ASB17 Antibody

What is ASB17 and what cellular functions is it involved in?

ASB17 is a member of the ankyrin repeat and SOCS box-containing protein (ASB) family, which has been characterized as an E3 ubiquitin ligase. Recent research has revealed that ASB17 plays significant roles in inflammatory signaling pathways, particularly through interaction with TNF receptor-associated factor 6 (TRAF6). ASB17 has been shown to facilitate lipopolysaccharide (LPS)-induced nuclear factor kappa B (NF-κB) activation by maintaining TRAF6 protein stability . This interaction occurs specifically via ASB17's aa177-250 segment, which binds with the Zn finger domain of TRAF6 . Additionally, ASB17 has been implicated in apoptotic processes in testicular tissue, where it mediates cell apoptosis by ubiquitylating and degrading proteins BCLW and MCL1 . These findings position ASB17 as an important modulator of both inflammatory responses and cell death mechanisms.

What types of ASB17 antibodies are available for research applications?

Several types of ASB17 antibodies are currently available for research purposes, targeting different epitopes and offering various conjugation options:

• Antibodies targeting specific amino acid regions:

  • ASB17 antibody (AA 30-130): Targets the N-terminal region

  • ASB17 antibody (AA 181-209, C-Term): Targets the C-terminal region

  • ASB17 antibody (AA 143-192): Intermediate region targeting

  • ASB17 antibody (AA 184-214, C-Term): Alternative C-terminal targeting

• Available conjugation formats:

  • Unconjugated antibodies for general applications

  • Biotin-conjugated for enhanced detection systems

  • Fluorescent conjugates including AbBy Fluor® 555, 488, 594, and 350 for immunofluorescence applications

These diverse options allow researchers to select antibodies best suited for their specific experimental requirements and detection systems.

Which experimental techniques can ASB17 antibodies be reliably used for?

ASB17 antibodies have been validated for multiple experimental techniques, with varying applications depending on the specific antibody:

When designing experiments, researchers should verify the validation status of their chosen antibody for their specific application and target species. Cross-reactivity profiles vary between antibodies, with some demonstrating broader species compatibility including dog, horse, monkey, rabbit, and bat samples for certain applications .

How does epitope selection impact experimental outcomes when studying ASB17-TRAF6 interactions?

When investigating ASB17-TRAF6 interactions, epitope selection is crucial for experimental success. Research has demonstrated that ASB17 interacts with TRAF6 specifically through its aa177-250 segment, which binds to the Zn finger domain of TRAF6 . Therefore, antibodies targeting the C-terminal region (such as AA 181-209) may interfere with or block this interaction . Conversely, N-terminal targeting antibodies (such as AA 30-130) would be less likely to disrupt the protein-protein binding .

For co-immunoprecipitation experiments studying ASB17-TRAF6 complexes, researchers should consider using antibodies targeting regions outside the interaction domain to avoid disrupting the natural binding. When performing immunofluorescence to study co-localization, epitope accessibility within the native protein complex must be evaluated. Preliminary experiments comparing multiple antibodies with different epitope targets are recommended to establish which provides the most accurate representation of the biological interaction in your specific experimental system.

What are the critical parameters for optimizing ASB17 antibody performance in inflammation research models?

When utilizing ASB17 antibodies in inflammation research, several critical parameters require optimization:

• Sample preparation and timing: ASB17 expression levels in bone marrow-derived dendritic cells (BMDCs) significantly increase following LPS stimulation . Therefore, time-course experiments are essential to capture optimal ASB17 expression windows. Studies have shown that ASB17 deficiency impairs the expression of LPS-induced pro-inflammatory cytokines including CCL2, IL-6, IL-1β, and IP-10 .

• Antibody concentration and incubation conditions: For immunofluorescence studies, research protocols indicate optimal results with overnight incubation at 4°C with primary ASB17 antibodies, followed by 2-hour room temperature incubation with appropriate secondary antibodies (anti-Mouse IgG DyLight 649 or anti-Rabbit IgG FITC) .

• Control selection: Implementing appropriate controls is critical, including:

  • Samples from ASB17 knockout models as negative controls

  • Untreated versus LPS-treated samples to evaluate induction

  • Isotype controls to assess non-specific binding

• Detection method sensitivity: When monitoring LPS-mediated NF-κB activation, techniques that can detect subtle changes in phosphorylation states are essential, as ASB17 deficiency has been shown to decrease the phosphorylation of NF-κB p65 .

How can researchers address potential data inconsistencies when ASB17 antibodies yield contradictory results across different experimental platforms?

When confronted with contradictory results across experimental platforms, researchers should implement a systematic troubleshooting approach:

• Antibody validation verification: Confirm antibody specificity through:

  • Western blotting with positive and negative controls (including ASB17 knockout samples if available)

  • Peptide competition assays to verify epitope specificity

  • Testing multiple antibodies targeting different epitopes to corroborate findings

• Platform-specific optimization: Each detection method requires specific optimization:

  • For Western blotting: Transfer conditions, blocking reagents, and detection systems should be optimized specifically for ASB17's molecular weight and expression level

  • For immunohistochemistry: Antigen retrieval methods significantly impact epitope accessibility in fixed tissues

  • For immunofluorescence: Fixation and permeabilization protocols (4% paraformaldehyde fixation for 15 minutes, followed by 0.5% Triton X-100 permeabilization for 5 minutes) have been successfully employed for ASB17 visualization

• Post-translational modification awareness: ASB17's function as an E3 ubiquitin ligase and its involvement in ubiquitination pathways suggests potential post-translational modifications that may affect antibody recognition in different contexts .

• Cell type and stimulation conditions: ASB17 expression profiles vary significantly between cell types and activation states. Particularly, BMDCs show inducible expression following LPS stimulation, which may not be observable in unstimulated states .

What is the recommended protocol for optimizing immunofluorescence detection of ASB17 in primary immune cells?

Based on published methodologies, the following protocol is recommended for immunofluorescence detection of ASB17 in primary immune cells:

• Cell preparation and stimulation:

  • For BMDCs, stimulate with LPS to induce optimal ASB17 expression

  • Process cells at multiple time points to determine peak expression

• Fixation and permeabilization:

  • Wash cells three times with pre-cold PBS

  • Fix with 4% paraformaldehyde for 15 minutes

  • Permeabilize with PBS containing 0.5% Triton X-100 for 5 minutes

• Blocking and antibody incubation:

  • Block with PBS containing 5% bovine serum albumin (BSA) for 45 minutes at room temperature

  • Incubate with primary ASB17 antibody at 4°C overnight

  • Wash thoroughly and incubate with appropriate fluorophore-conjugated secondary antibody at room temperature for 2 hours

• Nuclear counterstaining and imaging:

  • Incubate with DAPI for 5 minutes at 37°C for nuclear visualization

  • Analyze using confocal laser scanning microscopy

• Controls and validation:

  • Include ASB17-deficient cells as negative controls

  • Perform parallel experiments with multiple ASB17 antibodies targeting different epitopes

  • For co-localization studies with TRAF6, optimize signal intensity to prevent bleed-through artifacts

How should researchers establish appropriate experimental designs to study ASB17's role in inflammatory signaling cascades?

To properly investigate ASB17's role in inflammatory signaling, experimental designs should incorporate:

• Genetic modulation approaches:

  • ASB17 knockout models have demonstrated impaired expression of pro-inflammatory cytokines including CCL2, IL-6, IL-1β, and IP-10 in BMDCs when stimulated with LPS

  • Complementary overexpression systems, such as stable THP-1 cell lines expressing ASB17, provide validation through gain-of-function approaches

• Protein interaction studies:

  • Co-immunoprecipitation assays to confirm ASB17-TRAF6 interaction

  • Domain mapping experiments have identified the aa177-250 segment of ASB17 as critical for TRAF6 binding

  • Controls using truncated protein variants can confirm specificity

• Ubiquitination analysis:

  • ASB17 inhibits K48-linked TRAF6 polyubiquitination, thereby stabilizing TRAF6

  • Ubiquitination assays should include controls for different ubiquitin linkage types to distinguish between degradative (K48) and non-degradative (K63) polyubiquitination

• Downstream signaling assessment:

  • Monitor NF-κB activation through multiple readouts:

    • Phosphorylation status of signaling components (especially NF-κB p65)

    • Nuclear translocation of transcription factors

    • Reporter gene assays

    • Cytokine production profiling (including CCL2, IL-6, IL-1β, and IP-10)

• Time-course analyses:

  • Include multiple time points to capture the kinetics of ASB17's effects on inflammatory signaling

  • Early (30 minutes to 2 hours) and late (6-24 hours) time points can reveal different aspects of ASB17's regulatory function

What are the technical considerations for investigating ASB17-mediated protein stability through ubiquitination assays?

Investigating ASB17's role in regulating TRAF6 stability through ubiquitination requires specialized technical approaches:

• Ubiquitination assay design:

  • Include appropriate controls for different ubiquitin linkage types (K48, K63, K11, etc.)

  • Utilize linkage-specific antibodies to distinguish between degradative (K48) and non-degradative modifications

  • ASB17 has been shown to inhibit K48-linked TRAF6 polyubiquitination, which has direct implications for protein stability

• Protein stability assessment:

  • Cycloheximide chase assays to measure TRAF6 half-life in the presence or absence of ASB17

  • Proteasome inhibitors (such as MG132) can be used to confirm the proteasome-dependent degradation pathway

  • Comparison between wild-type ASB17 and function-disrupting mutants (particularly in the aa177-250 interaction region) can confirm specificity

• In vitro reconstitution:

  • Purified components can be used to determine if ASB17 directly inhibits TRAF6 ubiquitination or requires additional cofactors

  • Control reactions with other E3 ligases can establish specificity

• Mass spectrometry approaches:

  • Targeted mass spectrometry can identify specific ubiquitination sites on TRAF6

  • Quantitative approaches can measure the abundance of different ubiquitin chain linkages

• Biological outcome correlation:

  • Correlate ubiquitination patterns with downstream functional outcomes:

    • NF-κB activation status

    • Inflammatory cytokine production

    • Cellular responses to LPS stimulation

  • This correlation is essential to establish the biological significance of the observed ubiquitination changes

How might ASB17 antibodies be utilized to explore potential therapeutic applications in inflammatory diseases?

The involvement of ASB17 in promoting LPS-induced NF-κB activation suggests potential therapeutic applications in inflammatory diseases. Research strategies might include:

• Target validation in disease models:

  • ASB17 knockout models have demonstrated reduced inflammatory responses to LPS stimulation

  • ASB17 antibodies can be used to assess protein expression in various inflammatory disease tissues

  • Correlation of ASB17 expression levels with disease severity may identify conditions where ASB17-targeted therapies could be beneficial

• Monitoring therapeutic efficacy:

  • ASB17 antibodies can serve as tools to monitor target engagement in preclinical models

  • Quantitative assessment of ASB17-TRAF6 complexes could provide pharmacodynamic biomarkers

  • Changes in downstream cytokine production (CCL2, IL-6, IL-1β, and IP-10) can be assessed as functional readouts

• Development of blocking strategies:

  • Structure-function studies using ASB17 antibodies targeting the critical aa177-250 TRAF6 interaction region could help design peptide mimetics or small molecules that disrupt this interaction

  • High-throughput screening platforms using ASB17-TRAF6 interaction assays could identify novel inhibitors

• Personalized medicine approaches:

  • ASB17 expression profiling in patient samples could identify inflammatory disease subsets particularly dependent on this pathway

  • Stratification of patients based on ASB17 expression or activity might predict response to targeted therapies

What are the technical challenges in studying ASB17 expression across different tissue and cell types?

Studying ASB17 expression across diverse biological contexts presents several technical challenges:

• Variable expression levels:

  • ASB17 shows tissue-specific expression patterns, with highest expression in testis

  • Expression in immune cells like BMDCs is typically lower but inducible by inflammatory stimuli such as LPS

  • Detection methods must be optimized for both high and low abundance scenarios

• Antibody sensitivity and specificity considerations:

  • For low-expression tissues, more sensitive detection methods may be required

  • Multiple antibodies targeting different epitopes should be used to confirm expression patterns

  • Western blotting may require enrichment steps for low-abundance samples

• Post-translational modification impact:

  • ASB17's involvement in ubiquitination pathways suggests it may itself be subject to post-translational modifications

  • These modifications could affect antibody recognition and should be considered when interpreting negative results

• Inducible expression dynamics:

  • As demonstrated in BMDCs, ASB17 expression can be significantly upregulated by LPS stimulation

  • Experimental designs must include appropriate stimulation conditions and time points to capture this dynamic expression

  • Unstimulated samples may yield false negative results in certain cell types

• Single-cell versus population analysis:

  • Population heterogeneity may mask ASB17 expression in specific cell subsets

  • Single-cell approaches (flow cytometry, single-cell RNA-seq) may be necessary to identify ASB17-expressing cell populations within complex tissues