CTNNBL1 Antibody

What is CTNNBL1 and what experimental methods are most appropriate for detecting its expression in different cell types?

CTNNBL1 (Catenin Beta Like 1) is a widely expressed nuclear protein containing armadillo (ARM) motifs that functions as a novel nuclear localization sequence (NLS)-binding protein. It associates with the Prp19-CDC5L complex of the spliceosome and interacts with the antibody-diversifying enzyme AID .

Methodological approach:

• Western blotting: CTNNBL1 is detected at approximately 65 kDa. For optimal results, use protein lysates from tissues with high expression (skeletal muscle, placenta, heart, spleen, testis, thyroid) . Recommended dilutions range from 1:500-1:2400 .

• Immunohistochemistry: Effective for tissue sections with dilutions of 1:20-1:200; nuclear staining pattern is expected . Antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0 is recommended for optimal results .

• Immunofluorescence: Starting concentration of 20 μg/mL works well for nuclear visualization in most cell types .

How can researchers optimize co-immunoprecipitation protocols to study CTNNBL1 protein-protein interactions?

CTNNBL1 has been shown to interact with multiple proteins including components of the Prp19-CDC5L complex, Prp31, and AID, making co-immunoprecipitation a valuable technique for studying these interactions.

Methodological approach:

• Pre-clearing step: Incubate cell lysates with protein A/G beads for 1 hour at 4°C to reduce nonspecific binding.

• Antibody selection: For pull-downs, both monoclonal and polyclonal antibodies have shown efficacy, with monoclonals providing higher specificity .

• Buffer optimization: Use buffer D (composition described in search result 1) for initial lysis, which preserves CTNNBL1 interactions .

• Detection of interacting partners: When immunoprecipitating FLAG-tagged CTNNBL1 from transfected cells, components of the Prp19-CDC5L complex (CDC5L, Prp19, PLRG1) and other splicing factors like Prp31 can be detected as associated proteins .

• Crosslinking consideration: Mild formaldehyde crosslinking (0.1%) may help preserve transient interactions without disrupting antibody recognition sites.

What are the experimental contradictions regarding CTNNBL1's role in antibody diversification, and how can antibodies help resolve these discrepancies?

The research literature shows contradicting findings regarding CTNNBL1's role in antibody diversification processes:

Contradictory findings:

Methodological approaches to resolve contradictions:

• Comparative immunoprecipitation: Use CTNNBL1 antibodies to perform comparative IP-MS across different cell types (human, mouse, chicken) to identify potential species-specific interaction partners that might explain functional differences.

• Domain-specific antibodies: Develop antibodies targeting specific domains of CTNNBL1 to map which regions interact with AID and other factors in different cellular contexts.

• ChIP-seq experiments: Use CTNNBL1 antibodies for chromatin immunoprecipitation followed by sequencing to determine if CTNNBL1 associates with chromatin at immunoglobulin loci in different cell types.

• Proximity ligation assays: Combine CTNNBL1 and AID antibodies in proximity ligation assays to quantitatively assess their interaction in situ under different conditions.

How can researchers validate CTNNBL1 antibody specificity for their experimental systems?

Ensuring antibody specificity is critical for meaningful research outcomes, especially with proteins that have multiple isoforms or close homologs.

Methodological approach:

• Knockout/knockdown controls: The most definitive validation approach is using CTNNBL1-deficient cells created by gene targeting, as described in the literature where exons 7-10 were deleted .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide before application to demonstrate signal specificity.

• Multiple antibody verification: Use antibodies raised against different epitopes of CTNNBL1. The literature shows antibodies targeting N-terminal regions (aa 1-50) and C-terminal regions (aa 350 to C-terminus) are both effective.

• Cross-reactivity testing: Test against samples from multiple species - human and mouse CTNNBL1 show high homology and both are detected by many commercial antibodies .

• Isoform consideration: CTNNBL1 has at least four known isoforms; verify whether your antibody detects all isoforms or is specific to particular variants .

What are the optimal conditions for using CTNNBL1 antibodies in isothermal titration calorimetry (ITC) experiments to study NLS binding?

CTNNBL1 has been shown to bind nuclear localization sequences (NLSs) via its armadillo domain with specific binding preferences that differ from karyopherin αs.

Methodological approach:

• Protein preparation: For ITC experiments, use purified His-tagged CTNNBL1(Δ1–76) containing the ARM domain, dialyzed against 20 mM Hepes, pH 7.5, 50 mM NaCl .

• Peptide selection: Synthetic peptides corresponding to known NLS sequences (e.g., CDC5L NLS3: KKRKRKR) work effectively, with final concentrations determined by ninhydrin reaction .

• Equipment parameters: Use an ITC calorimeter with the cell containing approximately 150 μM CTNNBL1 and the syringe containing approximately 2.8 mM peptide solution .

• Injection protocol: Perform approximately 20 injections of 2 μl (spaced every 2 min) to a 4–5-fold molar excess .

• Data analysis: Fit titration curves to the data using appropriate software (e.g., ORIGIN software from MicroCal) to determine binding stoichiometry and dissociation constants (Kd) .

How do different fixation and sample preparation methods affect CTNNBL1 antibody performance in immunocytochemistry?

CTNNBL1 is primarily a nuclear protein, and proper fixation and preparation methods are crucial for accurate localization studies.

Methodological approach:

• Fixation methods comparison:

  • Paraformaldehyde (4%): Preserves nuclear structure while maintaining CTNNBL1 antigenicity

  • Methanol/acetone: May expose additional epitopes but can distort nuclear architecture

  • Glutaraldehyde: Not recommended as it can mask CTNNBL1 epitopes

• Permeabilization optimization:

  • For nuclear proteins like CTNNBL1, Triton X-100 (0.1-0.5%) is effective for accessing nuclear epitopes

  • Saponin (0.1%) provides gentler permeabilization but may require longer antibody incubation times

• Antigen retrieval techniques:

  • For paraffin sections, TE buffer pH 9.0 is suggested as the primary method

  • Citrate buffer pH 6.0 can serve as an alternative approach

  • Heat-induced epitope retrieval (95-100°C for 15-20 minutes) typically yields better results than enzymatic methods

• Blocking optimization:

  • 5% normal serum (species of secondary antibody) with 1% BSA in PBS

  • Extended blocking (2+ hours) may reduce background in nuclear regions

What are the most effective strategies for using CTNNBL1 antibodies to study its potential role in obesity-related pathways?

Genome-wide association studies have identified SNPs in the CTNNBL1 gene associated with variations in fat mass and body mass index (BMI) .

Methodological approach:

• Tissue-specific expression analysis:

  • Use immunohistochemistry with CTNNBL1 antibodies to compare expression patterns in adipose tissue from lean vs. obese subjects

  • Western blotting to quantify expression levels across multiple tissues (adipose, liver, muscle) relevant to metabolic regulation

• SNP-specific protein studies:

  • Generate antibodies specific to protein variants resulting from obesity-associated SNPs

  • Use proximity ligation assays to detect potential differences in protein-protein interactions between wildtype and variant CTNNBL1

• Chromatin association studies:

  • ChIP-seq using CTNNBL1 antibodies in adipocytes to identify potential regulatory targets

  • Compare chromatin association patterns between cells carrying different CTNNBL1 allelic variants

• Metabolic pathway interaction mapping:

  • Co-immunoprecipitation followed by mass spectrometry to identify novel interaction partners in metabolic tissues

  • Validate by reciprocal immunoprecipitation with antibodies against identified partners

How can researchers distinguish between CTNNBL1 isoforms using antibody-based techniques?

CTNNBL1 has multiple isoforms, with at least four being documented in the literature .

Methodological approach:

• Isoform-specific antibody selection:

  • Target unique regions present in specific isoforms

  • Use antibody combinations targeting different regions to create isoform-specific detection patterns

  • Note that many commercial antibodies will detect all isoforms except isoform b

• Western blot optimization:

  • Use high-resolution SDS-PAGE (8-10%) to separate closely migrating isoforms

  • Gradient gels (4-15%) can improve separation of differently sized variants

  • Extended running times at lower voltage improves resolution

• 2D electrophoresis approach:

  • Combine isoelectric focusing with SDS-PAGE to separate isoforms with similar molecular weights but different charges

  • Follow with western blotting using CTNNBL1 antibodies

• Mass spectrometry validation:

  • Immunoprecipitate with CTNNBL1 antibodies, then use mass spectrometry to identify unique peptides corresponding to specific isoforms

  • Compare peptide maps with theoretical digests of known isoforms

What are the critical controls needed when using CTNNBL1 antibodies in chromatin immunoprecipitation (ChIP) experiments?

When designing ChIP experiments to study CTNNBL1's potential chromatin associations:

Methodological approach:

• Essential negative controls:

  • IgG control: Perform parallel ChIP with matched isotype control IgG

  • No antibody control: Process chromatin without adding any antibody

  • CTNNBL1-deficient cells: Ideally, include a knockout/knockdown cell line as a biological negative control

• Critical positive controls:

  • Input chromatin: Always process an aliquot of pre-immunoprecipitation chromatin

  • Known targets: Include primers for genes associated with spliceosomal complexes where CTNNBL1 is expected to localize

  • Immunoprecipitation of known CTNNBL1-interacting factors (e.g., CDC5L) to confirm co-occupancy at target loci

• Validation approaches:

  • Sequential ChIP (re-ChIP): First immunoprecipitate with CTNNBL1 antibody, then with antibodies against interacting partners

  • Multiple antibody verification: Use antibodies targeting different epitopes of CTNNBL1

  • Spike-in normalization: Add chromatin from a different species as an internal control for technical variation

How can researchers optimize immunofluorescence protocols to detect CTNNBL1 in different subcellular compartments?

While CTNNBL1 is primarily nuclear, understanding its potential shuttling or specialized distribution requires optimized detection protocols.

Methodological approach:

• Fixation and permeabilization optimization:

  • Brief fixation (10 min, 4% PFA) preserves fine nuclear structures

  • Sequential permeabilization: first with digitonin (50 μg/ml, 5 min) to selectively permeabilize plasma membrane, then with Triton X-100 (0.1%, 5 min) for nuclear membrane

• Antibody selection and dilution:

  • Start with 20 μg/mL for immunofluorescence applications

  • Titrate antibody concentration to optimize signal-to-noise ratio

  • Use both N-terminal and C-terminal targeting antibodies to ensure complete detection

• Co-localization markers:

  • Nuclear speckle markers (SC35, SRSF2) to assess co-localization with splicing machinery

  • Nucleolar markers (fibrillarin) to distinguish specific subnuclear localizations

  • Nuclear envelope markers (Lamin B1) to assess perinuclear distribution

• Advanced imaging techniques:

  • Super-resolution microscopy (STED, STORM) for detailed subnuclear distribution

  • Live-cell imaging with fluorescently tagged antibody fragments to track dynamic changes

  • Fluorescence recovery after photobleaching (FRAP) to assess protein mobility in different compartments

What experimental approaches can resolve conflicting data about CTNNBL1's role in nuclear transport versus intranuclear targeting?

CTNNBL1 binds NLSs via its ARM domain but differs from karyopherin αs in its NLS preferences and interactions with import factors, suggesting a potential role in intranuclear targeting rather than canonical nuclear transport .

Methodological approach:

• Differential co-immunoprecipitation:

  • Use CTNNBL1 antibodies to immunoprecipitate from different nuclear fractions (soluble nucleoplasm vs. chromatin-bound)

  • Compare interacting partners to identify compartment-specific associations

• Domain-specific function analysis:

  • Develop antibodies against the N-terminal region that binds karyopherin αs

  • Use these in conjunction with antibodies against the ARM domain to separately track these regions' localizations and interactions

• Live-cell trafficking studies:

  • Combine antibody fragments with photoactivatable fluorescent proteins to track CTNNBL1 movement following cell activation

  • Correlate with co-transport of identified binding partners

• Nucleocytoplasmic fractionation:

  • Use stepwise extraction protocols with increasing salt concentrations

  • Western blot with CTNNBL1 antibodies to determine distribution and binding strength in different compartments

  • Compare with distribution patterns of known transport factors versus intranuclear structural proteins

How can researchers use CTNNBL1 antibodies to investigate its interactions with AID and potential role in antibody diversification mechanisms?

While some studies suggest CTNNBL1 interacts with AID and influences antibody diversification , others indicate it may be dispensable for class switch recombination (CSR) .

Methodological approach:

• Interaction mapping using mutant proteins:

  • Generate AID mutants at residues Arg19, Arg24, Arg50, and Arg112 (known to affect nuclear import and CTNNBL1 binding)

  • Perform co-immunoprecipitation with CTNNBL1 antibodies to map critical interaction regions

  • Compare binding patterns across multiple species (human, mouse, chicken) to identify conserved mechanisms

• In situ proximity detection:

  • Use proximity ligation assays combining CTNNBL1 and AID antibodies

  • Quantify interaction signals at immunoglobulin loci versus other genomic locations

  • Compare proximity patterns in cells actively undergoing CSR versus resting B cells

• Temporal dynamics analysis:

  • Chromatin immunoprecipitation with CTNNBL1 antibodies at different time points during B cell activation

  • Track association with switch regions and variable gene segments over the course of the activation response

  • Correlate with recruitment of other factors (UNG, MSH2/6) required for antibody diversification

• Structural interaction studies:

  • Use purified proteins and antibody fragments to map interaction surfaces via hydrogen-deuterium exchange mass spectrometry

  • Validate structural predictions using co-crystallization approaches with antibody-facilitated complex formation