DNASE2B Antibody, FITC conjugated

What is DNASE2B and why is it important in biological research?

DNASE2B (deoxyribonuclease II beta) shares considerable sequence similarity to, and is structurally related to DNase II. It functions as an endonuclease that catalyzes DNA hydrolysis in the absence of divalent cations at acidic pH. Unlike DNase II which is ubiquitously expressed, expression of DNASE2B is restricted to the salivary gland and lungs . The gene has been localized to chromosome 1p22.3 adjacent (and in opposite orientation) to the uricase pseudogene . Two transcript variants encoding different isoforms have been described for this gene.

DNASE2B is important in research related to DNA degradation pathways, cellular apoptosis, and potential roles in autoimmune conditions, as mutations in the related DNASE2 gene have been associated with type I interferon-mediated autoinflammation .

What is the molecular structure and function of DNASE2B protein?

DNASE2B is structurally similar to DNASE2 (DNase II alpha), with a molecular weight of approximately 42 kDa . The protein contains phospholipase D domains important for its catalytic function. The aspartate at position 121 falls within the N-terminal phospholipase D domain which, together with the histidine at position 130, likely plays an important role in DNase II catalytic function .

What are the key specifications of commercially available DNASE2B Antibody, FITC conjugated?

Based on available research materials, DNASE2B Antibody, FITC conjugated products typically have the following specifications:

How does FITC conjugation affect the functionality of DNASE2B antibodies?

FITC (Fluorescein isothiocyanate) conjugation provides direct visualization capabilities but may impact antibody functionality in several ways:

The conjugation process involves the reaction of FITC with primary amines (primarily lysine residues) on the antibody molecule . The fluorescein-to-protein (F/P) ratio is critical; overlabeling of proteins (molar F/P >6) usually results in increased non-specific binding (fluorescent background) and decreased quantum yield due to fluorophore self-quenching .

Optimal labeling conditions are essential, as excessive conjugation can alter antibody specificity, cause aggregation, and/or precipitation of the protein . The availability of amine groups varies greatly among proteins and even among different IgGs, resulting in variability of labeling levels .

When using FITC-conjugated antibodies, researchers should be aware that the conjugation might slightly reduce binding affinity compared to unconjugated versions, though properly conjugated antibodies maintain their specificity and functionality.

What are the validated applications for DNASE2B Antibody, FITC conjugated in research?

DNASE2B Antibody, FITC conjugated has been validated for several research applications:

• Western Blot (WB): Recommended dilutions typically range from 1:1000-1:5000 . Positive detection has been reported in Jurkat cells and A549 cells .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Recommended dilutions range from 1:10-1:100 . Successful detection has been reported in A549 cells .

• Immunohistochemistry (IHC): Though less common for FITC-conjugated antibodies, some products are validated for IHC with recommended dilutions of 1:20-1:200 .

• ELISA: FITC-conjugated antibodies can be used in certain ELISA formats, particularly those leveraging fluorescence detection .

• Flow Cytometry: The FITC fluorophore makes these antibodies particularly suitable for flow cytometry applications, though specific dilutions should be optimized for each experimental system.

How can DNASE2B Antibody, FITC conjugated be used in nucleic acid sensing research?

DNASE2B Antibody, FITC conjugated offers valuable tools for studying nucleic acid sensing pathways:

Based on studies of the related DNASE2 protein, researchers can investigate whether DNASE2B plays a similar role in preventing inappropriate activation of nucleic acid sensors. DNase II deficiency leads to type I interferon-mediated autoinflammation due to accumulation of undigested DNA .

Researchers can use the FITC-conjugated antibody to:

• Track DNASE2B localization in cells stimulated with various nucleic acid ligands

• Examine co-localization with nucleic acid sensors like cGAS or STING

• Investigate whether DNASE2B, like DNASE2, is involved in degradation of DNA from apoptotic cells or expelled erythroid nuclei

• Study potential interactions with TLR9, as DNase II has been shown to be required for TLR9 responses to certain DNA ligands

For such experiments, appropriate controls should include stimulation with TLR ligands like CpG-A and CpG-B, as well as comparative analysis with DNASE2-deficient cells.

What are the optimal storage and handling conditions for DNASE2B Antibody, FITC conjugated?

To maintain optimal activity of DNASE2B Antibody, FITC conjugated:

• Storage Temperature: Store at -20°C or -80°C .

• Buffer Composition: Typically stored in PBS with 0.02% sodium azide and 50% glycerol, pH 7.3 .

• Light Protection: FITC is sensitive to photobleaching; store and handle the antibody protected from light.

• Freeze-Thaw Cycles: Minimize repeated freeze-thaw cycles by aliquoting the antibody upon receipt.

• Long-term Storage: For longer storage, addition of 1% (w/v) BSA and 0.1% (w/v) sodium azide is recommended after determination of the F/P molar ratio .

• Shipping Conditions: Typically shipped on blue ice .

How should I determine the optimal concentration of DNASE2B Antibody, FITC conjugated for my specific experiment?

To determine the optimal concentration for your experiment:

• Titration Experiment: Perform a systematic titration using serial dilutions of the antibody (e.g., from 1:10 to 1:1000) on samples known to express DNASE2B.

• Signal-to-Background Analysis: For each dilution, calculate the ratio of specific signal to background. The optimal dilution provides the highest signal-to-background ratio while conserving antibody.

• Controls to Include:

  • Positive control: Sample known to express DNASE2B (e.g., salivary gland or lung tissue)

  • Negative control: Sample lacking DNASE2B expression

  • Isotype control: FITC-conjugated antibody of the same isotype but irrelevant specificity

• Fluorophore Density Consideration: Consider that the fluorescein-to-protein (F/P) ratio affects performance. High F/P ratios (>6) may increase non-specific binding and decrease quantum yield through self-quenching .

• Application-Specific Considerations:

  • For Western Blot: 1:1000-1:5000

  • For IF/ICC: 1:10-1:100

  • For IHC: 1:20-1:200

  • For Flow Cytometry: Start with 1:100 and adjust based on results

How can DNASE2B Antibody, FITC conjugated be used to investigate the relationship between DNASE2B and autoimmune conditions?

Based on research with the related DNASE2 protein, researchers can explore DNASE2B's potential role in autoimmunity:

• Comparative Expression Analysis: Use FITC-conjugated DNASE2B antibody to compare expression levels in tissues from healthy controls versus patients with autoimmune conditions using flow cytometry or fluorescence microscopy.

• Co-localization Studies: Perform dual labeling with markers of inflammation or autoimmunity to determine spatial relationships between DNASE2B and disease-relevant proteins.

• Cell-Type Specific Analysis: Identify which cell types express DNASE2B in autoimmune conditions using multi-parameter flow cytometry with lineage markers.

• Functional Studies in Disease Models: Investigate whether DNASE2B expression changes during disease progression in animal models of autoimmunity.

• Interferon Response Assessment: Given that DNASE2 deficiency leads to type I interferon-mediated autoinflammation , examine whether DNASE2B plays a similar role in regulating interferon responses by analyzing cells after interferon stimulation.

• DNA Clearance Analysis: Determine if DNASE2B contributes to clearance of extracellular DNA, which can be immunogenic when not properly degraded. This is particularly relevant given findings that DNase II deficiency prevents activation of autoreactive B cells .

What techniques can be used to validate the specificity of DNASE2B Antibody, FITC conjugated in experimental systems?

To ensure the specificity of DNASE2B Antibody, FITC conjugated, researchers should employ multiple validation techniques:

• Genetic Knockout/Knockdown Controls:

  • siRNA knockdown of DNASE2B in control cells

  • CRISPR/Cas9-mediated knockout of DNASE2B

  • Compare staining patterns between wild-type and modified cells

• Peptide Competition Assay:

  • Pre-incubate the antibody with the immunizing peptide (the synthetic peptide directed towards the middle region of human DNASE2B )

  • Compare staining with and without peptide competition

  • Specific staining should be blocked by the peptide

• Multiple Antibody Validation:

  • Compare staining patterns using antibodies targeting different epitopes of DNASE2B

  • Consistent localization patterns increase confidence in specificity

• Western Blot Validation:

  • Confirm detection of a single band at the expected molecular weight (~42 kDa)

  • Analyze samples with known differential expression of DNASE2B

• Tissue Expression Pattern:

  • Verify that strongest staining occurs in tissues known to express DNASE2B (salivary gland and lungs)

  • Minimal staining should be observed in tissues with low expression

• Recombinant Protein Controls:

  • Test antibody against purified recombinant DNASE2B protein

  • Include related proteins (e.g., DNASE2) to assess cross-reactivity

What are common issues encountered when using DNASE2B Antibody, FITC conjugated, and how can they be resolved?

Common issues and their solutions include:

• High Background Signal:

  • Cause: Over-labeling (high F/P ratio >6), insufficient blocking, or inadequate washing

  • Solution: Use optimal antibody dilution, increase blocking time/concentration, perform more stringent washing steps, and ensure the F/P ratio is within the optimal range (typically 2-4)

• Weak or No Signal:

  • Cause: Insufficient antigen, antibody degradation, or improper storage

  • Solution: Increase antibody concentration, verify target expression in positive control samples, check antibody activity, and ensure proper storage conditions

• Non-specific Binding:

  • Cause: High antibody concentration or cross-reactivity

  • Solution: Increase dilution, use more stringent washing, include additional blocking agents, and validate specificity with controls

• Photobleaching:

  • Cause: Excessive exposure to light

  • Solution: Minimize light exposure during storage and handling, use anti-fade mounting media, and capture images promptly

• Inconsistent Results:

  • Cause: Variability in experimental conditions or antibody quality

  • Solution: Standardize protocols, use the same antibody lot when possible, and include consistent positive and negative controls

• Reduced Antibody Activity After Storage:

  • Cause: Repeated freeze-thaw cycles or improper storage

  • Solution: Aliquot antibody upon receipt, store at recommended temperature (-20°C), and add stabilizers like BSA for long-term storage

How can the Fluorescein/Protein (F/P) ratio be determined and optimized for DNASE2B Antibody, FITC conjugated?

The F/P ratio is crucial for FITC-conjugated antibody performance and can be determined and optimized as follows:

• Spectrophotometric Determination:

  • Measure absorbance at 280 nm (A280) for protein concentration and 495 nm (A495) for FITC

  • Calculate F/P ratio using the formula: F/P = (A495 × Dilution Factor) ÷ (A280 - (0.35 × A495)) × 0.41

• Optimal F/P Range:

  • For most applications, aim for F/P ratios between 2-4

  • Ratios <2 may provide insufficient signal

  • Ratios >6 typically lead to increased background and reduced quantum yield due to self-quenching

• Optimization Approach:

  • Perform small-scale conjugations using different molar ratios (e.g., 5:1, 10:1, and 20:1 of FITC to antibody)

  • These reaction molar ratios typically result in F/P ratios of 1-2, 2-4, and 3-6, respectively

  • Evaluate each preparation in your specific application

  • Scale up using the optimal ratio for larger preparations

• Factors Affecting F/P Ratio:

  • pH of reaction buffer (optimal pH 9.0-9.5)

  • Reaction time and temperature

  • Concentration of reactants

  • Availability of amine groups on the antibody

• Quality Control:

  • Document F/P ratio for each preparation

  • Test functional activity through binding assays

  • Assess non-specific binding in negative control samples