ASB7 Antibody

What is ASB7 and why is it important in cellular research?

ASB7 is a member of the ankyrin repeat and SOCS box (ASB) family, characterized by a C-terminal SOCS box and an N-terminal ankyrin-related sequence of variable repeats. ASB7 functions as part of a Cullin 5-based E3 ubiquitin ligase complex that targets proteins like DDA3 for proteasomal degradation. This protein plays a critical role in regulating microtubule dynamics, spindle formation, and chromosome alignment during cell division . Research has shown that ASB7 depletion disrupts maturational progression and meiotic apparatus in mouse oocytes, causing abnormal spindle formation, misaligned chromosomes, and increased aneuploidy . Furthermore, significant reduction of ASB7 protein has been observed in oocytes from aged mice, suggesting its potential role in age-related fertility decline .

What detection techniques work best with ASB7 antibodies?

When selecting detection methods, consider that ASB7's interaction with microtubules and its role in cell division make immunofluorescence particularly valuable for studying its dynamic localization during mitosis or meiosis .

How should researchers validate ASB7 antibody specificity?

Antibody validation is critical for ensuring reliable results in ASB7 research. A multi-layered approach is recommended:

• Genetic knockdown validation: Comparing signal between control and ASB7-depleted samples is the gold standard. Western blotting should verify significant reduction of ASB7 protein in siRNA-injected samples, as demonstrated in previous studies where tubulin was used as a loading control .

• Peptide competition assay: Pre-incubate antibody with excess purified ASB7 peptide before application to samples. Specific binding should be blocked by the peptide.

• Cross-reactivity testing: Test antibody against related ASB family members to ensure specificity, particularly ASB9 which shares structural similarities.

• Multiple antibody verification: Use antibodies targeting different epitopes of ASB7 to confirm consistent localization patterns.

• Positive controls: Include samples with known ASB7 overexpression (e.g., FLAG-tagged ASB7 in HEK293T cells as used in previous studies) .

How can ASB7 antibodies be used to study ubiquitination pathways?

ASB7 antibodies can be powerful tools for investigating ubiquitination pathways through several sophisticated approaches:

• Co-immunoprecipitation studies: ASB7 antibodies can be used to pull down native protein complexes, followed by immunoblotting for ubiquitin or specific substrates like DDA3. Research has shown that ASB7 interacts selectively with Cullin 5 (Cul5) but not Cullin 2 (Cul2), forming a functional E3 ubiquitin ligase complex .

• Sequential immunoprecipitation: Perform first IP with ASB7 antibody, then a second IP with ubiquitin antibody to identify polyubiquitinated proteins in the ASB7 complex.

• Ubiquitination assays: Combine recombinant ASB7, E1, E2, and substrate (e.g., DDA3) with ubiquitin in vitro. ASB7 antibodies can help confirm components in the reaction and subsequent analysis.

• Domain-specific studies: Use antibodies targeting the SOCS box domain of ASB7 to study how this region mediates Cul5 recruitment. Previous studies have shown that mutant ASB7 lacking the SOCS box failed to interact with Cul5, confirming this domain's importance .

• Microtubule regulation analysis: ASB7 antibodies can reveal how microtubules affect DDA3 ubiquitination, as research has shown that addition of microtubules prevents polyubiquitination of DDA3 by ASB7 in a dose-dependent manner .

What approaches enable visualization of ASB7's role in spindle dynamics?

Visualizing ASB7's involvement in spindle dynamics requires specialized techniques:

• Dual immunofluorescence: Combining ASB7 antibodies with α-tubulin antibodies allows visualization of their spatial relationship during cell division. This approach has been used to demonstrate that ASB7 knockdown hinders spindle migration to cortical regions during oocyte maturation .

• Live-cell imaging: Complementing fixed-cell studies with GFP-tagged ASB7 enables real-time observation of protein dynamics during spindle formation.

• Super-resolution microscopy: Techniques like STED or STORM with appropriate ASB7 antibodies can resolve nanoscale localization patterns around the spindle apparatus.

• Proximity ligation assay: This technique can detect interactions between ASB7 and binding partners like DDA3 or Cul5 with spatial resolution in intact cells.

• Chromosome alignment analysis: Combined staining for ASB7, kinetochores, and chromosomes can quantify alignment defects. Previous studies measured metaphase plate width to determine chromosome misalignment, considering distances longer than 7 μm as misaligned .

Sample quantitative data from previous research:

How can protein-protein interactions of ASB7 be effectively studied?

Studying ASB7's interactions requires specialized techniques beyond basic antibody applications:

• Immunoprecipitation-mass spectrometry (IP-MS): ASB7 antibodies can be used to isolate protein complexes for mass spectrometric analysis. This approach successfully identified DDA3 peptides in FLAG-tagged ASB7 immunoprecipitates from HEK293T cells .

• Proximity-dependent biotin identification (BioID): Fusing ASB7 to a biotin ligase allows identification of proximal proteins in living cells, offering advantages over traditional IP for detecting transient interactions.

• FRET/BRET assays: These techniques can measure real-time interactions between ASB7 and potential binding partners in living cells.

• Structure-function analysis: Combining site-directed mutagenesis with co-IP and ASB7 antibodies can map interaction domains. Research has shown that the SOCS box domain is critical for Cul5 interaction .

• Microtubule cosedimentation assays: This technique can assess whether ASB7 directly binds to microtubules. Previous studies found that while DDA3 was present in both supernatant and microtubule pellet fractions, ASB7 and Cul5 were found only in the supernatant fraction, indicating ASB7 does not directly interact with microtubules .

Why might Western blots with ASB7 antibodies show inconsistent results?

When troubleshooting Western blot inconsistencies with ASB7 antibodies, consider these methodological solutions:

• Protein degradation: ASB7 is part of the ubiquitin-proteasome system and may be subject to rapid turnover. Include fresh protease inhibitors and deubiquitinase inhibitors (N-ethylmaleimide) in lysis buffers, and keep samples on ice.

• Cell cycle variability: ASB7 expression and localization change throughout the cell cycle, particularly during mitosis. Synchronize cells or sort populations based on cell cycle stage for consistent results.

• Antibody cross-reactivity: Some antibodies may cross-react with other ASB family members. Validate specificity with knockdown controls and blocking peptides.

• Sample preparation: ASB7's association with cytoskeletal elements may affect extraction efficiency. Compare different lysis buffers (RIPA vs. NP-40) and include cytoskeletal disrupting agents like nocodazole when appropriate.

• Loading controls: Standard loading controls like tubulin may be affected in experiments manipulating microtubule dynamics. Consider alternative loading controls like GAPDH or total protein staining.

What considerations are important when studying ASB7 in disease models?

When investigating ASB7 in disease contexts, researchers should address these methodological considerations:

• Tissue-specific expression: ASB7 expression varies across tissues. Antibody concentration and detection methods may need optimization for each tissue type.

• Pathological samples: Formalin-fixed samples may require antigen retrieval optimization for ASB7 detection. Test multiple methods (heat-induced vs. enzymatic) to determine optimal conditions.

• Quantification approaches: For comparing ASB7 levels between normal and pathological samples, use quantitative methods such as:

  • Densitometry with appropriate normalization

  • Fluorescence intensity measurements

  • Automated image analysis algorithms

• Context-dependent interactions: ASB7's binding partners may differ in disease states. Use unbiased approaches like IP-MS to identify context-specific interactors.

• Aging effects: ASB7 protein levels are significantly reduced in oocytes from aged mice, suggesting potential relevance to age-related pathologies . When studying age-related conditions, include age-matched controls and consider how ASB7 levels change with aging.

What protocols are recommended for studying ASB7 in oocyte meiosis?

Studying ASB7 in oocyte meiosis requires specialized approaches:

• Oocyte collection and culture: Fully grown GV oocytes should be collected from ovaries and cultured in M2 medium containing milrinone to prevent spontaneous maturation during microinjection procedures .

• Gene knockdown approach: For ASB7 depletion studies, microinject specifically designed ASB7 siRNAs into GV oocytes. After injection, maintain oocytes in milrinone-containing medium for 20 hours to allow sufficient time for mRNA degradation before inducing maturation .

• Phenotype assessment: Evaluate the following parameters after ASB7 knockdown:

  • GVBD (germinal vesicle breakdown) rate

  • Polar body extrusion rate

  • Incidence of symmetric division

  • Spindle morphology and chromosome alignment

  • Distance between spindle pole and plasma membrane

• Rescue experiments: To confirm specificity of knockdown phenotypes, co-inject siRNA-resistant ASB7 mRNA. Previous studies successfully established cell lines stably expressing siRNA-resistant ASB7 (wild-type or ΔSOCS box mutant) for rescue experiments .

• Aging studies: When investigating age-related effects, compare oocytes from young (6-8 weeks) and aged (42-45 weeks) female mice. Consider that increasing ASB7 expression via mRNA injection has been shown to partially rescue maternal age-induced meiotic defects .

How can machine learning approaches enhance ASB7 antibody-based research?

Machine learning can significantly advance ASB7 antibody research through:

• Active learning for optimizing experimental design: Recent research has shown that active learning strategies can improve experimental efficiency by up to 35% compared to random sampling approaches . For ASB7 antibody characterization, this could mean:

  • Prioritizing key epitopes for antibody development

  • Identifying optimal combinations of antibodies for multiplexed detection

  • Predicting cross-reactivity with other ASB family members

• Image analysis automation: Machine learning algorithms can analyze immunofluorescence images to:

  • Quantify ASB7 colocalization with microtubules or chromosomes

  • Measure spindle morphology and chromosome alignment defects

  • Track dynamic changes in ASB7 localization during cell division

• Protein-protein interaction prediction: Computational models trained on known interactions can predict potential ASB7 binding partners beyond the experimentally validated ones like DDA3 and Cul5.

• Out-of-distribution prediction challenges: As with antibody-antigen binding prediction, ASB7 interaction studies face challenges when predicting interactions with proteins not represented in training data . Approaches like those used in the Absolut! simulation framework could help address these limitations.

How can ASB7 antibodies be used in chromatin-associated studies?

Though ASB7 is primarily known for cytoplasmic functions, emerging evidence suggests potential nuclear roles that can be investigated using:

• Chromatin immunoprecipitation (ChIP): Modified protocols with optimized nuclear extraction can determine if ASB7 associates with chromatin during specific cell cycle phases.

• Proximity-based approaches: BioID or APEX2 fusions to ASB7 can identify chromatin-associated proteins that interact with ASB7 during mitosis when the nuclear envelope breaks down.

• Live-cell chromatin imaging: Combining fluorescently tagged ASB7 with chromatin markers enables real-time visualization of potential interactions during cell division.

• Chromosome misalignment analysis: ASB7 antibodies coupled with DNA stains can quantify chromosome alignment defects. In previous studies, knockdown of ASB7 significantly increased the proportion of cells with misaligned chromosomes (by 25–30%) relative to control cells .

• Kinetochore-microtubule interaction studies: ASB7 knockdown impairs kinetochore–microtubule interaction and provokes the spindle assembly checkpoint during oocyte meiosis . Combined immunostaining for ASB7, kinetochores, and microtubules can reveal the molecular basis of this phenotype.

What are the latest innovations in ASB7 antibody development for research?

Cutting-edge approaches for ASB7 antibody development include:

• Epitope-specific recombinant antibodies: Developing antibodies against specific domains (ankyrin repeats vs. SOCS box) enables more precise functional studies of ASB7.

• Single-domain antibodies (nanobodies): These smaller antibody fragments offer advantages for live-cell imaging and intracellular applications to track ASB7 dynamics.

• Bifunctional antibody conjugates: Antibodies linked to degradation-inducing moieties (e.g., PROTAC technology) can enable acute depletion of ASB7 for temporal studies of function.

• Conformation-specific antibodies: Developing antibodies that recognize specific ASB7 conformational states could reveal activity-dependent changes during cell cycle progression.

• Computationally designed antibodies: Machine learning approaches like those used for antibody-antigen binding prediction could be applied to develop antibodies with enhanced specificity for ASB7 over other ASB family members.