TULP4 Antibody

What is TULP4 and why is it important to study?

TULP4 (Tubby Like Protein 4) is a member of the TUB protein family with a molecular weight of approximately 169 kDa and consists of 1543 amino acid residues in its canonical human form. TULP4 functions as a substrate-recognition component of a SCF-like ECS (Elongin-Cullin-SOCS-box protein) E3 ubiquitin ligase complex, mediating ubiquitination and subsequent proteasomal degradation of target proteins . This protein is also known by several synonyms, including tubby super-family protein, tubby superfamily protein, and tubby-related protein 4.

TULP4 has gained research interest due to its potential roles in various cellular processes through its ubiquitination functions. Recent research has implicated circular RNA derived from the TULP4 gene (circTulp4) in Alzheimer's disease pathogenesis, suggesting that TULP4 may play indirect roles in neurodegenerative conditions . Understanding TULP4's function requires specific antibodies that can accurately detect the protein across various experimental contexts.

What is the expression pattern of TULP4 in different tissues?

TULP4 demonstrates a notable tissue-specific expression pattern, being predominantly expressed in four major tissue types:

• Brain tissue (with potential implications for neurological research)

• Skeletal muscle

• Testis

• Kidney

The expression of TULP4 appears to be regulated by a circular RNA (circTulp4) derived from the same gene. Research has shown that the expression pattern of Tulp4 mirrors that of circTulp4 in mouse brain tissue, and both are upregulated during neuronal development . This co-expression pattern suggests important functional relationships between the circular RNA and its parent gene.

TULP4 orthologs have been reported in multiple species, including mouse, rat, bovine, frog, chimpanzee, and chicken, making it a conserved protein across various vertebrate lineages . This conservation suggests evolutionary importance of the protein's function.

What applications are TULP4 antibodies most commonly used for?

TULP4 antibodies are utilized in several key applications for detecting and studying this protein:

• Western Blot (WB): The most widely used application for TULP4 antibodies, allowing researchers to detect and quantify TULP4 protein in tissue or cell lysates . Western blotting is particularly valuable for distinguishing between the canonical form and different isoforms of TULP4.

• Enzyme-Linked Immunosorbent Assay (ELISA): A common application for quantitative detection of TULP4 in various sample types .

• Immunohistochemistry (IHC): Some TULP4 antibodies are validated for IHC applications, allowing for the visualization of TULP4 distribution in tissue sections .

• Chromatin Immunoprecipitation (ChIP) assays: Used in more advanced applications to study the interactions between TULP4, circular RNA, and DNA regulatory elements .

When selecting TULP4 antibodies for these applications, researchers should prioritize antibodies with validated reactivity to species of interest. Many commercially available TULP4 antibodies demonstrate cross-reactivity with human, mouse, rabbit, rat, bovine, dog, guinea pig, hamster, and zebrafish TULP4 proteins .

How do TULP4 antibodies differ in their detection capabilities?

TULP4 antibodies vary in their detection capabilities based on several key factors:

How can researchers effectively study the relationship between circTulp4 and TULP4 expression?

Studying the relationship between circTulp4 (circular RNA from the TULP4 gene) and TULP4 protein expression requires sophisticated methodological approaches:

• RNA knockdown studies: SiRNA-mediated knockdown of circTulp4 has been shown to reduce both TULP4 mRNA and protein levels in primary hippocampal neurons, suggesting regulatory relationships. Researchers should design siRNAs targeting the unique back-splice junction of circTulp4 to ensure specificity .

• Overexpression experiments: Conversely, overexpression of circTulp4 increases TULP4 mRNA and protein levels. Researchers should use circular RNA expression vectors that can generate the proper circular structure when studying these effects .

• Nuclear/cytoplasmic fractionation: Since circTulp4 is predominantly localized in the nucleus, proper subcellular fractionation protocols are essential for studying its regulatory effects on TULP4 expression .

• RNA Antisense Purification (RAP): This technique can be employed to identify RNA-RNA interactions involving circTulp4, such as its binding to U1 snRNA, which appears to mediate its effects on TULP4 expression .

• Chromatin Immunoprecipitation (ChIP): ChIP assays with antibodies against U1A (a component of U1 snRNP) have revealed that U1 snRNP specifically binds to the TULP4 promoter region, suggesting a mechanism by which circTulp4 regulates TULP4 transcription .

• Chromatin Isolation by RNA Purification (ChIRP): This technique has shown that circTulp4 occupies a region approximately 500 bp upstream of the transcriptional start site of TULP4, further elucidating the regulatory mechanism .

These methodologies collectively suggest that circTulp4 recruits U1 snRNP to the TULP4 promoter region, thereby regulating TULP4 transcription. Researchers investigating this relationship should consider designing experiments that account for this nuclear regulatory mechanism.

What are the key validation steps for TULP4 antibodies before experimental use?

Proper validation of TULP4 antibodies is crucial for generating reproducible research data. Researchers should follow these key validation steps:

• Western blot validation: Confirm the antibody detects a band of the expected molecular weight (approximately 169 kDa for canonical TULP4) . Multiple bands may indicate detection of different isoforms (up to 2 have been reported for TULP4) or potential cross-reactivity.

• Positive and negative controls: Include tissue samples known to express TULP4 (brain, skeletal muscle, testis, kidney) as positive controls, and tissues or cell lines with minimal TULP4 expression as negative controls .

• Knockdown/knockout validation: Use siRNA knockdown or CRISPR knockout of TULP4 to confirm antibody specificity. The signal should be reduced or eliminated in knockdown/knockout samples.

• Recombinant protein controls: When available, use purified recombinant TULP4 protein as a positive control to confirm antibody specificity.

• Cross-reactivity assessment: If working with non-human models, validate the antibody against the species-specific TULP4 ortholog, as sequence variations may affect antibody binding .

• Application-specific validation: Validate the antibody specifically for each experimental application (WB, ELISA, IHC) as performance can vary between applications.

• Preabsorption test: Incubate the antibody with purified antigen prior to use; this should eliminate specific binding if the antibody is truly specific.

These validation steps are critical for ensuring that experimental results with TULP4 antibodies are reliable and reproducible across different research contexts, addressing known issues with antibody reproducibility in the research community .

How should researchers interpret conflicting results between different TULP4 antibody types?

When researchers encounter conflicting results using different TULP4 antibody types (polyclonal vs. monoclonal, or antibodies targeting different epitopes), the following analytical approach is recommended:

• Epitope accessibility analysis: Different antibodies may target epitopes with varying accessibility depending on protein conformation or post-translational modifications. Polyclonal antibodies, with their multi-epitope binding properties, may detect TULP4 in contexts where specific epitopes are masked .

• Denaturation conditions assessment: The method of sample preparation affects epitope exposure. Some antibodies perform better in native conditions while others are optimized for denatured proteins. Researchers should compare results using multiple sample preparation methods .

• Isoform detection capability: TULP4 has up to two reported isoforms . Antibodies targeting different regions may detect distinct isoform subsets, leading to apparently conflicting results that actually reveal isoform-specific expression patterns.

• Lot-to-lot variation analysis: For polyclonal antibodies, batch variations can significantly impact results. When using different lots, researchers should maintain validation samples for standardization .

• Orthogonal validation approach: When results conflict, employ alternative, non-antibody-based techniques (such as mass spectrometry or RNA analysis) to resolve discrepancies.

• Cross-reactivity evaluation: Perform immunoprecipitation followed by mass spectrometry to identify all proteins captured by each antibody, revealing potential cross-reactive targets.

• Functional validation: For studies examining TULP4's role in ubiquitination, functional assays measuring ubiquitination activity can help resolve conflicting antibody results.

This systematic approach helps researchers distinguish between true biological variations and technical artifacts when interpreting conflicting antibody results.

How are TULP4 antibodies utilized in Alzheimer's disease research?

TULP4 antibodies are emerging as valuable tools in Alzheimer's disease research, particularly in light of recent findings connecting circTulp4 to AD pathogenesis:

• Expression pattern analysis: TULP4 antibodies enable researchers to track changes in TULP4 protein expression in various brain regions during Alzheimer's disease progression. Research has shown that circTulp4 expression is markedly lower in 9-month-old APP/PS1 mice (an Alzheimer's disease model) compared to wild-type mice, suggesting potential dysregulation of the TULP4 pathway in AD .

• Co-localization with pathological markers: Immunohistochemistry using TULP4 antibodies, combined with markers for amyloid plaques or neurofibrillary tangles, helps researchers understand spatial relationships between TULP4 expression and AD pathology.

• Protein-protein interaction studies: Immunoprecipitation with TULP4 antibodies allows identification of TULP4's interaction partners in healthy versus AD brain tissue, potentially revealing disrupted protein networks.

• Ubiquitination pathway analysis: Given TULP4's role in the E3 ubiquitin ligase complex , antibodies help researchers investigate whether dysfunctional protein degradation involving TULP4 contributes to protein aggregation in AD.

• Mechanistic studies: The finding that circTulp4 regulates TULP4 expression and is dysregulated in AD models suggests a mechanistic link worth exploring . Antibodies enable researchers to track how interventions targeting circTulp4 affect TULP4 protein levels.

• Biomarker development: While still exploratory, TULP4 antibodies are being used to evaluate whether altered TULP4 expression or post-translational modifications could serve as biomarkers for AD progression.

These applications highlight how TULP4 antibodies contribute to understanding the complex molecular mechanisms of Alzheimer's disease, potentially revealing new therapeutic targets in the ubiquitin-proteasome system.

What experimental protocols are recommended for studying TULP4's role in the ubiquitin-proteasome pathway?

To investigate TULP4's function as a substrate-recognition component of a SCF-like ECS (Elongin-Cullin-SOCS-box protein) E3 ubiquitin ligase complex , researchers should consider these methodological approaches:

• Ubiquitination assays: In vitro ubiquitination assays using immunoprecipitated TULP4 can help identify substrates targeted for degradation. The protocol should include:

  • Immunoprecipitation of TULP4 using validated antibodies

  • Addition of E1, E2 enzymes, ubiquitin, and ATP

  • Western blot analysis for ubiquitinated proteins

  • Mass spectrometry to identify modified substrates

• Proteasome inhibition studies: Treating cells with proteasome inhibitors (e.g., MG132) before TULP4 immunoprecipitation can help identify transient ubiquitinated substrates that would otherwise be rapidly degraded.

• TULP4 domain mapping: Using antibodies recognizing different TULP4 domains helps determine which regions are critical for substrate recognition versus ubiquitin ligase complex formation.

• Co-immunoprecipitation assays: To identify components of the TULP4-containing E3 ligase complex, researchers should:

  • Immunoprecipitate TULP4 using specific antibodies

  • Perform western blotting for known SCF-like complex components

  • Use mass spectrometry for unbiased identification of associated proteins

• Pulse-chase experiments: To study the dynamics of TULP4-mediated protein degradation:

  • Pulse-label cells with radioactive amino acids

  • Immunoprecipitate potential substrates at different chase timepoints

  • Compare degradation rates between wild-type cells and TULP4 knockdown/knockout cells

• Subcellular fractionation: Since TULP4's subcellular localization is primarily cytoplasmic , fractionation followed by western blotting with TULP4 antibodies can reveal where ubiquitination occurs within the cell.

These methodological approaches provide a comprehensive framework for elucidating TULP4's specific role in the ubiquitin-proteasome pathway and identifying its physiologically relevant substrates.

What are the optimal protocols for TULP4 detection by Western blotting?

For optimal detection of TULP4 by Western blotting, researchers should consider the following protocol recommendations:

• Sample preparation:

  • Use tissues with known TULP4 expression (brain, skeletal muscle, testis, kidney)

  • Include protease inhibitors in lysis buffers to prevent degradation

  • Add deubiquitinase inhibitors (N-ethylmaleimide) if studying ubiquitinated forms

  • Sonicate samples to shear DNA and reduce viscosity

• Gel electrophoresis:

  • Use 6-8% gels due to TULP4's large size (169 kDa)

  • Run at lower voltage (80-100V) for better resolution of high molecular weight proteins

  • Include molecular weight markers spanning 100-250 kDa range

• Transfer conditions:

  • Use wet transfer systems for large proteins like TULP4

  • Transfer at 30V overnight at 4°C for optimal transfer of high molecular weight proteins

  • Use 0.45μm PVDF membrane rather than 0.2μm for larger proteins

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk or BSA in TBST

  • Incubate with primary TULP4 antibody at dilutions of 1:500-1:1000 (optimize for specific antibody)

  • Extend primary antibody incubation to overnight at 4°C for better signal

  • Use TBS with 0.1% Tween-20 for all wash steps

• Detection optimization:

  • Use high-sensitivity ECL substrates for enhanced detection

  • Consider signal amplification systems for low abundance detection

  • Perform longer exposures (5-10 minutes) due to TULP4's large size and potential low abundance

• Controls:

  • Include positive control lysates from tissues with high TULP4 expression

  • Use TULP4 knockdown samples as negative controls

  • Include loading controls appropriate for large proteins (e.g., vinculin rather than GAPDH)

This optimized Western blotting protocol accounts for the specific challenges associated with detecting a large protein like TULP4 and should help researchers obtain clear, specific signals for analysis.

How can RNA-protein interaction studies be designed to investigate circTulp4 and TULP4 relationships?

To effectively study the regulatory relationship between circTulp4 and TULP4 expression, researchers should employ these RNA-protein interaction methodologies:

• RNA Antisense Purification (RAP):

  • Design biotinylated probes targeting the unique back-splice junction of circTulp4

  • Include control probes targeting unrelated sequences

  • Use streptavidin beads for pulldown

  • Analyze captured RNAs by qRT-PCR or RNA sequencing

  • This technique has successfully shown that circTulp4 can bind to U1 snRNA

• RNA Immunoprecipitation (RIP):

  • Use antibodies against RNA-binding proteins (e.g., U1A, SNRNP70)

  • Extract and analyze associated RNAs by qRT-PCR

  • Include appropriate controls (e.g., IgG, RNA polymerase II)

  • This approach has demonstrated that U1 snRNP components interact with circTulp4

• Chromatin Isolation by RNA Purification (ChIRP):

  • Design biotinylated probes tiling the circTulp4 sequence

  • After crosslinking and pulldown, analyze associated DNA by qPCR or sequencing

  • Focus on the TULP4 promoter region (~500 bp upstream of TSS)

  • This method has revealed that circTulp4 occupies the TULP4 promoter region

• Double Fluorescence In Situ Hybridization (FISH):

  • Design probes specific to circTulp4 (targeting the back-splice junction)

  • Use differently labeled probes for U1 snRNA or TULP4 mRNA

  • Perform in neuronal cells or brain tissue sections

  • Analyze co-localization using confocal microscopy

  • This technique has shown nuclear co-localization of circTulp4 and U1 snRNA

• Functional manipulation studies:

  • Design siRNAs targeting circTulp4 junction sequences

  • Measure effects on TULP4 mRNA and protein levels

  • Create circTulp4 overexpression constructs

  • Analyze changes in TULP4 expression using validated antibodies

  • These experiments have demonstrated that circTulp4 knockdown reduces TULP4 levels

These methodological approaches provide complementary data for understanding how circTulp4 regulates TULP4 expression through recruitment of U1 snRNP to the TULP4 promoter region, advancing our understanding of this novel regulatory mechanism.