirm the expected subcellular distribution of DNASE1L1.

• Image acquisition parameters: Use confocal microscopy with appropriate z-stack imaging to properly visualize the three-dimensional distribution of DNASE1L1 within the endoplasmic reticulum network.

For quantitative analysis of subcellular localization, employ specific software tools that can measure co-localization coefficients (Pearson's, Mander's) between DNASE1L1 and organelle markers to provide objective measures of spatial distribution.

How can DNASE1L1 antibodies be effectively employed in immunoprecipitation experiments to study protein interactions?

Immunoprecipitation (IP) with DNASE1L1 antibodies requires careful consideration of several methodological aspects to effectively study protein interactions:

• Antibody selection: For IP applications, polyclonal antibodies like 13653-1-AP have been validated for successful pulldown of DNASE1L1 . Use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate as a starting point .

• Tissue/sample selection: Mouse skeletal muscle tissue has been confirmed as positive for DNASE1L1 immunoprecipitation . This makes it an ideal positive control tissue when establishing IP protocols.

• Lysis buffer optimization: Since DNASE1L1 is an endoplasmic reticulum-associated protein , use lysis buffers containing mild detergents (e.g., 1% NP-40 or 0.5% CHAPS) that effectively solubilize membrane proteins while preserving protein-protein interactions.

• Cross-linking considerations: For transient or weak interactions, consider using chemical cross-linkers (e.g., DSP, formaldehyde) prior to cell lysis to stabilize protein complexes.

• RNase/DNase treatment: Since DNASE1L1 has DNA-binding properties, treating lysates with nucleases may help distinguish between direct protein-protein interactions and nucleic acid-mediated associations.

• Elution conditions: Test different elution strategies (competitive elution with immunizing peptide, low pH, or SDS) to determine which provides the highest yield with minimal antibody contamination.

• Validation approaches: Confirm successful immunoprecipitation by Western blotting a small portion of the IP sample with the same or different DNASE1L1 antibody. For identified interacting partners, validate with reciprocal IP and/or proximity ligation assays.

When studying DNASE1L1 interactions with actin or other cytoskeletal proteins, take special care with buffer compositions to preserve these interactions, as DNASE1 family members have known interactions with actin that may be conserved in DNASE1L1 .

What strategies can be employed to validate DNASE1L1 antibody specificity in knockout/knockdown models?

Validating DNASE1L1 antibody specificity using knockout/knockdown models is a critical step in ensuring experimental reliability. Several methodological approaches can be implemented:

• CRISPR/Cas9 knockout validation: Generate DNASE1L1 knockout cell lines using CRISPR/Cas9 technology targeting early exons. The absence of signal in knockout cells compared to wild-type cells across multiple applications (WB, IF, IHC) provides strong evidence for antibody specificity. Published applications have confirmed the utility of DNASE1L1 antibodies in KO/KD experimental designs .

• siRNA/shRNA knockdown approach: For transient validation, use multiple siRNA sequences targeting different regions of DNASE1L1 mRNA. Quantify the reduction in signal intensity by Western blotting or immunofluorescence, which should correlate with the degree of knockdown measured by qRT-PCR.

• Rescue experiments: Re-express DNASE1L1 in knockout cells, preferably with a small epitope tag that doesn't interfere with antibody binding. Recovery of antibody signal provides compelling evidence for specificity.

• Tissue-specific analysis: Compare antibody staining patterns across tissues with known differential expression of DNASE1L1 (high in muscle, lower in other tissues) . Signal intensity should correlate with known expression levels.

• Cross-species validation: Test the antibody across human, mouse, and rat samples (with which 13653-1-AP has confirmed reactivity) . Conservation of the expected molecular weight and tissue-specific expression pattern across species supports specificity.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide or recombinant DNASE1L1 protein before application. Specific antibodies will show diminished or abolished staining.

Document all validation results thoroughly, including positive and negative controls, to provide a comprehensive assessment of antibody specificity that can inform experimental design and data interpretation.

How can researchers optimize DNASE1L1 antibody-based assays for studying the protein's role in apoptosis and DNA degradation?

Studying DNASE1L1's role in apoptosis and DNA degradation requires carefully optimized antibody-based assays:

• Temporal analysis during apoptosis: Design time-course experiments to track DNASE1L1 localization, expression, and activity following apoptotic stimuli. Use the validated antibodies (e.g., 13653-1-AP or KK-M3) in combination with established apoptosis markers (cleaved caspases, PARP cleavage).

• Subcellular fractionation combined with immunoblotting: Since DNASE1L1 is primarily localized to the endoplasmic reticulum , monitor its potential translocation during apoptosis by isolating nuclear, cytoplasmic, and ER fractions followed by Western blotting with DNASE1L1 antibodies at the recommended dilutions (1:500-1:1000) .

• Immunofluorescence co-localization: Perform dual staining with DNASE1L1 antibodies (dilution 1:10-1:100) and DNA fragmentation markers (TUNEL) or chromatin markers (H2AX) to visualize the spatial relationship between DNASE1L1 and DNA degradation sites.

• Chromatin immunoprecipitation (ChIP): Adapt IP protocols (using 0.5-4.0 μg antibody per 1.0-3.0 mg protein) to perform ChIP assays assessing DNASE1L1 association with chromatin during apoptosis, followed by analysis of bound DNA fragments.

• In situ activity assays: Combine immunolocalization of DNASE1L1 with in situ DNase activity assays to correlate protein presence with functional activity in fixed cells or tissues.

• Flow cytometry applications: Develop intracellular staining protocols with DNASE1L1 antibodies for flow cytometry to quantitatively assess protein levels in relation to apoptotic markers (Annexin V, propidium iodide) across cell populations.

• Live-cell imaging: For dynamic studies, consider creating fluorescently tagged DNASE1L1 constructs and validating their localization pattern against antibody staining to enable real-time monitoring during apoptosis.

When comparing DNASE1L1's role to other family members, ensure differential detection by careful antibody selection and validation, as some functions may overlap with DNASE1 and DNASE1L3 .

What are common challenges in DNASE1L1 antibody-based experiments and how can they be addressed?

Researchers working with DNASE1L1 antibodies might encounter several challenges that require methodological adjustments:

• Low signal intensity:

  • Cause: Insufficient antibody concentration or low DNASE1L1 expression in sample

  • Solution: Increase antibody concentration within recommended ranges (WB: 1:500-1:1000; IHC: 1:20-1:200; IF/ICC: 1:10-1:100) ; use positive control tissues (skeletal/cardiac muscle) with known high expression

  • Methodological approach: Test signal amplification systems (e.g., biotin-streptavidin) or more sensitive detection reagents

• Non-specific bands in Western blotting:

  • Cause: Cross-reactivity with related DNase family members or insufficient blocking

  • Solution: Increase blocking time/concentration; perform peptide competition assays

  • Methodological approach: Include DNASE1L1 knockdown controls; test alternative antibodies like KK-M3 monoclonal alongside polyclonal options

• Inconsistent immunoprecipitation results:

  • Cause: Inefficient extraction of membrane-associated DNASE1L1 from the endoplasmic reticulum

  • Solution: Optimize lysis conditions using buffers containing 0.5-1% NP-40 or CHAPS

  • Methodological approach: Increase antibody amount (up to 4.0 μg for 1.0-3.0 mg protein) and extend incubation time

• Variable immunostaining patterns:

  • Cause: Differences in fixation affecting epitope accessibility

  • Solution: Compare multiple fixation methods (cross-linking vs. precipitating fixatives)

  • Methodological approach: Test antigen retrieval with both recommended buffers (TE buffer pH 9.0 and citrate buffer pH 6.0)

• Issues with reproducibility:

  • Cause: Antibody lot-to-lot variation or sample handling differences

  • Solution: Validate each new antibody lot against previous results

  • Methodological approach: Create standardized positive controls (overexpression lysates) that can be used across experiments

For all troubleshooting scenarios, methodically document conditions tested and results obtained to systematically identify optimal parameters for your specific experimental system.

How do polyclonal and monoclonal DNASE1L1 antibodies compare in different applications, and which is preferable for specific research questions?

Polyclonal and monoclonal DNASE1L1 antibodies offer distinct advantages that should be considered when designing experiments:

Polyclonal Antibodies (e.g., 13653-1-AP):

• Application versatility: Validated for multiple applications including WB, IHC, IF/ICC, IP, and ELISA

• Species reactivity: Demonstrated cross-reactivity with human, mouse, and rat samples

• Epitope recognition: Recognize multiple epitopes, potentially increasing detection sensitivity

• Methodological advantage: Better at detecting native protein in applications where epitope conformation matters

• Research scenarios where preferable:

  • Initial characterization of DNASE1L1 expression patterns across tissues

  • Detection of denatured protein in Western blotting

  • Applications requiring detection across multiple species

  • Immunoprecipitation studies due to multiple epitope recognition

Monoclonal Antibodies (e.g., KK-M3):

• Application specificity: Validated for more limited applications (WB, IP, ELISA)

• Species restriction: Primarily detects human DNASE1L1

• Epitope specificity: Recognizes a single epitope, potentially reducing cross-reactivity

• Methodological advantage: Greater lot-to-lot consistency, critical for longitudinal studies

• Research scenarios where preferable:

  • Highly specific detection of human DNASE1L1

  • Studies requiring consistent reagents over extended time periods

  • Experiments where cross-reactivity with related DNase family members is a concern

  • Quantitative assays where standardization is critical

Comparative performance by application:

• Western blotting: Both antibody types perform well, with polyclonals potentially offering higher sensitivity

• Immunoprecipitation: Both are effective; choice depends on whether breadth of epitope recognition (polyclonal) or specificity (monoclonal) is prioritized

• Immunohistochemistry/Immunofluorescence: Polyclonals like 13653-1-AP have more extensive validation

• ELISA: Monoclonals may provide more consistent quantitative results

The optimal choice depends on the specific research question, required applications, target species, and whether sensitivity or specificity is the primary concern.

How can researchers effectively distinguish between DNASE1L1 and related family members (DNASE1, DNASE1L3) in experimental systems?

Distinguishing between DNASE1L1 and related family members requires strategic experimental design:

• Antibody selection and validation:

  • Verify immunogen sequences to ensure they target non-conserved regions among family members

  • Test antibodies on overexpression systems for each family member separately

  • Methodological approach: Run comparative Western blots with recombinant DNASE1, DNASE1L1, and DNASE1L3 proteins to assess cross-reactivity

• Expression pattern analysis:

  • Leverage distinct tissue expression profiles: DNASE1L1 is highly expressed in cardiac and skeletal muscle , while DNASE1 is more broadly expressed in exocrine and endocrine organs

  • Methodological approach: Use tissue panels in Western blotting or IHC to create expression fingerprints for each family member

• Subcellular localization differentiation:

  • DNASE1L1: Predominantly in the endoplasmic reticulum

  • DNASE1: Secreted into body fluids

  • DNASE1L3: Has both secreted and intracellular forms

  • Methodological approach: Perform subcellular fractionation followed by Western blotting or immunofluorescence co-localization with organelle markers

• Functional assays to distinguish activities:

  • DNASE1: Preferentially cleaves protein-free DNA and is involved in NET degradation

  • DNASE1L1: Functions in apoptotic DNA fragmentation

  • DNASE1L3: Also involved in NET degradation

  • Methodological approach: Design assays that measure activity under different salt conditions or on different DNA substrates

• Gene expression knockdown/knockout controls:

  • Create single and combination knockdowns of family members

  • Methodological approach: Use siRNA targeting specific regions of each family member with minimal sequence homology

• Mass spectrometry validation:

  • Following immunoprecipitation with DNASE1L1 antibodies, perform mass spectrometry analysis

  • Methodological approach: Look for unique peptides that definitively distinguish between family members

• Protein interaction partners:

  • Identify differential binding partners for each family member

  • Methodological approach: Compare IP-mass spectrometry results between DNASE1, DNASE1L1, and DNASE1L3 pulldowns

By combining multiple approaches, researchers can confidently distinguish between these related family members and correctly attribute observed functions to specific proteins.

What are the potential applications of DNASE1L1 antibodies in studying the protein's role in disease pathogenesis?

DNASE1L1 antibodies can be valuable tools for investigating this protein's role in various disease contexts:

• Barth Syndrome investigations:

  • Given the association between DNASE1L1 and Barth Syndrome , antibodies can be used to examine expression patterns in patient-derived samples

  • Methodological approach: Compare DNASE1L1 levels and localization in control versus patient cardiac tissues using IHC (1:20-1:200 dilution)

• Human Monocytic Ehrlichiosis studies:

  • Investigate DNASE1L1's potential role in this infectious disease's pathogenesis

  • Methodological approach: Examine DNASE1L1 expression and localization in infected versus healthy immune cells using IF/ICC protocols (1:10-1:100 dilution)

• Cancer research applications:

  • Since DNASE1L1 functions in DNA degradation and apoptosis , explore its potential dysregulation in cancer

  • Methodological approach: Create tissue microarrays of tumor and matching normal tissues for IHC analysis with DNASE1L1 antibodies

• Muscle-specific pathologies:

  • Given DNASE1L1's high expression in cardiac and skeletal muscle , investigate its role in myopathies

  • Methodological approach: Perform Western blot analysis (1:500-1:1000 dilution) on muscle biopsies from patients with various myopathies

• Autoimmune disorder research:

  • Explore whether DNASE1L1, like other DNase family members, may be involved in preventing autoimmunity by clearing self-DNA

  • Methodological approach: Examine DNASE1L1 expression and activity in experimental autoimmune models using multiple antibody-based techniques

• X-linked disease studies:

  • Since the DNASE1L1 gene is located on the X chromosome , investigate its potential involvement in X-linked disorders

  • Methodological approach: Compare DNASE1L1 protein levels between male and female tissues and in X-chromosome abnormalities

By applying DNASE1L1 antibodies in these disease contexts with appropriate experimental design and controls, researchers can gain insights into both pathogenesis mechanisms and potential therapeutic targets.

How can DNASE1L1 antibodies be integrated into multi-omics approaches to understand the protein's broader biological context?

Integrating DNASE1L1 antibodies into multi-omics approaches enables comprehensive understanding of this protein's functional context:

• Proteomics integration:

  • Use DNASE1L1 antibodies for immunoprecipitation (0.5-4.0 μg per 1.0-3.0 mg protein) followed by mass spectrometry to identify interaction partners

  • Methodological approach: Compare interactomes across different tissues (skeletal muscle versus others) and under various conditions (normal versus apoptotic)

  • Cross-reference findings with publicly available protein-protein interaction databases

• Transcriptomics correlation:

  • Correlate DNASE1L1 protein levels (detected by Western blotting) with mRNA expression profiles

  • Methodological approach: Perform parallel analysis of protein (by Western blot) and mRNA (by RNA-seq) from the same samples to identify potential post-transcriptional regulation

• Epigenomics applications:

  • Investigate whether DNASE1L1's DNA degradation activity influences chromatin accessibility and epigenetic patterns

  • Methodological approach: Combine DNASE1L1 immunofluorescence with DNA methylation or histone modification staining to assess spatial relationships

• Phosphoproteomics analysis:

  • Determine if DNASE1L1 undergoes post-translational modifications affecting its function

  • Methodological approach: Immunoprecipitate DNASE1L1 followed by phosphorylation-specific proteomics analysis under different cellular conditions

• Metabolomics correlation:

  • Explore relationships between DNASE1L1 expression/activity and cellular metabolic states

  • Methodological approach: Correlate DNASE1L1 protein levels (by Western blot) with metabolomic profiles in matched samples

• Spatial transcriptomics/proteomics:

  • Map DNASE1L1 distribution in tissue contexts alongside other molecular markers

  • Methodological approach: Use DNASE1L1 antibodies in multiplexed immunofluorescence or imaging mass cytometry to create spatial protein expression maps

• Systems biology modeling:

  • Incorporate DNASE1L1 protein quantification data into pathway models

  • Methodological approach: Use antibody-based protein quantification to parameterize mathematical models of DNA degradation pathways

By methodically integrating antibody-based DNASE1L1 detection with multiple omics approaches, researchers can develop a systems-level understanding of this protein's functions and regulatory networks in normal physiology and disease states.