DNASE2 Antibody

What is DNASE2 and what are its primary functions in cells?

DNASE2 (deoxyribonuclease II, lysosomal) is a lysosomal endonuclease that hydrolyzes DNA under acidic conditions with a preference for double-stranded DNA. It plays crucial roles in:

• Degradation of DNA from apoptotic nuclei in phagocytes

• Clearance of DNA during erythropoiesis in fetal liver and bone marrow

• Maintenance of skin cell homeostasis

• Prevention of autoinflammation by clearing nucleic acids

The enzyme hydrolyzes DNA specifically under acidic conditions, explaining its effectiveness in the acidic environment of lysosomes where it degrades DNA expelled from erythroid precursor cells and DNA from engulfed apoptotic cells .

What are the validated applications for DNASE2 antibodies?

DNASE2 antibodies have been validated for multiple applications according to manufacturer data:

Multiple cell lines have shown positive results with DNASE2 antibodies, including HEK-293 cells, HeLa cells, LNCaP cells, and MDA-MB-231 cells for Western blot applications . For immunoprecipitation, U-87 MG cells and HeLa cells have been successfully used .

What species reactivity is available for DNASE2 antibodies?

Most commercially available DNASE2 antibodies show reactivity with:

• Human

• Mouse

• Rat

Some antibodies have been specifically tested and validated with human, mouse, and rat samples as noted in product information sheets . Researchers should check specific product information for cross-reactivity when planning experiments involving other species.

How can I verify the specificity of my DNASE2 antibody?

To verify specificity of DNASE2 antibodies:

• Positive controls: Use cell lines with known DNASE2 expression (HeLa, HEK-293) as positive controls .

• Knockdown/knockout validation:

  • Compare staining in wild-type versus DNASE2 knockout/knockdown cells

  • CRISPR-Cas9 edited HEK293FT cells with DNASE2 gene disruption can serve as negative controls .

• Immunoprecipitation validation: Perform IP followed by Western blot to confirm the antibody pulls down the correct protein.

• Blocking peptide: Use the immunizing peptide to block antibody binding and confirm specificity .

• Multiple antibodies: Compare results using antibodies targeting different epitopes of DNASE2.

Research by Chan et al. generated specific anti-mouse DNASE2 monoclonal antibodies by immunizing DNASE2-deficient mice with wild-type DNASE2 protein, screening using Ba/F3 cells expressing DNASE2 on their surface .

Why does DNASE2 appear at different molecular weights in Western blots?

DNASE2 is detected at varying molecular weights due to proteolytic processing and post-translational modifications:

• Calculated molecular weight: 40 kDa (360 amino acids)

• Pro-enzyme form: ~40-45 kDa

• Processed forms: 30 kDa and 23 kDa forms detected in lysosomes

Research has shown that DNASE2 undergoes proteolytic processing in vivo. Western blot detection using a carboxyl-terminal antibody revealed two predominant forms (30 kDa and 23 kDa) in lysosomes, rather than the expected 45 kDa form . This processing is significantly altered in mice lacking cathepsin L, indicating that this protease is involved in DNASE2 maturation .

The extracellularly secreted DNASE2 is detected as a pro-form that becomes activated under acidic conditions, suggesting that proteolytic processing occurs in lysosomes and is necessary for full enzymatic activity .

What controls should be used when working with DNASE2 antibodies?

For robust DNASE2 antibody experiments, include these controls:

• Positive tissue controls:

  • Human spleen tissue lysate (recommended for antibody PA5-19900)

  • Mouse kidney tissue (for IHC applications)

• Negative controls:

  • Primary antibody omission

  • Isotype control (rabbit IgG)

  • DNASE2 knockout/knockdown samples

  • Blocking peptide competition

• Loading controls for Western blot:

  • Housekeeping proteins (β-actin, GAPDH)

  • Total protein stain methods

• Expression controls:

  • DNASE2-transfected cells compared to non-transfected cells

  • Recombinant DNASE2 protein

• Functional controls:

  • DNase activity assay to correlate protein detection with enzymatic function

How can DNASE2 antibodies be used to study DNA clearance mechanisms?

DNASE2 antibodies are valuable tools for investigating DNA clearance mechanisms:

• Colocalization studies: Use IF/ICC with DNASE2 antibodies and DNA dyes to:

  • Track undegraded DNA in lysosomes

  • Monitor nuclear DNA export to lysosomes

  • Study autophagy-mediated DNA delivery to lysosomes

• Phagocytosis assays: Use DNASE2 antibodies to:

  • Track DNASE2-mediated degradation of engulfed apoptotic cells

  • Study professional phagocytes like macrophages and dendritic cells

• Disease models:

  • Autoimmunity: Examine DNASE2 function in models of autoimmune disease

  • Inflammation: Study DNASE2's role in preventing activation of cytoplasmic DNA sensors

  • Erythropoiesis: Investigate DNASE2 in models of erythroid maturation

Research by Eckard et al. showed that DNASE2-deficient mice develop autoantibodies against DNA-associated antigens. Using bifunctional immunoglobulins with DNA binding domains, they demonstrated that DNASE2-deficient B cells fail to respond to DNA-containing complexes, suggesting a role for DNASE2 in properly processing DNA-associated autoantigens .

How do DNASE2 antibodies help investigate subcellular localization and trafficking?

DNASE2 antibodies enable detailed subcellular localization studies:

• Lysosomal localization:

  • Use DNASE2 antibodies with lysosomal markers (LAMP1/2) to confirm localization

  • Monitor DNASE2 trafficking to lysosomes under different conditions

• Subcellular fractionation:

  • Use DNASE2 antibodies to detect different processed forms in distinct cellular compartments

  • Research shows the 23 kDa and 30 kDa forms are primarily localized in lysosomes

• Autophagy pathway:

  • Track DNASE2 interaction with autophagosomes using co-immunoprecipitation

  • Research indicates damaged nuclear DNA requires autophagy-mediated delivery to lysosomes for DNASE2-dependent degradation

• Nuclear DNA export:

  • Use DNASE2 antibodies to study mechanisms of nuclear DNA export to lysosomes

  • Evidence suggests nuclear export and autophagy are required for DNASE2-mediated clearance of damaged nuclear DNA

The study by Lan et al. demonstrated that DNASE2 is essential for clearance of damaged nuclear DNA through an autophagy-dependent mechanism. They observed co-localization of DNA with lysosomes in DNASE2-deficient cells, suggesting that DNASE2 acts in lysosomes to degrade DNA delivered there by autophagy .

What methods are effective for detecting endogenous DNASE2 in tissue samples?

Detection of endogenous DNASE2 requires specific approaches:

• Enrichment strategies:

  • Use Con A Sepharose for partial purification before Western blotting

  • The protocol described by Shiokawa and Tanuma includes:

    • Incubation with Con A Sepharose

    • Elution with 0.5 M α-methyl-D-mannoside

    • SDS-PAGE and Western blotting

• IHC optimization:

  • Antigen retrieval: Use TE buffer pH 9.0 or citrate buffer pH 6.0

  • Dilution range: 1:1000-1:4000

  • Mouse kidney tissue has been validated as positive control tissue

• IF/ICC protocols:

  • Fixation: 4% paraformaldehyde is typically effective

  • Permeabilization: Use 0.1% Triton X-100 for accessing intracellular antigens

  • Co-staining: Combine with lysosomal markers like LAMP1

• Activity correlation:

  • DNase II activity assay can be performed using:

    • Incubation of plasmid DNA with sample in acetate buffer pH 4.7

    • Visualization of DNA degradation by agarose gel electrophoresis

How do mutations in DNASE2 affect protein detection and function?

DNASE2 mutations impact both antibody detection and functional studies:

• Loss-of-function mutations:

  • Studies identified biallelic loss-of-function mutations in patients with type I interferon-mediated autoinflammation

  • Specific mutations include c.347G>C (p.Gly116Ala) and c.362A>T (p.Asp121Val)

• Antibody epitope considerations:

  • Mutations may affect antibody recognition if they occur within the epitope region

  • Antibodies targeting different regions may show differential detection of mutant proteins

• Functional impact assessment:

  • DNase activity assays reveal enzyme activity loss in mutant proteins

  • Cellular accumulation of undegraded DNA can be visualized by DNA dyes

• Protein stability effects:

  • Some mutations may affect protein stability or processing

  • Western blot may reveal altered molecular weight patterns or reduced protein levels

Research by Rodero et al. demonstrated that biallelic mutations in DNASE2 lead to autoinflammation due to activation of the STING-dependent interferon pathway by undegraded DNA .

How can DNASE2 antibodies be used to study autoinflammatory disorders?

DNASE2 antibodies are valuable tools for investigating autoinflammatory mechanisms:

• Patient sample analysis:

  • Compare DNASE2 expression and localization in patient vs. control samples

  • Assess DNASE2 processing in affected tissues

• DNA accumulation studies:

  • Use DNASE2 antibodies with DNA dyes to visualize DNA accumulation

  • Track DNA localization in lysosomes and cytosol

• Signaling pathway activation:

  • Study STING pathway activation downstream of DNASE2 deficiency

  • Monitor type I interferon production and signaling

• Therapeutic testing:

  • Evaluate potential therapies that might compensate for DNASE2 deficiency

  • Monitor changes in DNA accumulation and inflammatory markers

Rodero et al. identified patients with biallelic DNASE2 mutations who presented with severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, and arthropathy. They demonstrated that these mutations resulted in loss of DNase II function, leading to activation of the interferon pathway by undegraded DNA .

What is the role of DNASE2 in immune function and how can antibodies help study this?

DNASE2 antibodies can illuminate several aspects of immune function:

• Phagocytosis studies:

  • Track DNASE2-mediated degradation of DNA in phagocytes

  • Compare wild-type and DNASE2-deficient phagocytes

• TLR9 interaction:

  • Research indicates DNASE2 is required for DNA sensing by TLR9

  • Chan et al. found that DNase II digestion of DNA in endolysosomes is necessary for proper TLR9 activation

• Innate immunity:

  • DNASE2 knockdown in Drosophila resulted in decreased hemocyte numbers and increased susceptibility to bacterial infection

  • Antibodies can help visualize DNASE2 in immune cells

• B cell activation:

  • DNASE2-deficient B cells show impaired responses to DNA-containing immune complexes

  • Eckard et al. demonstrated that DNASE2 is required for proper B cell activation by DNA-containing antigens

The research by Chan et al. demonstrated that DNASE2 digestion of DNA in endolysosomes is required for proper TLR9 sensing, revealing a previously unappreciated role for DNASE2 in innate immune recognition of DNA .

How does DNASE2 deficiency impact cellular processes, and how can antibodies visualize these effects?

DNASE2 antibodies help visualize multiple cellular consequences of DNASE2 deficiency:

• DNA accumulation patterns:

  • DNASE2-deficient cells show increased DNA content by flow cytometry and microscopy

  • Both phagocytic and non-phagocytic cells accumulate DNA

• Subcellular DNA localization:

  • DNASE2 antibodies combined with DNA dyes reveal DNA accumulation in:

    • Lysosomes

    • Cytoplasmic compartments

    • Extra-nuclear spaces

• Autophagy pathway visualization:

  • Co-staining with autophagy markers reveals:

    • Impaired clearance of damaged nuclear DNA

    • Defective autophagy-lysosome pathway function

• Inflammation activation:

  • DNASE2 antibodies help track:

    • DNA sensor activation (STING pathway)

    • Inflammatory signaling cascade initiation

Lan et al. observed that DNASE2-deficient cells accumulate DNA not only from phagocytosed material but also from damaged nuclear DNA. They demonstrated that this accumulation triggers inflammation via the STING cytosolic DNA-sensing pathway .

What are the optimal storage conditions for DNASE2 antibodies?

Proper storage is critical for maintaining DNASE2 antibody performance:

According to manufacturer guidelines, DNASE2 antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. They remain stable for one year after shipment when stored at -20°C .

What are common issues when using DNASE2 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with DNASE2 antibodies:

• Multiple band detection:

  • Issue: Detection of multiple bands (23 kDa, 30 kDa, 40 kDa)

  • Solution: Understand that DNASE2 undergoes proteolytic processing; different bands represent different processed forms

• Low signal intensity:

  • Issue: Weak detection of endogenous DNASE2

  • Solution: Enrich DNASE2 using Con A Sepharose before Western blotting

• High background in IF/ICC:

  • Issue: Non-specific staining

  • Solution: Optimize blocking (5% BSA or serum), increase washing steps, and validate specificity with knockout controls

• Variability between tissue types:

  • Issue: Different tissues show varying detection patterns

  • Solution: Optimize protocols for each tissue type; consider tissue-specific processing differences

• Cross-reactivity concerns:

  • Issue: Potential detection of related proteins

  • Solution: Validate with DNASE2 knockout samples or blocking peptides

Shiokawa and Tanuma demonstrated that partial purification using Con A Sepharose significantly improved detection of endogenous DNASE2 forms by Western blotting, enabling visualization of the distinct 30 kDa and 23 kDa processed forms .

How should experimental conditions be optimized for different DNASE2 antibody applications?

Application-specific optimization strategies for DNASE2 antibodies:

Western Blot Optimization:

• Sample preparation: Consider enrichment with Con A Sepharose for endogenous detection

• Recommended dilutions: 1:1000-1:8000

• Loading control: β-actin is commonly used

• Expected bands: 32-40 kDa for the pro-enzyme; 23-30 kDa for processed forms

Immunohistochemistry Optimization:

• Antigen retrieval: TE buffer pH 9.0 or citrate buffer pH 6.0

• Recommended dilutions: 1:1000-1:4000

• Positive control: Mouse kidney tissue

• Visualization system: HRP or AP-based detection systems

Immunofluorescence Optimization:

• Fixation: 4% paraformaldehyde (10-15 minutes)

• Permeabilization: 0.1-0.5% Triton X-100

• Recommended dilutions: 1:50-1:500

• Counterstaining: DAPI for nuclei, specific organelle markers as needed

Immunoprecipitation Optimization:

• Lysate preparation: Use 1.0-3.0 mg of total protein

• Antibody amount: 0.5-4.0 μg per IP reaction

• Beads: Protein A/G for rabbit polyclonal antibodies

• Elution: Use SDS sample buffer for Western blot analysis

For detection of extracellular DNASE2, researchers have successfully used Con A Sepharose followed by immunoprecipitation with anti-FLAG antibody, as described by Shiokawa and Tanuma .