ASB6 Antibody

What is ASB6 and what cellular functions has it been associated with?

ASB6 (ankyrin repeat and SOCS box-containing 6) belongs to a family of ankyrin repeat proteins that contain a C-terminal SOCS box motif. Growing evidence suggests that the SOCS box acts as a bridge between specific substrate-binding domains and the more generic proteins that comprise a large family of E3 ubiquitin protein ligases . ASB6 has been identified as part of the CUL5-ASB6 complex that promotes p62/SQSTM1 ubiquitination and degradation, thereby regulating cell proliferation and autophagy . Recent research has also implicated ASB6 as an independent prognostic biomarker for colorectal cancer progression, with involvement in lymphatic invasion and immune infiltration .

What are the key specifications of available ASB6 antibodies?

ASB6 antibodies are typically available as rabbit polyclonal antibodies that target human ASB6 protein. The specifications typically include:

The antibodies are generally purified using antigen affinity chromatography methods .

What are the recommended storage conditions for ASB6 antibodies?

To maintain optimal activity of ASB6 antibodies, researchers should:

• Store the antibody at -20°C

• Ensure stability for one year after shipment when properly stored

• For liquid formulations, aliquoting is unnecessary for -20°C storage

• For lyophilized antibodies, reconstitute in PBS buffer with 2% sucrose to a final concentration of 1 mg/mL

• Avoid multiple freeze-thaw cycles to prevent degradation

• Some formulations may contain 0.02% sodium azide and 50% glycerol (pH 7.3) as preservatives

What are the recommended dilutions and protocols for using ASB6 antibodies in Western Blot experiments?

For optimal results in Western Blot applications using ASB6 antibodies, researchers should:

• Use a dilution range of 1:500-1:1000 for standard Western Blot applications

• Titrate the antibody in each testing system to obtain optimal signal-to-noise ratio

• Be aware that optimal dilutions may be sample-dependent

• When detecting endogenous ASB6, positive signals have been confirmed in L02 cells and HEK-293 cells

• Following manufacturer-specific protocols is recommended for consistent results

It is essential to include appropriate positive and negative controls to validate specificity, and to optimize blocking conditions to minimize background signal.

How can researchers validate the specificity of ASB6 antibodies in their experimental systems?

Validating antibody specificity is crucial for reliable research outcomes. For ASB6 antibodies, consider these methodological approaches:

• Positive and negative controls: Use cells known to express ASB6 (e.g., L02 cells, HEK-293 cells) as positive controls, and cells with low/no expression as negative controls

• Knockdown/Knockout validation: Utilize siRNA knockdown or CRISPR-Cas9 knockout of ASB6 in your experimental system to confirm antibody specificity

• Multiple antibody approach: Compare results using multiple antibodies targeting different epitopes of ASB6

• Epitope blocking: Pre-incubate the antibody with the immunizing peptide to confirm binding specificity

• Molecular weight verification: Confirm that the detected band corresponds to the expected molecular weight of ASB6 (approximately 47 kDa)

Published validation data should be consulted when available, as referenced in the antibody product information.

What factors should be considered when designing experiments to study ASB6's role in the ubiquitin-proteasome pathway?

When investigating ASB6's function in the ubiquitin-proteasome pathway, researchers should consider:

• Complex formation analysis: Design co-immunoprecipitation experiments to study the interaction between ASB6 and CUL5, as they form a complex that promotes p62/SQSTM1 ubiquitination

• Ubiquitination assays: Implement in vitro and in vivo ubiquitination assays to assess ASB6's role in protein ubiquitination

• Proteasome inhibition: Include proteasome inhibitors (e.g., MG132) to distinguish between ubiquitination and degradation effects

• Substrate identification: Use mass spectrometry-based approaches to identify novel substrates of the ASB6-containing E3 ligase complex

• Domain mutation analysis: Create mutants with altered SOCS box or ankyrin repeat domains to define functional regions essential for ubiquitin ligase activity

• Cellular context: Evaluate ASB6 function across multiple cell types, as its role may vary depending on the cellular context

These methodological considerations will help establish a comprehensive understanding of ASB6's role in the ubiquitin-proteasome system.

How does the CUL5-ASB6 complex regulate p62/SQSTM1 ubiquitination and autophagy?

The CUL5-ASB6 complex functions as an E3 ubiquitin ligase that specifically targets p62/SQSTM1 for ubiquitination and subsequent degradation, which has significant implications for both cell proliferation and autophagy regulation .

To investigate this mechanism:

• Proximity ligation assays can be used to confirm the in situ interaction between ASB6, CUL5, and p62/SQSTM1

• Domain mapping experiments help identify specific binding interfaces between complex components

• Ubiquitination site analysis through mass spectrometry can pinpoint the exact lysine residues on p62/SQSTM1 that are ubiquitinated by this complex

• Autophagy flux assays using LC3-II/I ratios and p62 accumulation measurements reveal the functional impact on autophagic processes

• Cell proliferation studies with ASB6 knockdown/overexpression can demonstrate the biological significance of this regulatory mechanism

Understanding this pathway provides insights into how cells coordinate protein degradation systems and may offer potential therapeutic targets for diseases with dysregulated autophagy.

What methodological approaches can effectively assess ASB6's role as a prognostic biomarker in colorectal cancer?

Given ASB6's potential as an independent prognostic biomarker for colorectal cancer progression , researchers should consider these methodological approaches:

• Tissue microarray analysis: Evaluate ASB6 expression across large cohorts of colorectal cancer samples and matched normal tissues

• Multiplex immunohistochemistry: Assess ASB6 expression in relation to immune cell infiltration markers to understand the tumor microenvironment

• Survival analysis: Apply Kaplan-Meier curves and Cox regression models to correlate ASB6 expression levels with patient outcomes

• Lymphatic invasion assessment: Implement lymphatic vessel-specific staining alongside ASB6 to examine correlations with lymphatic invasion

• Functional studies: Use in vitro and in vivo models to investigate the mechanistic link between ASB6 expression and tumor progression

• Multivariate analysis: Include established prognostic markers to determine if ASB6 provides independent prognostic information

These approaches enable comprehensive evaluation of ASB6's clinical utility as a prognostic biomarker in colorectal cancer, potentially identifying patient subgroups that might benefit from targeted therapies.

How can researchers design experiments to investigate potential ASB6 interaction partners beyond known associations?

To discover novel protein interactions with ASB6, researchers can employ these methodological strategies:

• Proximity-dependent biotin identification (BioID): Fuse ASB6 to a biotin ligase to identify proteins in close proximity within the cellular environment

• Affinity purification coupled with mass spectrometry (AP-MS): Use tagged ASB6 to pull down interaction partners and identify them through mass spectrometry

• Yeast two-hybrid screening: Screen cDNA libraries to identify direct protein-protein interactions with ASB6

• Co-immunoprecipitation followed by proteomics: Immunoprecipitate endogenous ASB6 and identify co-precipitating proteins

• Domain-specific interaction mapping: Create truncated variants of ASB6 to map which domains are responsible for specific protein interactions

• In silico prediction and validation: Use computational tools to predict potential interactions based on structural homology, followed by experimental validation

Validating interactions through multiple complementary methods increases confidence in the results and helps establish functional significance.

How can researchers address common technical challenges when working with ASB6 antibodies?

When using ASB6 antibodies, researchers may encounter several technical challenges. Here are methodological solutions:

• High background in Western blot:

  • Increase blocking time or concentration (5% BSA or milk)

  • Try alternative blocking agents

  • Decrease primary antibody concentration (try 1:1000 instead of 1:500)

  • Include 0.1% Tween-20 in wash buffers and increase washing frequency

• Weak or no signal:

  • Confirm ASB6 expression in your sample (use positive control cells like L02 or HEK-293)

  • Increase protein loading amount

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use enhanced chemiluminescence detection systems

• Multiple bands:

  • Validate bands using knockout/knockdown controls

  • Optimize SDS-PAGE conditions for better separation

  • Confirm expected molecular weight (47 kDa for ASB6)

  • Consider potential post-translational modifications or splice variants

• Antibody cross-reactivity:

  • Use more stringent washing conditions

  • Perform pre-adsorption with related proteins

  • Test alternative ASB6 antibodies targeting different epitopes

What are the considerations for selecting the appropriate immunogen when generating ASB6 antibodies?

When selecting or evaluating immunogens for ASB6 antibody generation, researchers should consider:

• Epitope uniqueness: Select regions of ASB6 with minimal homology to related proteins, especially other ASB family members, to reduce cross-reactivity

• Protein structure awareness: Avoid regions buried within the protein structure; prioritize surface-exposed regions

• Post-translational modification sites: Consider whether the antibody should recognize specific post-translationally modified forms of ASB6

• Fusion protein design: When using fusion proteins as immunogens (as in product 21449-1-AP), ensure the fusion partner doesn't interfere with antibody specificity

• Synthetic peptide alternatives: Consider synthetic peptides from unique regions of ASB6 as alternative immunogens

• Species conservation: For cross-species reactivity, select epitopes conserved across species of interest

Current commercial ASB6 antibodies use fusion proteins as immunogens, such as ASB6 fusion protein Ag13863, which has demonstrated good specificity for human samples .

How should researchers interpret variations in ASB6 expression across different tissue and cell types?

When analyzing ASB6 expression patterns, consider these methodological principles:

• Establish baseline expression levels: Determine ASB6 expression in normal tissues to establish reference points before comparing diseased states

• Quantitative analysis: Use quantitative Western blot or qRT-PCR with appropriate housekeeping genes for normalization

• Single-cell analysis: Consider single-cell RNA-seq to identify cell-type specific expression patterns that might be masked in bulk tissue analysis

• Subcellular localization: Assess whether ASB6 shows different subcellular distributions across tissues using fractionation or immunofluorescence

• Correlation with function: Interpret expression differences in the context of tissue-specific functions, particularly regarding ubiquitination activity

• Disease context interpretation: In cancer studies, interpret ASB6 expression changes in relation to clinical parameters such as stage, grade, and patient outcomes

These approaches provide a framework for robust interpretation of ASB6 expression data across different experimental contexts.

What statistical approaches are most appropriate for analyzing ASB6 expression data in cancer biomarker studies?

For rigorous analysis of ASB6 as a potential cancer biomarker, researchers should employ these statistical methods:

• Survival analysis:

  • Kaplan-Meier curves with log-rank tests to compare survival outcomes between ASB6-high and ASB6-low expression groups

  • Cox proportional hazards models to assess ASB6 as an independent prognostic factor while controlling for clinical covariates

• Expression comparison:

  • Paired t-tests or Wilcoxon signed-rank tests for comparing ASB6 expression between matched tumor and normal samples

  • ANOVA or Kruskal-Wallis tests for comparing expression across multiple cancer stages or subtypes

• Biomarker performance metrics:

  • ROC curve analysis to determine sensitivity and specificity of ASB6 as a diagnostic or prognostic marker

  • Calculation of positive and negative predictive values in the context of the specific cancer population

• Multivariate analysis:

  • Principal component analysis or clustering to identify patterns of expression with other markers

  • Multiple regression models to evaluate ASB6's contribution to predictive models

• Meta-analysis approaches:

  • Forest plots and random effects models when combining data from multiple studies

  • Publication bias assessment using funnel plots

These statistical approaches provide robust frameworks for evaluating ASB6's potential as a biomarker in colorectal cancer and other malignancies .

How can researchers evaluate antibody quality when published validation data is limited?

When faced with limited published validation data for ASB6 antibodies, researchers should implement these methodological approaches:

• In-house validation protocols:

  • Perform Western blots on positive control samples (e.g., L02 cells, HEK-293 cells) to confirm band size at 47 kDa

  • Include negative controls lacking ASB6 expression

  • Conduct siRNA knockdown experiments to verify signal reduction

• Cross-antibody validation:

  • Compare results using multiple antibodies targeting different epitopes of ASB6

  • Correlate protein expression results with mRNA expression data

• Orthogonal technique comparison:

  • Verify protein expression using mass spectrometry-based proteomics

  • Compare antibody-based detection with genetic reporter systems

• Application-specific validation:

  • For each application (WB, IHC, ICC), perform application-specific controls

  • Determine optimal conditions through titration experiments

• Batch testing and consistency:

  • Test antibody performance across different lots

  • Establish reproducible standard operating procedures

These approaches help researchers independently validate antibodies when published data is insufficient, ensuring reliable experimental results .

What are promising areas for future research on ASB6's role in cancer progression and immune infiltration?

Based on recent findings linking ASB6 to colorectal cancer progression and immune infiltration , several promising research directions emerge:

• Mechanistic studies: Investigate the molecular mechanisms by which ASB6 influences lymphatic invasion in colorectal cancer

• Immune microenvironment: Characterize how ASB6 expression affects tumor-infiltrating immune cell populations and their functional states

• Therapeutic targeting: Develop and test small molecule inhibitors or degraders targeting the ASB6-CUL5 complex

• Biomarker development: Validate ASB6 as part of multi-marker panels for improved prognostic accuracy in colorectal cancer

• Resistance mechanisms: Explore whether ASB6 expression correlates with resistance to standard therapies in colorectal cancer

• Broader cancer relevance: Extend studies to other cancer types to determine if ASB6's prognostic value extends beyond colorectal cancer

These research directions could significantly advance our understanding of ASB6's biological functions and its potential as a therapeutic target.

How might advanced antibody engineering approaches improve ASB6 antibody performance in research applications?

Advanced antibody engineering techniques offer opportunities to enhance ASB6 antibody performance:

• Recombinant antibody production: Develop recombinant ASB6 antibodies with defined epitope binding to improve batch-to-batch consistency over traditional polyclonal antibodies

• Fragment-based approaches: Engineer Fab or scFv fragments for applications requiring better tissue penetration or reduced background

• Phage display selection: Utilize phage display to select high-affinity antibodies against specific epitopes of ASB6

• Epitope-focused design: Design antibodies targeting functionally significant domains of ASB6 (e.g., SOCS box or ankyrin repeat domains)

• Deep learning approaches: Apply deep learning algorithms to optimize antibody sequence design, as demonstrated in recent research on antibody generation

In a recent study, researchers used deep learning to generate antibody sequences with low redundancy and improved developability characteristics. This approach showed that in-silico generated antibodies exhibited comparable biophysical properties to marketed antibodies, suggesting similar techniques could be applied to develop improved ASB6 antibodies .