TOB1 Antibody, Biotin conjugated

What is TOB1 protein and why is it significant for immunological research?

TOB1 (Transducer of ERBB2-1) is a member of the antiproliferative (APRO) family of proteins that controls cell cycle progression in several cell types. This protein contains a nuclear localization signal (NLS) and a nuclear export signal (NES) that enable translocation between nucleus and cytoplasm at different stages of the cell cycle. TOB1 has been implicated in diverse cellular mechanisms including embryonic dorsal development and T helper 17 (Th17) cell function. Recent evidence has linked TOB1 function to experimental and human immune-related disorders, underscoring its potential as both a biomarker and therapeutic target in conditions such as multiple sclerosis .

In T lymphocytes, TOB1 is constitutively expressed in unstimulated cells but strongly down-regulated after activation. When expressed, TOB1 inhibits T cell proliferation by suppressing transcription of cytokines (IL-2, IL-4, IFNγ) and positive regulators of the cell cycle such as cyclin E and cyclin A .

What are the key specifications of commercially available TOB1 Antibody, Biotin conjugated?

Commercial TOB1 Antibody, Biotin conjugated preparations typically have the following specifications:

This information helps researchers select the appropriate antibody preparation for their specific experimental needs .

How does TOB1 function at the molecular level in cell cycle regulation?

TOB1 functions as a negative regulator at multiple levels of cellular processes:

• Transcriptional regulation: In T lymphocytes, TOB1 associates with Smad2 and Smad4, enhancing Smad4 DNA binding and Smad-dependent transcription. Interestingly, in osteoblasts, TOB1 shows the opposite effect, inhibiting Smad-mediated transcription despite enhancing DNA binding .

• Translational control: TOB1 can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 (via its N-terminal domain) and cytoplasmic poly(A)-binding proteins (via its C-terminal domain), effectively enhancing mRNA decay and blocking translation of target genes .

• Cell cycle inhibition: TOB1 promotes transcription of p27 (CDKN1B), a cyclin-dependent kinase inhibitor that blocks cell cycle progression .

• Protein degradation: TOB1 levels are regulated by Skp2, which promotes TOB1 degradation via the ubiquitin-proteasome pathway, allowing cell cycle progression when TOB1 inhibition is no longer needed .

These molecular mechanisms collectively contribute to TOB1's antiproliferative effects and its importance in maintaining T cell quiescence.

What are the recommended protocols for using TOB1 Antibody, Biotin conjugated in ELISA applications?

When using TOB1 Antibody, Biotin conjugated for ELISA applications, researchers should follow these methodological guidelines:

• Coating: Coat ELISA plates with capture antibody (anti-TOB1) or target antigen (recombinant TOB1) at 1-10 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C.

• Blocking: Block non-specific binding sites with 2-5% BSA or non-fat milk in PBS for 1-2 hours at room temperature.

• Sample preparation: Prepare cell or tissue lysates using non-denaturing buffers to preserve native protein conformation. Typical working dilutions for TOB1 Antibody, Biotin conjugated range from 1:500 to 1:5000, depending on the specific antibody concentration and application .

• Detection system: Utilize streptavidin-HRP or streptavidin conjugated to another reporter molecule, with dilutions ranging from 1:1000 to 1:10,000 depending on the sensitivity required.

• Controls: Include both positive controls (samples known to express TOB1) and negative controls (samples lacking TOB1 expression or isotype control antibodies) to validate assay specificity .

Always optimize antibody concentration, incubation times, and detection systems for each specific experimental context.

How can TOB1 Antibody be utilized to study T cell activation dynamics?

TOB1 plays a crucial role in T cell quiescence and activation. Researchers can leverage TOB1 Antibody, Biotin conjugated to investigate these processes using the following approaches:

• Temporal expression analysis: Monitor TOB1 expression at various time points after T cell stimulation (0, 6, 12, 24, 48, 72 hours) using ELISA or Western blotting. TOB1 levels typically decrease significantly following activation with anti-CD3/CD28 antibodies or other stimuli .

• Correlation with activation markers: Perform dual-parameter analysis correlating TOB1 expression with established T cell activation markers (CD25, CD69) and proliferation markers (Ki-67, CFSE dilution).

• Pathway analysis: Investigate the relationship between TOB1 and TGFβ signaling by analyzing Smad phosphorylation and nuclear translocation in relation to TOB1 expression levels .

• Subset-specific analysis: Compare TOB1 expression across T cell subsets (Th1, Th17, Treg) to understand its differential regulation. Recent research indicates higher TOB1 expression in Th17 cells compared to Th1 cells, with potential implications for subset-specific functions .

These approaches provide insights into how TOB1 regulates T cell quiescence and activation thresholds.

What strategies can be used to validate antibody specificity for TOB1 detection?

Ensuring antibody specificity is critical for obtaining reliable experimental results. Researchers should implement the following validation strategies:

• Genetic validation: Compare staining patterns in wild-type versus TOB1 knockout or knockdown samples. Complete absence of signal in knockout samples confirms specificity .

• Peptide competition: Pre-incubate the antibody with excess immunizing peptide (amino acids 42-175 of human TOB1) before application. Specific binding should be blocked by this competition .

• Multiple antibody validation: Compare results using antibodies targeting different TOB1 epitopes. Concordant results increase confidence in specificity .

• Western blot analysis: Verify a single band of the expected molecular weight (~38 kDa for human TOB1). Multiple or incorrectly sized bands may indicate cross-reactivity .

• Recombinant protein controls: Include purified recombinant TOB1 as a positive control in assay development and optimization .

These validation approaches are essential when implementing TOB1 Antibody in new experimental systems or when troubleshooting unexpected results.

How can TOB1 antibodies be used to investigate the relationship between TOB1 and autoimmune disorders?

Recent research has established important connections between TOB1 and autoimmune disorders, particularly multiple sclerosis (MS). Researchers can investigate these relationships using TOB1 antibodies through several approaches:

• Biomarker studies: Quantify TOB1 expression in peripheral blood T cells from patients with autoimmune disorders compared to healthy controls. Downregulation of TOB1 has been associated with higher risk of disease activity in MS patients .

• Experimental autoimmune models: Compare TOB1 expression levels before and during disease development in experimental autoimmune encephalomyelitis (EAE) and other animal models. TOB1-deficient mice exhibit earlier disease onset and more aggressive EAE progression .

• T cell subset analysis: Examine TOB1 expression in specific T cell subsets (Th1, Th17, Treg) isolated from patients with autoimmune disorders. The balance between these subsets is often disrupted in autoimmune conditions, with potential links to TOB1 expression patterns .

• Correlation with clinical parameters: Analyze relationships between TOB1 expression levels and clinical measures such as disease severity, progression, and response to therapy. This approach may identify patient subgroups for whom TOB1-targeted interventions might be beneficial .

These methodologies can provide insights into TOB1's role in autoimmune pathogenesis and its potential as a therapeutic target.

What approaches can be used to study TOB1's role in mRNA decay and translational regulation?

TOB1 regulates gene expression post-transcriptionally through its interactions with the CCR4-CAF1 deadenylase complex and poly(A)-binding proteins. Researchers can investigate these processes using:

• RNA immunoprecipitation: Use TOB1 Antibody to pull down associated mRNAs, followed by sequencing or qPCR to identify specific transcripts regulated by TOB1 .

• Decay rate analysis: Measure half-lives of candidate mRNAs in systems with normal versus altered TOB1 expression using actinomycin D chase experiments.

• P-body localization: Perform co-immunofluorescence studies to examine TOB1 localization to RNA processing bodies (P-bodies) under various cellular conditions .

• Polysome profiling: Analyze the translation efficiency of TOB1-regulated transcripts by examining their distribution across non-translating and actively translating ribosome fractions.

• Protein-protein interaction studies: Investigate TOB1's interactions with translation and deadenylation machinery components using co-immunoprecipitation with TOB1 Antibody followed by mass spectrometry or Western blotting .

These approaches can elucidate TOB1's complex roles in post-transcriptional gene regulation.

How can researchers investigate the cell type-specific effects of TOB1 in different biological contexts?

TOB1 exhibits context-dependent functions across different cell types. In T lymphocytes, TOB1 enhances Smad-dependent transcription, while in osteoblasts, it inhibits Smad-mediated transcription despite enhancing DNA binding. To investigate these cell type-specific effects:

• Comparative expression analysis: Use TOB1 Antibody to quantify expression levels across different cell types under various stimulation conditions .

• Interactome mapping: Perform immunoprecipitation with TOB1 Antibody followed by mass spectrometry to identify cell type-specific binding partners .

• ChIP-seq analysis: Use chromatin immunoprecipitation with TOB1 Antibody to map genomic binding sites in different cell types, revealing cell-specific transcriptional targets .

• Conditional knockout models: Generate cell type-specific TOB1 knockout animals to assess function in distinct lineages without systemic effects.

• Single-cell approaches: Implement single-cell RNA-seq with TOB1 protein quantification to correlate expression with cell state and differentiation status .

These methodological approaches can reveal how TOB1 function is modulated by cellular context, providing insights into its diverse biological roles.

What are common challenges when working with TOB1 Antibody, Biotin conjugated, and how can they be addressed?

Researchers may encounter several technical challenges when working with TOB1 Antibody, Biotin conjugated:

• Low signal intensity: This may result from insufficient antibody concentration, degraded target protein, or epitope masking. Optimize by:

  • Titrating antibody concentration (typical working dilutions range from 1:500 to 1:5000)

  • Using freshly prepared samples with protease inhibitors

  • Testing alternative sample preparation methods to preserve epitope accessibility

• High background: This common problem can be addressed by:

  • Optimizing blocking conditions (2-5% BSA or non-fat milk)

  • Increasing washing steps (number and duration)

  • Diluting antibody and detection reagents appropriately

  • For tissue samples, block endogenous biotin using avidin/biotin blocking kits

• Cross-reactivity: Validate specificity using:

  • Peptide competition assays with the immunizing peptide (amino acids 42-175)

  • Genetic controls (TOB1 knockout or knockdown samples)

  • Western blot analysis to confirm correct target molecular weight

• Storage and stability issues: Maintain antibody performance by:

  • Storing at -20°C or -80°C as recommended

  • Avoiding repeated freeze-thaw cycles by preparing working aliquots

  • Checking for precipitation or contamination before use

These troubleshooting approaches can significantly improve experimental outcomes when working with TOB1 Antibody, Biotin conjugated.

How can researchers optimize sample preparation for different experimental applications of TOB1 detection?

Sample preparation is critical for successful TOB1 detection across different applications:

• For ELISA applications:

  • Use non-denaturing lysis buffers (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40) with protease inhibitors

  • Determine optimal protein concentration (typically 1-10 μg/ml)

  • For cell-based ELISAs, fix cells with 4% paraformaldehyde and permeabilize with 0.1% Triton X-100

• For Western blotting:

  • Use denaturing buffers containing SDS to fully expose epitopes

  • Load 25-50 μg of total protein per lane

  • Transfer efficiency is critical; optimize transfer conditions for proteins in TOB1's molecular weight range (~38 kDa)

• For immunohistochemistry/immunofluorescence:

  • Optimize fixation conditions (4% paraformaldehyde for 10-15 minutes generally works well)

  • For formalin-fixed tissues, perform antigen retrieval (citrate buffer pH 6.0)

  • Permeabilize with 0.1-0.5% Triton X-100 for intracellular TOB1 detection

• For flow cytometry:

  • Fix cells with 2-4% paraformaldehyde

  • Permeabilize with saponin-based buffers for intracellular staining

  • Use appropriate blocking to reduce non-specific binding

Optimizing these parameters for each specific application will significantly improve detection sensitivity and specificity.

How can researchers interpret conflicting results when studying TOB1 across different experimental contexts?

When faced with seemingly contradictory data regarding TOB1 function or expression:

These analytical frameworks can help researchers reconcile apparently contradictory findings and develop more nuanced understanding of TOB1 biology.

What is the current understanding of TOB1's role in T cell subset differentiation and function?

Recent research has provided important insights into TOB1's role in T cell subset differentiation:

• Differential expression across T cell subsets: Higher expression of TOB1 has been observed in IL-17 producing CD4+ T helper (Th17) cells compared to IFN-γ producing Th1 cells .

• Regulatory circuit with IL4I1: A significant positive correlation exists between IL-4 induced gene 1 (IL4I1) and TOB1 mRNA expression in human Th17 cells, suggesting a regulatory network that limits TCR-mediated expansion of these cells .

• Impact on regulatory T cells: Lower proportions of CD4+CD25+FoxP3+ T regulatory (Treg) cells have been observed in Tob1-deficient mice, indicating TOB1's role in maintaining the balance between effector and regulatory T cell populations .

• Effects on proliferation thresholds: TOB1 sets activation thresholds for T cells, with its downregulation necessary for full T cell activation and proliferation. This mechanism appears differentially regulated across T cell subsets .

These findings highlight TOB1's complex role in maintaining immune homeostasis through differential effects on T cell subset development and function.

How has TOB1 been implicated in multiple sclerosis pathogenesis?

Evidence linking TOB1 to multiple sclerosis (MS) pathogenesis has emerged from both human studies and experimental models:

• Transcriptome analysis in MS patients: Genome-wide transcriptome analysis revealed that TOB1 downregulation (7-fold, the largest differential expression of any transcript) was associated with higher risk of disease activity in patients with MS .

• Molecular signature of disease risk: The high-risk signature in MS patients included downregulation of TOB1 along with pro-apoptotic genes and cell cycle inhibitors, suggesting T cells were poised to enter a proliferative state triggering concomitant disease flares .

• Experimental models: Tob1-deficient mice experienced earlier disease onset and more aggressive experimental autoimmune encephalomyelitis (EAE, a murine model of MS) when immunized with myelin oligodendrocyte glycoprotein peptide .

• T cell proliferation and differentiation: T cells from Tob1-deficient mice proliferated more vigorously and showed higher proportions of pro-inflammatory Th1 and Th17 cells with concomitant reduction in regulatory T cells .

• Adoptive transfer experiments: Transfer of Tob1-deficient CD4+ T cells into Rag1 knockout mice was sufficient to reproduce enhanced EAE symptoms, highlighting the importance of TOB1 specifically in CD4+ T cells .

• Spontaneous disease development: Crossing Tob1-deficient mice with transgenic mice expressing a myelin-specific T cell receptor resulted in spontaneous EAE development in approximately 50% of offspring .

These findings collectively establish TOB1 as an important regulator of autoimmune pathogenesis with particular relevance to MS.

What are promising future research directions for TOB1 antibodies in biomedical research?

Several promising research directions for TOB1 antibodies in biomedical research include:

• Biomarker development: TOB1 expression levels in peripheral blood T cells could serve as biomarkers for disease activity, progression, or therapeutic response in autoimmune disorders. TOB1 antibodies with improved sensitivity and specificity would facilitate clinical implementation .

• Single-cell applications: Adapting TOB1 antibodies for single-cell protein analysis would enable correlation of TOB1 expression with cell state at unprecedented resolution. This could reveal heterogeneity within T cell populations and identify specific subsets with disease-promoting potential .

• Multiplex imaging approaches: Developing TOB1 antibodies compatible with multiplexed tissue imaging technologies would allow spatial analysis of TOB1 expression in complex tissues such as inflammatory lesions .

• Therapeutic target validation: TOB1 antibodies could help validate this protein as a therapeutic target through in vivo imaging of TOB1 expression and function in preclinical models .

• Structural and functional studies: Epitope-specific TOB1 antibodies could help map functional domains and post-translational modifications critical for TOB1's diverse biological activities .

• Interaction proteomics: TOB1 antibodies optimized for immunoprecipitation would facilitate comprehensive mapping of TOB1's interactome across different cell types and disease states .

These research directions could significantly advance our understanding of TOB1 biology and its potential as a therapeutic target in autoimmune and inflammatory diseases.