TSC22D4 Antibody

What is TSC22D4 and why is it important in research?

TSC22D4 (also known as TILZ2, THG-1) is a transcriptional repressor and leucine zipper-containing protein that is highly conserved evolutionarily . It belongs to the TSC22 protein family which contains the evolutionarily conserved TSC box with a leucine zipper motif. This protein family consists of 4 members (TSC22D1, TSC22D2, TSC22D3, and TSC22D4) with alternatively spliced isoforms .

TSC22D4 is important in research because it:

• Regulates cellular processes including cell proliferation and cellular senescence

• Functions as a suppressor in tumorigenesis

• Plays a critical role in hepatic glucose and lipid metabolism

• Interacts with Akt1, a crucial mediator of insulin/PI3K signaling

• Is transcriptionally upregulated by various stimuli including anti-cancer drugs and growth inhibitors

What species reactivity do commercially available TSC22D4 antibodies demonstrate?

Based on the available data, commercial TSC22D4 antibodies show reactivity with:

What are the recommended applications for TSC22D4 antibodies?

TSC22D4 antibodies have been validated for multiple research applications:

The optimal dilution may vary depending on the specific experimental conditions and should be determined empirically.

How should TSC22D4 antibodies be stored and handled?

For optimal performance and longevity, TSC22D4 antibodies should be stored according to these guidelines:

• Short-term storage: 4°C for up to two weeks is recommended for immediate use

• Long-term storage: -20°C or -80°C in aliquots of no less than 20 μL

• Avoid repeated freeze-thaw cycles

• Many antibodies are supplied in PBS with stabilizers such as:

  • PBS (pH 7.2) with 40% glycerol and 0.02% sodium azide

  • PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• Some hybridoma products may contain the antimicrobial ProClin

How does TSC22D4 interact with Akt1 and what research methods best capture this interaction?

TSC22D4 has been identified as a novel protein kinase B/Akt1 interacting protein, which is a critical mediator of insulin/PI3K signaling pathway implicated in diseases including type 2 diabetes, obesity, and cancer . The interaction between TSC22D4 and Akt1 is not constitutive but regulatory and responds to various metabolic and stress signals.

Key findings about this interaction:

• Glucose and insulin stimulation or refeeding impairs hepatic TSC22D4-Akt1 interaction

• Mitochondrial inhibition and oxidative stress promote TSC22D4-Akt1 interaction

• TSC22D4 interacts specifically with Akt1 rather than Akt2

• The interaction involves the intrinsically disordered region of TSC22D4, particularly domain 2 (D2)

Research methods to capture this interaction:

• Co-immunoprecipitation (Co-IP) with endogenous proteins from tissue lysates

• Co-IP with tagged recombinant proteins in cell culture systems

• Domain mapping using deletion mutants

• Genetic reconstitution experiments in mouse models

What technical challenges might researchers encounter when using TSC22D4 antibodies for detecting endogenous protein?

Several technical challenges may arise when detecting endogenous TSC22D4:

• Antibody specificity issues: Given that TSC22D4 belongs to a family of proteins with conserved domains, cross-reactivity with other family members (TSC22D1, TSC22D2, TSC22D3) could occur . Researchers should validate antibody specificity through:

  • Protein arrays (as conducted with Sigma-Aldrich's antibody against 364 human recombinant protein fragments)

  • Knockout/knockdown validation controls

• Protein expression level variability: TSC22D4 expression is regulated by various stimuli and metabolic states , potentially resulting in variable detection levels depending on:

  • Nutritional status (fed vs. fasted)

  • Stress conditions

  • Disease states (e.g., cancer cachexia, liver damage)

• Detection of multiple isoforms: Given the presence of alternatively spliced isoforms , researchers might observe multiple bands with varying molecular weights.

• Post-translational modifications: TSC22D4 might undergo post-translational modifications that affect antibody recognition or apparent molecular weight.

How can researchers effectively validate TSC22D4 knockdown or knockout models?

Validation of TSC22D4 genetic models is critical for establishing reliable research systems. Based on the provided literature, researchers have successfully used several approaches:

• Protein level validation:

  • Western blot analysis using validated antibodies shows significantly reduced TSC22D4 protein levels in knockdown/knockout samples compared to controls

  • Quantitative comparison of band intensity normalized to loading controls

• mRNA level validation:

  • qRT-PCR to confirm reduction in TSC22D4 mRNA levels (>60% decrease observed in AAV-mediated knockdown)

• Functional validation:

  • Assessment of known TSC22D4-dependent phenotypes, such as:

    • Serum triglyceride levels (increased in TSC22D4-deficient models)

    • Glucose tolerance (improved in TSC22D4 hepatocyte-specific knockout mice on high-fat diet)

    • Insulin-induced Akt/GSK3β/FoxO1 phosphorylation (enhanced in knockout primary hepatocytes)

• Control experiments:

  • Use of non-specific shRNA/miRNA controls alongside TSC22D4-specific constructs

  • Rescue experiments with TSC22D4 reconstitution to confirm specificity

What experimental conditions optimize TSC22D4-Akt1 interaction studies?

To optimize experimental conditions for studying TSC22D4-Akt1 interactions, researchers should consider:

• Metabolic state manipulation:

  • Fasting conditions enhance the interaction

  • Insulin stimulation reduces the interaction

  • Refeeding disrupts the interaction in liver tissue

• Stress induction:

  • Mitochondrial inhibitors (antimycin/rotenone) promote the interaction

  • Oxidative stress (H₂O₂ treatment) enhances the interaction

• Buffer composition for co-immunoprecipitation:

  • Use buffers that preserve protein-protein interactions while minimizing non-specific binding

  • Include appropriate protease and phosphatase inhibitors to prevent degradation

• Protein domain considerations:

  • The D2 domain alone successfully interacts with Akt1

  • The D2+TSC combined domains exhibit stronger interaction with Akt1 compared to wild-type TSC22D4

  • The TSC box alone fails to interact with Akt1

What are the key differences between monoclonal and polyclonal TSC22D4 antibodies for research applications?

When selecting between monoclonal and polyclonal TSC22D4 antibodies, researchers should consider these differences:

For TSC22D4 research, the choice between monoclonal and polyclonal antibodies depends on the specific application needs:

• Use monoclonal antibodies when high specificity and reproducibility are critical

• Consider polyclonal antibodies for applications requiring higher sensitivity or when detecting potentially denatured proteins

How should researchers design experiments to study TSC22D4 domain function and protein interactions?

Based on the literature, effective experimental design for studying TSC22D4 domains and interactions includes:

• Domain mapping strategy:

  • Create systematic deletion mutants covering key domains :

    • R1 and R2 regions in the N-terminus

    • Intrinsically disordered regions (D1, D2)

    • TSC box domain

  • Generate truncation mutants containing only specific domains (e.g., D2 alone, TSC box alone, D2+TSC)

  • Use epitope tags (e.g., Flag) for detection and immunoprecipitation

• Interaction analysis approach:

  • Co-transfection of tagged constructs (e.g., Flag-TSC22D4 variants and HA-Akt1)

  • Co-immunoprecipitation followed by western blot analysis to detect interactions

  • Consider both forward and reverse co-IP to confirm interactions

  • Include appropriate controls (IgG control, empty vector)

• Functional validation:

  • Assess how domain mutations affect known TSC22D4 functions:

    • Akt1 binding capacity

    • Regulation of metabolic parameters

    • Response to cellular stressors

• Considerations for intrinsically disordered regions:

  • TSC22D4 contains a relatively long stretch of intrinsically disordered regions that play important roles in protein interactions

  • These regions may adopt different conformations depending on binding partners and conditions

  • Special care should be taken with experimental conditions that might affect these regions

What controls should be implemented when using TSC22D4 antibodies in chromatin immunoprecipitation (ChIP) studies?

When using TSC22D4 antibodies for ChIP applications (such as with PCRP-TSC22D4-3E10 ), researchers should implement these critical controls:

• Antibody specificity controls:

  • IgG isotype control to assess non-specific binding

  • Use of TSC22D4 knockout/knockdown samples as negative controls

  • Pre-adsorption with immunizing peptide/antigen when available

• Input controls:

  • Analysis of chromatin samples before immunoprecipitation (typically 1-10% of starting material)

  • Normalization of ChIP data to input signals

• Positive and negative genomic region controls:

  • Include primers for genomic regions known to be bound by TSC22D4 (positive control)

  • Include primers for genomic regions known not to be bound by TSC22D4 (negative control)

• Procedural controls:

  • Appropriate sonication/fragmentation verification

  • Cross-linking efficiency assessment

  • Multiple biological replicates to ensure reproducibility

• Validation strategies:

  • Confirmation of identified binding sites using independent methods (e.g., reporter assays)

  • Cross-validation using different TSC22D4 antibodies when available

  • Follow-up functional studies to confirm biological relevance of binding sites

What are the best practices for using TSC22D4 antibodies in studies of metabolic disease models?

When investigating TSC22D4 in metabolic disease models, researchers should follow these best practices:

• Model selection considerations:

  • High-fat diet models have shown TSC22D4-dependent effects on glucose handling

  • Cancer cachexia models reveal roles in liver metabolism reprogramming

  • Consider both acute (viral-mediated knockdown) and chronic (genetic knockout) approaches

• Experimental conditions optimization:

  • Control feeding/fasting status as it affects TSC22D4-Akt1 interactions

  • Consider both basal and insulin-stimulated conditions

  • Assess multiple timepoints to capture dynamic changes

• Comprehensive metabolic assessment:

  • Measure multiple parameters including:

    • Glucose tolerance

    • Insulin sensitivity

    • Serum triglycerides and cholesterol

    • Hepatic lipid content

    • Insulin signaling pathway components (Akt, GSK3β, FoxO1, S6K1 phosphorylation)

• Tissue-specific considerations:

  • Focus on liver as a primary site of TSC22D4 metabolic function

  • Consider potential roles in other metabolically active tissues

  • Use tissue-specific gene manipulation approaches (e.g., AAV with liver-specific promoters)

• Validation in human samples:

  • Correlate findings with human disease states where possible

  • Previous studies found that elevated TSC22D4 expression in human livers positively correlates with insulin resistance