DNASE2B Antibody

What is DNASE2B and what are its primary biological functions?

DNASE2B (deoxyribonuclease II beta) is a lysosomal endonuclease that functions as the primary acid DNase essential for nuclear DNA degradation during lens cell differentiation. It shares considerable sequence similarity to and is structurally related to DNase II, which catalyzes DNA hydrolysis in the absence of divalent cations at acidic pH. Unlike DNase II which is ubiquitously expressed, DNASE2B expression is more restricted, with notable presence in the salivary gland and lungs .

The protein has:

• Molecular weight of approximately 42 kDa

• 361 amino acids in its canonical form

• Gene ID (NCBI): 58511

• UniProt ID: Q8WZ79

Recent research has identified DNASE2B's potential involvement in cancer cell proliferation and cell cycle regulation, particularly in non-small cell lung cancer .

How do I select the appropriate DNASE2B antibody for my research applications?

When selecting a DNASE2B antibody, consider these key parameters based on your experimental objectives:

When evaluating technical specifications, review the antibody datasheet for:

• Documented reactivity with specific cell lines (e.g., Jurkat cells, A549 cells)

• Validation data for your intended application

• Recommended positive control tissues (e.g., human tonsillitis tissue)

• Storage conditions and stability information

What are the optimal experimental conditions for Western blot detection of DNASE2B?

For optimal Western blot detection of DNASE2B, follow these methodological guidelines:

Sample preparation:

• Human cell lines showing consistent DNASE2B expression include Jurkat cells, A549 cells, HEK-293 cells, and HeLa cells

• Use standard lysis buffers with protease inhibitors

• Load 20-50 μg total protein per lane

Protocol optimization:

• Use recommended dilutions: 1:1000-1:4000 for Proteintech antibody (22652-1-AP) or 1:500-1:2000 for Abbexa antibody

• Expect detection at approximately 42 kDa molecular weight

• Include positive controls (e.g., A549 cell lysate) validated for DNASE2B expression

• For troubleshooting non-specific bands, increase blocking time or adjust antibody concentration

Research by Zhou et al. demonstrated successful DNASE2B protein detection in A549 cells using Western blot, validating DNASE2B knockdown experiments with clear band reduction at the expected molecular weight .

How should I optimize immunohistochemistry protocols for DNASE2B detection in tissue samples?

For effective DNASE2B detection in tissue sections:

Tissue preparation and antigen retrieval:

• Positive control tissues: human tonsillitis tissue or human prostate hyperplasia tissue

• Recommended antigen retrieval: TE buffer at pH 9.0 (primary) or citrate buffer at pH 6.0 (alternative)

• Use 4-6 μm thick sections from formalin-fixed paraffin-embedded tissues

Staining protocol optimization:

• Antibody dilution: Start with 1:20-1:200 range for most DNASE2B antibodies

• Incubation conditions: Overnight at 4°C for primary antibody is typically optimal

• Detection systems: HRP/DAB-based detection systems are commonly effective

• Counterstaining: Hematoxylin provides good nuclear contrast

• Always include validated positive and negative controls

For challenging samples, consider testing multiple antigen retrieval methods or extending primary antibody incubation time to enhance signal without increasing background.

How do I interpret conflicting DNASE2B expression data between Western blot and immunofluorescence results?

When encountering discrepancies between Western blot and immunofluorescence results for DNASE2B:

Systematic analysis approach:

• Verify antibody specificity:

  • Confirm the antibody recognizes the same epitope in both applications

  • Review validation data for cross-reactivity with related proteins (DNASE2/DNASE2A)

• Evaluate protein localization effects:

  • DNASE2B is primarily lysosomal, which may affect accessibility in fixed cells

  • Subcellular fractionation may reveal pools of protein not detected in whole cell lysates

• Consider post-translational modifications:

  • Glycosylation or other modifications may affect epitope recognition differently in native vs. denatured conditions

  • Western blot uses denatured proteins while IF observes proteins in their cellular context

• Experimental verification strategies:

  • Perform knockdown experiments to confirm antibody specificity

  • Use multiple antibodies targeting different epitopes

  • Include controls with known DNASE2B expression patterns

Research shows that DNASE2B has been successfully detected in A549 cells using both Western blot and immunofluorescence techniques , suggesting both approaches can be valid with proper optimization.

What are common pitfalls in DNASE2B antibody-based experiments and how can they be addressed?

Common technical challenges when working with DNASE2B antibodies include:

For reliable results, verify antibody performance through:

• Positive and negative controls in each experiment

• Validation with genetic approaches (siRNA/shRNA knockdown)

• Correlation with mRNA expression data when possible

How can DNASE2B antibodies be employed to investigate its role in cell cycle regulation and cancer progression?

Based on recent research by Zhou et al. (2018), DNASE2B plays a significant role in cell cycle regulation in non-small cell lung cancer cells . To investigate this function:

Experimental approach for cell cycle studies:

• DNASE2B knockdown using shRNA or CRISPR:

  • Transfect cells with shDNASE2B lentivirus (verified with antibody detection)

  • Confirm knockdown efficiency via Western blot and qPCR (>70% reduction)

• Cell cycle analysis:

  • Flow cytometry revealed DNASE2B knockdown caused significant S-phase depletion with G1 and G2/M accumulation

  • Cells with shDNASE2B showed only 2.75% S-phase cells compared to 17.37% in control cells (p<0.001)

• Proliferation analysis:

  • Colony formation assay with crystal violet staining

  • Cell counting using Celigo Cell Counting application

  • MTT assay for metabolic activity assessment

• Molecular mechanism investigation:

  • Analyze cell cycle regulators (Ki-67, PCNA) using DNASE2B antibodies in combination with antibodies against these markers

  • Correlate DNASE2B levels with cancer cell proliferation markers

Zhou et al. demonstrated that DNASE2B knockdown significantly reduced colony formation and proliferation in A549 cells, suggesting its potential as a therapeutic target in NSCLC .

How does DNASE2B function compare to DNASE2 (DNASE2A) in immunological contexts, and how can antibodies help differentiate their roles?

DNASE2 and DNASE2B share sequence similarity but have distinct biological functions that can be investigated using specific antibodies:

Comparative analysis approach:

Differential research strategies:

• Co-immunoprecipitation studies:

  • Use specific antibodies against DNASE2 and DNASE2B to identify unique binding partners

  • Identify differential protein complexes in various cell types

• Knockout model comparisons:

  • THP1-Dual™ KO-DNase2 cells provide a model system for DNASE2 functional studies

  • Compare with DNASE2B knockdown models in relevant cell types

• Immunofluorescence co-localization:

  • Use dual staining with specific antibodies to determine subcellular localization differences

  • Analyze co-localization with lysosomal markers

• Functional assays:

  • Measure DNase activity in different pH conditions

  • Analyze DNA degradation patterns in different cellular contexts

Research has shown DNASE2 is associated with systemic lupus erythematosus (SLE), with decreased serum DNase activity correlating with antinucleosome antibody levels (rs = -0.256, p<0.01) , while DNASE2B has been implicated in cancer cell proliferation .

What are the most effective strategies for measuring DNASE2B enzymatic activity alongside protein expression levels?

To comprehensively evaluate both DNASE2B protein levels and its enzymatic function:

Integrated experimental approach:

• Protein expression quantification:

  • Western blot with densitometry using DNASE2B antibody (1:1000-1:4000 dilution)

  • Immunofluorescence for subcellular localization (1:10-1:100 dilution)

  • Flow cytometry for single-cell analysis of expression levels

• Enzymatic activity measurement:

  • Adapt ELISA-based DNase activity assays similar to those used for DNASE2

  • Use acidic pH (4.5-5.5) buffer conditions optimal for DNASE2B activity

  • Measure DNA degradation using fluorescently labeled substrates

• Correlation analysis:

  • Compare expression levels with enzymatic activity across:

    • Different cell types

    • Normal vs. pathological conditions

    • Wild-type vs. mutant DNASE2B variants

• Validation strategies:

  • Genetic manipulation (knockdown/overexpression) followed by activity assays

  • Use of specific inhibitors to confirm assay specificity

  • Recombinant DNASE2B protein as positive control

This integrated approach provides more comprehensive insights than protein detection alone and can reveal functional implications of DNASE2B expression changes in different research contexts.

How can DNASE2B antibodies be used to investigate potential biomarker applications in non-small cell lung cancer?

Based on findings showing DNASE2B's role in NSCLC cell proliferation , researchers can explore its biomarker potential using these methodological approaches:

Biomarker validation strategy:

• Tissue microarray analysis:

  • Use DNASE2B antibodies (1:20-1:200 dilution) on NSCLC tissue microarrays

  • Compare expression in tumor vs. adjacent normal tissue

  • Correlate with clinicopathological parameters and survival data

• Multi-marker panel development:

  • Combine DNASE2B with established lung cancer markers

  • Perform multiplexed IHC with DNASE2B and cell cycle markers (Ki-67, PCNA)

  • Develop scoring system based on intensity and distribution patterns

• Functional validation:

  • Correlate DNASE2B expression with S-phase depletion patterns

  • Analyze relationship between DNASE2B levels and treatment response

• Liquid biopsy development:

  • Investigate circulating antibodies against DNASE2B in patient sera

  • Explore DNASE2B in extracellular vesicles from lung cancer patients

The research by Zhou et al. provides a foundation, showing DNASE2B knockdown significantly affects cell cycle with S-phase depletion (2.75% vs. 17.37% in controls, p<0.001) and reduced colony formation in A549 cells , suggesting its potential utility as a proliferation marker.

What methodological approaches can differentiate between normal and pathological DNASE2B functions in tissue samples?

To distinguish between physiological and pathological DNASE2B functions:

Comprehensive analytical framework:

• Quantitative expression analysis:

  • Use calibrated immunohistochemistry with DNASE2B antibodies

  • Implement digital pathology quantification methods

  • Compare expression levels across:

    • Normal vs. disease tissues

    • Different disease stages

    • Treatment-responsive vs. non-responsive samples

• Subcellular localization analysis:

  • Use high-resolution immunofluorescence to detect aberrant localization

  • Co-stain with lysosomal markers to assess proper compartmentalization

  • Evaluate nuclear vs. cytoplasmic distribution ratios

• Context-specific functional assays:

  • Assess DNA degradation capacity in tissue extracts

  • Measure acid DNase activity in normal vs. pathological samples

  • Correlate with cell proliferation markers in matched samples

• Multi-omics integration:

  • Combine antibody-based detection with:

    • Transcriptomic data (RNA-seq)

    • Proteomic profiles

    • Genetic variant analysis (particularly in DNASE2B gene region)

This approach can help establish DNASE2B's normal baseline function in tissues like lung and identify pathological alterations associated with disease processes like cancer.

How can DNASE2B antibodies contribute to understanding the relationship between DNA degradation and inflammation pathways?

While DNASE2 has established roles in inflammation and autoimmunity , DNASE2B's potential involvement remains less explored. Researchers can investigate this connection using:

Investigative approach:

• Parallel analysis of DNASE2 and DNASE2B:

  • Use specific antibodies to compare expression patterns in inflammatory conditions

  • Analyze co-expression with inflammatory markers in various tissue contexts

  • Study expression changes during inflammatory stimulation

• Genetic manipulation models:

  • Compare inflammatory responses in:

    • DNASE2 knockout models (e.g., THP1-Dual™ KO-DNase2 cells)

    • DNASE2B knockdown models

    • Double knockdown/knockout models

• Nucleic acid sensing pathway analysis:

  • Investigate DNASE2B's potential role in preventing cytosolic DNA accumulation

  • Analyze interactions with cGAS-STING pathway components

  • Measure type I interferon responses in DNASE2B-deficient cells

• Translational investigation:

  • Examine DNASE2B expression in inflammatory conditions like SLE

  • Compare with findings on DNASE2, where decreased activity correlates with antinucleosome antibodies (rs = -0.256, p<0.01)

  • Evaluate potential compensatory mechanisms between DNASE2 and DNASE2B

This approach leverages insights from DNASE2 research while exploring DNASE2B's unique contributions to DNA clearance and inflammation regulation.

What are the most promising experimental designs for studying DNASE2B's role in cellular differentiation beyond lens cells?

To expand understanding of DNASE2B's role in cellular differentiation:

Experimental framework:

• Expression profiling across differentiation models:

  • Use DNASE2B antibodies to analyze protein levels during:

    • Epithelial cell differentiation

    • Lung cell differentiation (given DNASE2B's expression in lungs)

    • Erythrocyte differentiation (comparing with DNASE2 function)

• Temporal dynamics analysis:

  • Time-course studies of DNASE2B expression during differentiation

  • Correlation with chromatin remodeling markers

  • Analysis of DNA degradation patterns at different differentiation stages

• CRISPR-based functional genomics:

  • Generate tissue-specific DNASE2B knockout models

  • Perform lineage tracing with simultaneous DNASE2B detection

  • Analyze differentiation capacity in DNASE2B-deficient cells

• Mechanistic investigations:

  • Study DNASE2B's involvement in programmed nuclear degradation

  • Compare with other DNases in differentiation contexts

  • Analyze epigenetic consequences of DNASE2B deficiency

These approaches can reveal whether DNASE2B's established role in lens cell differentiation extends to other cellular contexts, potentially identifying new biological functions and therapeutic targets.