TBL1Y Monoclonal Antibody

What is TBL1Y and what are its known cellular functions?

TBL1Y (Transducin beta-like 1Y) is a Y-linked homologue of TBL1X that functions as an F-box-like protein involved in the recruitment of the ubiquitin/19S proteasome complex to nuclear receptor-regulated transcription units. It plays an essential role in transcription activation mediated by nuclear receptors. Molecular evidence indicates that TBL1Y likely acts as an integral component of corepressor complexes that mediate the recruitment of the 19S proteasome complex, leading to the subsequent proteasomal degradation of transcription repressor complexes, thereby allowing cofactor exchange .

Unlike other members of the TBL1 family (TBL1X and TBLR1), TBL1Y shows distinct expression patterns and functional characteristics. Research has demonstrated that while TBL1X and TBLR1 act as corepressor/coactivator exchangers for several nuclear receptors and transcription factors, TBL1Y exhibits different functional properties in gene regulation .

What are the typical expression patterns of TBL1Y in human tissues?

TBL1Y demonstrates a tissue-specific expression pattern. RT-PCR analysis has revealed that TBL1Y is expressed in multiple tissues but notably absent in leukocytes. Among cell lines tested, expression was only detected in NT2/D1 cells and in lymphoblasts transformed with Epstein Barr virus .

Immunohistochemical studies using TBL1Y-specific antibodies have demonstrated expression in the human cochlea, particularly in cochlear spiral ganglion neurons and in outer and inner hair cells. Additionally, TBL1Y expression has been documented in the prostate, which is consistent with its involvement in both hearing-related functions and potentially prostate biology .

In fetal development, TBL1Y mRNA expression has been detected in thyroid gland, brain, and kidney, while adult expression appears more restricted .

What types of TBL1Y antibodies are available for research and what are their key characteristics?

Several types of TBL1Y antibodies are available for research applications:

When selecting a TBL1Y antibody, researchers should consider the specific experimental application, required species reactivity, and whether TBL1Y-specific detection or broader family detection is desired for their research questions .

What are the validated applications for TBL1Y monoclonal antibodies and their recommended working dilutions?

TBL1Y monoclonal antibodies have been validated for multiple research applications, each with specific recommended working dilutions:

For optimal results, researchers should:

• Perform preliminary titration experiments to determine the optimal antibody concentration for their specific sample type

• Include appropriate positive and negative controls

• Consider using different antibody clones if cross-reactivity with other TBL family members (TBL1X, TBL1XR1) is a concern

• Validate the expected molecular weight (approximately 56 kDa for TBL1Y) when performing Western blot analysis

How should researchers design experiments to distinguish between TBL1Y and its homologues (TBL1X and TBL1XR1)?

Distinguishing between TBL1Y and its homologues requires careful experimental design:

• Antibody Selection Strategy:

  • Use TBL1Y-specific antibodies that have been validated against recombinant proteins of all three family members

  • Consider using antibodies raised against non-conserved regions of TBL1Y

  • When absolute specificity is required, validate antibody specificity using recombinant proteins or knockout/knockdown controls

• Control Design:

  • Include recombinant protein controls for TBL1Y, TBL1X, and TBL1XR1 in Western blot experiments

  • Use tissues with differential expression patterns (e.g., male-specific tissues for TBL1Y)

  • Consider using Y-chromosome-negative cells as negative controls for TBL1Y

• Expression Analysis:

  • Perform sqRNA-PCR to quantitatively compare expression levels of TBL1Y, TBL1X, and TBL1XR1 in your samples of interest

  • For protein detection, use antibodies that can distinguish between the family members, as demonstrated in Western blot validation where distinct bands are observed for TBL1Y, TBL1X, and TBL1R proteins

• Functional Validation:

  • TBL1Y shows different functional properties in reporter assays compared to other family members, which can be used as a functional discrimination method

What methodological approaches should be used to investigate TBL1Y expression in tissue samples?

For comprehensive analysis of TBL1Y expression in tissue samples:

• Tissue Preparation and Fixation:

  • For IHC-P applications, use formalin-fixed, paraffin-embedded tissues

  • Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining

• Multi-method Verification Approach:

  • Combine protein detection (IHC, IF, WB) with mRNA analysis (RT-PCR, qPCR)

  • For cochlear and neural tissues, counterstain using neuronal markers such as parvalbumin to identify specific cell populations expressing TBL1Y

• Visualization Techniques:

  • For IHC applications: Use HRP Polymer for detection and hematoxylin for counterstaining

  • For IF applications: Use appropriate secondary antibodies (e.g., Alexa Fluor® 555-conjugated anti-rabbit IgG) and DAPI for nuclear counterstaining

• Quantification Methods:

  • Use sqRNA-PCR for comparative quantification of TBL1Y versus TBL1X and TBL1XR1

  • For protein quantification in Western blots, normalize to appropriate housekeeping proteins

  • For IHC quantification, use digital image analysis to score staining intensity and distribution

How can TBL1Y antibodies be used to investigate protein-protein interactions within transcriptional regulatory complexes?

TBL1Y functions within transcriptional regulatory complexes, and antibodies can be instrumental in elucidating these interactions:

• Co-immunoprecipitation (Co-IP) Protocols:

  • Use TBL1Y antibodies for immunoprecipitation followed by Western blotting for known or suspected interaction partners

  • Example validated approach: TBL1Y (Myc-tagged) can be co-immunoprecipitated with SMRT (Flag/Myc-tagged) co-repressor, demonstrating their interaction within the same complex

• Sequential ChIP Approach:

  • Perform chromatin immunoprecipitation with TBL1Y antibodies followed by secondary IP with antibodies against other transcriptional regulators

  • This can identify genomic regions where TBL1Y co-localizes with other factors

• Proximity Ligation Assay:

  • Combine TBL1Y antibodies with antibodies against suspected interaction partners

  • This technique allows visualization of proteins that are in close proximity (<40 nm) in situ

• Mass Spectrometry Integration:

  • Use TBL1Y antibodies for immunoprecipitation followed by mass spectrometry to identify novel interaction partners

  • Compare interaction profiles between wild-type and mutant TBL1Y to understand how mutations affect complex formation

What experimental approaches can elucidate the role of TBL1Y in hearing loss pathogenesis?

Research has implicated TBL1Y in syndromic hearing loss, necessitating specialized experimental approaches:

• Mutation Analysis Protocol:

  • Screen for known mutations (e.g., r.206A>T;p.Asp69Val) in patients with Y-linked hearing loss

  • Use Sanger sequencing to confirm mutations identified by whole-exome sequencing

  • Apply prediction tools (PolyPhen-2, Mutation Taster, Provean) to assess potential pathogenicity of novel variants

• Protein Stability Assessment:

  • Compare wild-type and mutant TBL1Y protein stability using cycloheximide chase assays

  • Analyze ubiquitination patterns through immunoprecipitation with TBL1Y antibodies followed by detection with ubiquitin-specific antibodies

  • Examine protein degradation kinetics with and without proteasome inhibitors (e.g., MG132)

• Functional Consequence Analysis:

  • Use reporter gene assays with thyroid response elements (TRE) to compare transcriptional effects of wild-type versus mutant TBL1Y

  • Assess co-repressor recruitment efficiency through quantitative co-immunoprecipitation experiments

  • Analyze protein conformation differences using limited proteolysis assays (e.g., in vitro trypsin digestion profiles)

• Cochlear Expression Studies:

  • Perform immunohistochemistry on human cochlea slices using TBL1Y-specific antibodies

  • Combine with neuronal markers (e.g., parvalbumin) to pinpoint cell-specific expression

  • Compare expression patterns between normal and affected tissues when available

How should researchers approach the investigation of TBL1Y's role in nuclear receptor-mediated transcriptional regulation?

To effectively investigate TBL1Y's role in nuclear receptor-mediated transcriptional regulation:

• Reporter Gene Assay Design:

  • Construct reporter plasmids containing natural thyroid response elements (TRE) driving luciferase expression

  • Co-transfect with expression vectors for wild-type or mutant TBL1Y

  • Measure transcriptional activity in response to hormone treatment and with/without proteasome inhibitors (e.g., MG132)

• Proteasome Recruitment Analysis:

  • Investigate TBL1Y's role in recruiting the 19S proteasome complex to nuclear receptor-regulated transcription units

  • Use co-immunoprecipitation to detect interactions between TBL1Y and proteasome components

  • Compare recruitment efficiency between wild-type and mutant TBL1Y proteins

• Co-repressor Complex Dynamics:

  • Analyze the assembly and disassembly of co-repressor complexes containing TBL1Y

  • Compare SMRT interaction efficiency between wild-type and mutant TBL1Y

  • Investigate how TBL1Y mutations affect transcriptional repression and activation transitions

• Comparative Analysis with Family Members:

  • Unlike GAL4DBD-fused TBL1X and TBLR1, GAL4DBD-fused TBL1Y does not repress promoter activity in dual luciferase assays

  • Design experiments to directly compare the functional differences between TBL1Y, TBL1X, and TBLR1 in the same experimental system

What methodological strategies can resolve contradictory data regarding TBL1Y function across different experimental systems?

When facing contradictory data about TBL1Y function:

• Cell Type Dependence Resolution:

  • Compare TBL1Y function across multiple cell types (e.g., HEK293, NT2/D1, EB-transformed lymphoblasts)

  • Document cell type-specific expression levels of TBL1Y and potential cofactors

  • Note that TBL1Y expression has been observed in NT2/D1 cells and EB-transformed lymphoblasts but not in many other cell lines

• Species-Specific Function Analysis:

  • Compare TBL1Y function between human and model organism systems

  • Document species-specific differences in protein sequence, expression, and interaction partners

  • Consider Y-chromosome evolution and the absence of direct orthologs in some model organisms

• Isoform-Specific Investigation:

  • Identify and characterize potential TBL1Y splice variants

  • Document the effect of the IVS7+1G>C polymorphism, which may cause splicing errors

  • Compare the function of different isoforms in the same experimental system

• Protocol Harmonization Approach:

  • Standardize experimental conditions across laboratories (cell density, transfection methods, reagent concentrations)

  • Document the effects of specific reagents (e.g., MG132) on experimental outcomes

  • Establish a consensus on positive and negative controls for TBL1Y function assays

What are the most common technical challenges when working with TBL1Y antibodies and how can they be addressed?

Researchers commonly encounter several technical challenges when working with TBL1Y antibodies:

How can researchers optimize experimental conditions for detecting low-abundance TBL1Y protein in various tissue samples?

For optimal detection of low-abundance TBL1Y:

• Sample Preparation Enhancement:

  • For protein extraction, use specialized buffers containing protease inhibitors and potentially proteasome inhibitors

  • Enrich nuclear fractions, as TBL1Y predominantly localizes to the nucleus

  • Consider using phosphatase inhibitors, as phosphorylation may affect epitope recognition

• Signal Amplification Strategies:

  • For IHC applications, implement tyramide signal amplification (TSA) to enhance sensitivity

  • For Western blot, use high-sensitivity ECL substrates or fluorescent secondary antibodies

  • Consider using biotin-streptavidin amplification systems for very low abundance detection

• Protocol Modifications:

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

  • Optimize blocking conditions to reduce background while preserving specific signal

  • For Western blot, load higher protein amounts (50-100 μg) and use gradient gels for better resolution

• Specialized Detection Methods:

  • Consider proximity ligation assay (PLA) for in situ detection with enhanced sensitivity

  • Use immunoprecipitation followed by Western blot for enrichment of low-abundance TBL1Y

  • For cellular localization, combine with nuclear markers to confirm expected subcellular distribution

What quality control measures should be implemented when validating a new lot of TBL1Y monoclonal antibody?

Comprehensive quality control for TBL1Y antibodies should include:

• Specificity Validation Tests:

  • Western blot against recombinant TBL1Y, TBL1X, and TBL1XR1 proteins to assess cross-reactivity

  • Analysis using tissues/cells known to express or not express TBL1Y (e.g., prostate as positive control)

  • Peptide competition assays with the immunizing peptide to confirm binding specificity

• Performance Metrics Assessment:

  • Titration experiments to determine optimal working dilutions for each application

  • Sensitivity testing to determine minimum detectable amount of target protein

  • Reproducibility testing across multiple experimental runs

• Application-Specific Validation:

  • For WB: Confirm correct molecular weight (~56 kDa) and band pattern

  • For IHC: Verify expected cellular and subcellular localization patterns

  • For IF: Confirm nuclear localization consistent with TBL1Y function

• Lot-to-Lot Comparison Protocol:

  • Run side-by-side comparisons with previous antibody lot

  • Document any differences in sensitivity, specificity, or background

  • Maintain reference samples for consistent comparison across antibody lots