ASB11 Antibody

What is the biological function of ASB11?

ASB11 functions as a substrate-recognition component of a SCF-like ECS (Elongin-Cullin-SOCS-box protein) E3 ubiquitin-protein ligase complex. This complex mediates the ubiquitination and subsequent proteasomal degradation of target proteins . ASB11 has been specifically identified as a novel endoplasmic reticulum-associated ubiquitin ligase with the ability to interact and promote the ubiquitination of Ribophorin 1, an integral protein of the oligosaccharyltransferase (OST) glycosylation complex . Expression studies demonstrate that ASB11 is present in multiple tissues including muscle, heart, eye, intestine, gills, testis, pancreas, and brain, indicating functionality beyond the nervous system .

What are the recommended applications for ASB11 antibodies?

Based on commercially available options, ASB11 antibodies have been validated for several applications:

When selecting an antibody, researchers should verify that it has been specifically validated for their intended application and species of interest .

What sample types can be used with ASB11 antibodies?

ASB11 antibodies have been validated for detection in multiple sample types:

• Cell culture supernatant

• Plasma

• Serum

• Tissue homogenate

For optimal results, sample preparation should follow manufacturer recommendations for the specific antibody and application being used. For tissue samples, proper fixation and antigen retrieval methods may be necessary to ensure specific signal detection.

How should I design experiments to study ASB11 expression patterns?

When designing experiments to study ASB11 expression patterns, consider using a multi-method approach:

• Transcript-level analysis: Use reverse transcription-PCR as demonstrated in zebrafish studies where d-asb11 expression was characterized across multiple tissues . This approach allows for sensitive detection of gene expression.

• Protein-level analysis: Implement Western blotting using validated ASB11 antibodies with appropriate positive and negative controls.

• Cellular localization: For tissue or cell localization studies, immunohistochemistry or immunofluorescence can be used with careful consideration of antibody specificity. Co-localization studies with known markers can provide additional validation of expression patterns.

• Quantification methods: For precise quantification, consider ELISA-based methods with standard curves using recombinant proteins .

For developmental studies, temporal analysis at different stages is essential as ASB11 has demonstrated roles in embryonic development and regenerative processes .

What controls should I include when using ASB11 antibodies?

Robust experimental design requires appropriate controls:

• Positive tissue controls: Include tissues known to express ASB11 (e.g., brain, muscle, heart) .

• Negative controls:

  • Omission of primary antibody

  • Non-expressing tissues/cells

  • Isotype controls to assess non-specific binding

• Peptide competition assays: Pre-incubation of the antibody with the immunizing peptide should abolish specific signals .

• Knockdown validation: If possible, include ASB11 knockdown samples (siRNA, shRNA) to demonstrate antibody specificity.

• Overexpression validation: Samples with overexpressed ASB11 can serve as positive controls, especially when using epitope-tagged versions for co-detection .

How can I optimize immunoprecipitation experiments with ASB11 antibodies?

For successful immunoprecipitation of ASB11 and its interaction partners:

• Cell lysis conditions: Use buffers containing 1% NP-40 or CHAPS to maintain protein interactions while ensuring efficient extraction.

• Pre-clearing: Remove non-specific binding proteins by pre-clearing lysates with protein A/G beads.

• Antibody amount optimization: Titrate antibody concentration to determine optimal amounts (typically 1-5 μg per mg of total protein).

• Cross-linking consideration: For stable complexes, consider cross-linking the antibody to beads to prevent co-elution.

• Washing stringency: Balance between removing non-specific interactions and maintaining specific ones by testing different wash buffer compositions.

• Elution methods: Compare different elution methods (pH, competitive peptide elution) based on downstream applications.

• Validation by mass spectrometry: Follow protocols like those described in the ASB family-wide proteomic screen to identify interaction partners .

How can I investigate the ubiquitination activity of ASB11 on target proteins?

To investigate ASB11-mediated ubiquitination:

• In vitro ubiquitination assays: Reconstitute the ubiquitination reaction using:

  • Purified E1 ubiquitin-activating enzyme

  • Appropriate E2 conjugating enzyme

  • Recombinant ASB11 within its E3 ligase complex

  • Potential substrate (e.g., Ribophorin 1)

  • Ubiquitin (consider using tagged versions for easier detection)

  • ATP regeneration system

• Cell-based ubiquitination assays:

  • Co-express ASB11 with epitope-tagged ubiquitin and potential substrates

  • Use proteasome inhibitors (MG132) to prevent degradation of ubiquitinated proteins

  • Immunoprecipitate the substrate and detect ubiquitin modifications by Western blot

  • Alternative approach: immunoprecipitate ubiquitinated proteins and detect the substrate

• Protein turnover assays: As demonstrated with Ribophorin 1, expression of ASB11 can increase target protein turnover in vivo . Design cycloheximide chase experiments to compare protein half-life in the presence and absence of ASB11.

• Proximity ligation assays (PLA): This technique can detect protein interactions in situ, as described in studies with ASB11 and Ribophorin 1 using U2OS cells .

What approaches can be used to study ASB11's role in muscle regeneration and development?

Given ASB11's expression in muscle tissue and potential role in regeneration :

• Developmental studies:

  • Use timed sample collection during embryonic development

  • Implement lineage tracing with ASB11 as a marker

  • Analyze co-expression with muscle developmental markers

• Regeneration models:

  • Induce muscle injury (cardiotoxin, freeze injury) in model organisms

  • Monitor ASB11 expression during different phases of regeneration

  • Assess satellite cell activation and proliferation in relation to ASB11 expression

• Cell culture models:

  • Use C2C12 or primary myoblast cultures to study ASB11 during differentiation

  • Implement knockdown and overexpression approaches

  • Analyze fusion index, differentiation markers, and proliferation

• Co-localization with satellite cell markers:

  • Perform immunofluorescence studies using ASB11 antibodies with Pax7 (satellite cell marker) as described in previous research

  • Quantify BrdU incorporation to assess proliferation rates

How can I address contradictory data when studying ASB11 expression or function?

When facing contradictory results:

• Antibody validation evaluation:

  • Verify antibody specificity through multiple methods

  • Consider using different antibody clones targeting distinct epitopes

  • Compare results with transcript-level data (qPCR, RNA-seq)

• Context-dependent expression analysis:

  • Evaluate different developmental stages, as ASB11 shows distinct expression patterns during development

  • Consider tissue-specific or cell type-specific variation

  • Assess potential isoform differences that might be recognized differently by antibodies

• Functional redundancy assessment:

  • Examine potential compensation by related ASB family members

  • Note that zebrafish Asb11 shows homology with both mammalian ASB9 and ASB11, suggesting potential functional overlap

• Cross-species validation:

  • ASB proteins show high pan-chordate conservation (50% similarity between human ASB11 and its orthologue in Ciona intestinalis)

  • Compare findings across model organisms while considering evolutionary differences

• Technical approach diversification:

  • Employ multiple techniques (Western blot, immunofluorescence, mass spectrometry)

  • Use genetic approaches (CRISPR, siRNA) alongside antibody-based methods

What are common pitfalls when using ASB11 antibodies and how can they be addressed?

Common challenges with ASB11 antibody applications include:

• Non-specific binding:

  • Optimize blocking conditions (use alternative blockers like 2% goat serum, 1% BSA, and 1% DMSO as described in protocols)

  • Increase washing stringency

  • Verify antibody dilution (start with manufacturer recommendations, then optimize)

  • Pre-absorb antibody with non-specific proteins (fish powder for zebrafish studies)

• Low signal strength:

  • Optimize antigen retrieval methods for fixed samples

  • Adjust antibody concentration

  • Extend incubation time (overnight at 4°C is often recommended)

  • Explore signal amplification methods

• Background issues:

  • Include appropriate blocking of endogenous peroxidases for IHC

  • Test different detection systems

  • Use highly purified antibody preparations

  • Consider specialized blocking for certain tissues (mouse-on-mouse blocking for mouse tissues)

• Batch-to-batch variability:

  • Request data on lot-specific validation

  • Test new lots against previous successful lots

  • Maintain consistent experimental conditions

What are the best practices for storing and handling ASB11 antibodies?

For optimal antibody performance:

• Storage conditions:

  • Store according to manufacturer recommendations, typically:

    • Short-term (up to 2 weeks): 2-8°C

    • Long-term: -20°C in small aliquots to prevent freeze-thaw cycles

• Aliquoting strategy:

  • Create single-use aliquots upon receipt

  • Document date and number of freeze-thaw cycles

  • Avoid repeated freeze-thaw cycles

• Working dilution preparation:

  • Prepare fresh working dilutions for each experiment

  • Use appropriate diluent buffers with stabilizers

  • Consider carrier proteins for very dilute solutions

• Quality monitoring:

  • Include positive controls in each experiment to monitor antibody performance over time

  • Document lot numbers and performance characteristics

How can I validate the specificity of an ASB11 antibody for my experimental system?

A comprehensive validation strategy includes:

• Western blot analysis:

  • Verify single band at expected molecular weight (approximately 35.4 kDa for human ASB11)

  • Compare with recombinant protein standard

  • Assess samples with known differential expression

  • Include knockout/knockdown controls if available

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide

  • Signal should be significantly reduced or eliminated

• Cross-species reactivity assessment:

  • Test antibody against target protein from different species

  • Consider sequence homology in epitope regions

• Orthogonal validation:

  • Correlate protein detection with mRNA expression data

  • Use multiple antibodies targeting different epitopes

  • Compare with tagged overexpression systems

• Application-specific validation:

  • For immunohistochemistry/immunofluorescence: assess cellular/subcellular localization patterns

  • For ELISA: evaluate linearity, recovery, and precision

  • For immunoprecipitation: verify enrichment of target protein

How can ASB11 antibodies be used to investigate neural development pathways?

ASB11 has demonstrated roles in neural development, particularly in regulating the size of the neural progenitor pool :

• Developmental time course analysis:

  • Track ASB11 expression during neural development stages

  • Correlate with neurogenesis markers (sox2, sox3, neurogenin1, HuC)

  • Implement spatial analysis across neural tube regions

• Cell fate determination studies:

  • Examine co-localization with progenitor vs. differentiated neuron markers

  • Analyze expression in relation to cell cycle exit markers

  • Study expression in neurogenic vs. gliogenic phases

• Signaling pathway integration:

  • Investigate ASB11 regulation by neural development pathways

  • Analyze co-immunoprecipitation with signaling components

  • Study post-translational modifications of ASB11 during development

• Gain/loss-of-function approach:

  • Correlate antibody detection with phenotypic outcomes

  • Compare results with studies showing premature neuronal differentiation upon ASB11 knockdown

What are emerging techniques for studying ASB11 protein interactions beyond traditional methods?

Advanced methods for protein interaction studies include:

• BioID or TurboID proximity labeling:

  • Fuse ASB11 to a biotin ligase

  • Allow in vivo biotinylation of proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

  • Provides spatial context to interactions

• APEX2 proximity labeling:

  • Similar to BioID but with faster kinetics

  • Particularly useful for capturing transient interactions

• Förster resonance energy transfer (FRET):

  • Label ASB11 and potential interacting proteins with appropriate fluorophores

  • Detect energy transfer as indicator of close proximity

  • Can be performed in living cells

• Single-molecule pull-down (SiMPull):

  • Combine immunoprecipitation with single-molecule fluorescence imaging

  • Allows determination of complex stoichiometry

  • Can detect heterogeneity in protein complexes

• Cryo-electron microscopy:

  • Obtain structural information about ASB11-containing complexes

  • Particularly relevant for understanding E3 ligase complex architecture

How might ASB11 antibodies contribute to understanding diseases related to protein degradation pathways?

ASB11's role in ubiquitination links it to protein degradation pathways relevant to disease:

• Neurodegenerative disease research:

  • Study ASB11 expression in models of neurodegeneration

  • Investigate potential roles in protein quality control

  • Assess interactions with disease-relevant proteins

• Cancer biology applications:

  • Analyze ASB11 expression in tumor samples

  • Correlate with ubiquitination patterns of known oncoproteins

  • Examine potential roles in cell proliferation regulation

• Developmental disorders:

  • Investigate ASB11 in conditions with aberrant cell differentiation

  • Study potential links to congenital disorders affecting tissues where ASB11 is expressed

• Therapeutic target identification:

  • Use antibodies to screen compounds that modulate ASB11 activity

  • Develop tools for monitoring pharmacodynamic responses

  • Establish biomarker potential for related pathways