TNIP1 Antibody, Biotin conjugated

What is TNIP1 and what cellular functions does it regulate?

TNIP1 (TNFAIP3 Interacting Protein 1), also known as ABIN-1, is a 72 kDa protein that plays crucial roles in multiple cellular pathways. It primarily functions as a key repressor of inflammatory signaling by inhibiting nuclear factor-κB (NF-κB) activation through its interaction with TNF alpha-induced protein 3 (TNFAIP3/A20) . TNIP1 is found in both nuclear and cytoplasmic compartments, where it performs distinct functions. In the nucleus, it acts as a corepressor of ligand-bound retinoic acid receptors (RARs) and peroxisome proliferator acid receptors (PPARs). In the cytoplasm, it interacts with numerous proteins including HIV-encoded proteins (nef and matrix), modulates signaling downstream of epidermal growth factor receptor (EGFR) via interactions with ERK2, and participates in autophagy regulation . Recent studies have also identified TNIP1 as a selective autophagy receptor involved in recruiting signaling molecules like MyD88 and IRAK1 to autophagosomes, thereby regulating Toll-like receptor signaling and inflammatory responses .

What applications are biotin-conjugated TNIP1 antibodies typically used for?

Biotin-conjugated TNIP1 antibodies offer enhanced detection sensitivity and versatility across multiple experimental applications. These antibodies are particularly valuable in flow cytometry, immunohistochemistry/immunofluorescence, ELISA, and protein pull-down experiments. The biotin conjugation allows for signal amplification through the strong biotin-streptavidin interaction, enabling detection of low-abundance TNIP1 proteins in complex tissue samples. Most commonly, these conjugated antibodies are employed in multi-color flow cytometry panels to identify TNIP1-expressing cell populations, in immunohistochemistry for visualization of TNIP1 distribution in tissue sections with amplified signal, and in chromatin immunoprecipitation (ChIP) assays to study TNIP1's role in transcriptional regulation . When designing experiments with biotin-conjugated TNIP1 antibodies, researchers should consider potential endogenous biotin interference in certain sample types and implement appropriate blocking steps.

What is the reactivity profile of commercially available TNIP1 antibodies?

Commercial TNIP1 antibodies show varying species reactivity profiles depending on the specific clone and manufacturer. Based on the available data, most TNIP1 antibodies demonstrate confirmed reactivity with human and mouse samples . For instance, the polyclonal antibody (15104-1-AP) has been tested and validated for both human and mouse reactivity across multiple applications including Western blot (WB), immunohistochemistry (IHC), immunofluorescence/immunocytochemistry (IF/ICC), immunoprecipitation (IP), and ELISA . The mouse monoclonal variant (68387-1-Ig) has been validated primarily in human samples, with positive Western blot detection in U2OS, A549, and HeLa cell lines . When selecting a TNIP1 antibody for your research, it's critical to verify both the tested and cited reactivity profiles for your species of interest, particularly for less common research models. Cross-reactivity testing is recommended when working with species not explicitly validated by the manufacturer.

How should I optimize biotin-conjugated TNIP1 antibody concentration for immunofluorescence studies?

Optimizing biotin-conjugated TNIP1 antibody concentration for immunofluorescence requires a systematic titration approach to maximize specific signal while minimizing background. Begin with a wide concentration range (typically 1:100 to 1:2000 dilutions) based on the antibody's reported working concentration. Conduct parallel experiments on positive control samples (cells or tissues known to express TNIP1, such as U2OS, A549, or HeLa cells) and negative controls (either TNIP1-knockout samples or by omitting the primary antibody).

What controls should be included when using biotin-conjugated TNIP1 antibodies in functional studies of autophagy pathways?

When investigating TNIP1's role in autophagy pathways using biotin-conjugated antibodies, a comprehensive set of controls is essential to ensure reliable data interpretation. First, include cellular controls that represent different autophagy states: basal autophagy (normal conditions), induced autophagy (starvation or rapamycin treatment), and autophagy inhibition (bafilomycin A1 treatment) . Since TNIP1 shows altered localization and function during autophagy regulation, these controls help establish baseline dynamics.

For antibody validation, include:

• Positive control: Cells with confirmed TNIP1 expression (U2OS, A549, HeLa)

• Negative control: TNIP1-knockout cells or siRNA-mediated knockdown

• Epitope competition: Pre-incubate antibody with excess TNIP1 fusion protein/peptide

• Isotype control: Biotin-conjugated IgG of the same isotype (rabbit or mouse IgG depending on your antibody)

For colocalization studies investigating TNIP1's interaction with autophagy proteins (LC3B, ATG5, ATG7), include both single-stained controls to assess bleed-through and samples co-stained with established autophagy markers. Additionally, confirm the specificity of detected interactions using co-immunoprecipitation. When evaluating autophagy flux, combine TNIP1 staining with LC3-II/LC3-I ratio assessments by Western blot, with and without autophagy inhibitors like bafilomycin A1, as demonstrated in recent TNIP1 studies .

How can I design experiments to investigate TNIP1's role in TLR7-mediated autoimmunity using biotin-conjugated antibodies?

To investigate TNIP1's role in TLR7-mediated autoimmunity, design a multi-faceted experimental approach that leverages the advantages of biotin-conjugated TNIP1 antibodies. Begin with cellular models that recapitulate aspects of TLR7-mediated autoimmunity, such as B cells or plasmacytoid dendritic cells from autoimmune-prone mouse models or patient-derived cells.

First, establish TNIP1 expression and localization patterns in these cells using immunofluorescence with biotin-conjugated antibodies and streptavidin-fluorophore detection. Compare cells with wild-type TNIP1 to those expressing disease-associated variants (such as Q333P) , examining TNIP1 localization changes in response to TLR7 stimulation with R848.

For signaling studies, design co-localization experiments to track TNIP1's association with MyD88 and IRAK1 in autophagosomes following TLR7 activation. Recent research indicates that TNIP1 Q333P shows reduced colocalization with MyD88 in autophagosomes and diminished interaction with IRAK1 . Include appropriate controls for autophagosome formation (ATG5, LC3B staining).

To investigate functional outcomes, measure type I interferon (IFN1) responses in cells expressing wild-type versus mutant TNIP1, following TLR7 stimulation. This can be done using reporter assays or by directly measuring IFNβ production. Design parallel experiments with TLR7 antagonists and MyD88 inhibitors to confirm the pathway specificity.

For in vivo studies, utilize biotin-conjugated TNIP1 antibodies in immunohistochemistry to examine TNIP1 expression and localization in tissues from autoimmune models, particularly focusing on B cell-rich regions in secondary lymphoid organs and sites of inflammation like salivary glands .

How can I address non-specific binding when using biotin-conjugated TNIP1 antibodies in tissue samples?

Non-specific binding is a common challenge when using biotin-conjugated TNIP1 antibodies, particularly in tissue samples with high endogenous biotin content. To address this issue, implement a systematic troubleshooting approach. First, always incorporate an avidin-biotin blocking step before primary antibody incubation. Commercial avidin-biotin blocking kits are effective, but ensure complete blocking by testing various incubation times (15-30 minutes per component).

Second, optimize your blocking solution by testing different blockers beyond standard BSA or normal serum. For tissues with persistent background, try a combination of 1% BSA, 5% normal serum (matching the species of your secondary reagent), 0.1% Tween-20, and 0.1% Triton X-100. Additionally, include 0.1% cold fish skin gelatin which effectively reduces non-specific hydrophobic interactions.

If background persists, consider these additional strategies:

• Use a biotin-free detection system temporarily to determine if the issue is biotin-specific

• Increase washing stringency with higher salt PBS (up to 500 mM NaCl) or add 0.1% Tween-20 to wash buffers

• Reduce primary antibody concentration and extend incubation time (overnight at 4°C)

• Pre-adsorb the antibody against tissues from TNIP1-knockout models or with tissue powder

For critical applications, validate staining patterns by comparing results with a non-conjugated TNIP1 antibody using conventional detection methods. This comparative approach can help distinguish true TNIP1 signals from biotin-related artifacts.

What might cause conflicting results when analyzing TNIP1's effects on NF-κB versus type I interferon pathways?

Conflicting results when analyzing TNIP1's effects on NF-κB versus type I interferon pathways can stem from several methodological and biological factors. Recent research has revealed that TNIP1 variants can differentially impact these pathways. For instance, the Q333P variant impairs TNIP1's ability to repress interferon-β production while maintaining normal inhibition of NF-κB signaling . This domain-specific effect occurs because different TNIP1 domains regulate distinct signaling pathways – the ubiquitin-binding region primarily regulates NF-κB, while the AHD3 domain (containing the Q333P variant) is essential for IFN1 regulation through interactions with TAX1BP1 and TBK1 .

To resolve conflicting results:

• Ensure pathway-specific readouts: Use separate reporter systems for NF-κB (using consensus NF-κB response elements) and IFN1 (using IFNβ promoter elements)

• Validate pathway activation with multiple measures: For NF-κB, assess IκBα phosphorylation/degradation, p65 nuclear translocation, and target gene expression; for IFN1, measure IRF3 phosphorylation, IFNβ production, and ISG expression

• Consider cell type-specific effects: TNIP1 may regulate these pathways differently in various cell types (B cells versus macrophages versus epithelial cells)

• Account for temporal differences: NF-κB activation typically occurs earlier than IFN1 responses

• Evaluate stimulus specificity: Different TLR ligands (CpG-B, R848, LPS) can activate TNIP1 differently

When comparing studies, pay careful attention to the specific TNIP1 domains being studied, experimental readouts, and cellular contexts, as these can significantly influence observed regulatory effects on each pathway.

Why might I observe discrepancies in TNIP1 molecular weight between Western blot and immunoprecipitation experiments?

Discrepancies in TNIP1 molecular weight between Western blot and immunoprecipitation experiments can occur due to several factors related to both the protein's biology and experimental conditions. TNIP1 has a calculated molecular weight of 72 kDa , but post-translational modifications, protein activation state, and experimental conditions can all affect its observed molecular weight.

TNIP1 undergoes phosphorylation upon activation by TLR signaling, which is detected as a higher molecular weight band . This activation-induced shift is particularly prominent following stimulation with TLR ligands like R848 (TLR7/8 agonist) in B cells and plasmacytoid dendritic cells. Additionally, activation can lead to subsequent degradation of TNIP1, particularly through autophagy pathways, which may result in lower molecular weight bands.

To address such discrepancies:

• Always run positive control lysates from cells known to express TNIP1 (U2OS, A549, HeLa)

• Include both stimulated and unstimulated samples to observe activation-dependent shifts

• Use freshly prepared samples with protease and phosphatase inhibitors to prevent degradation

• For immunoprecipitation experiments, compare results using different lysis buffers (RIPA versus NP-40) as buffer composition can affect protein conformation and epitope accessibility

• Validate with multiple TNIP1 antibodies targeting different epitopes

If discrepancies persist, consider:

• Performing phosphatase treatment on a portion of your samples to determine if shifts are phosphorylation-dependent

• Using 2D gel electrophoresis to separate TNIP1 isoforms by both isoelectric point and molecular weight

• Confirming TNIP1 identity by mass spectrometry analysis of the immunoprecipitated protein bands

How should I interpret changes in TNIP1 localization during autophagy and inflammatory responses?

When analyzing immunofluorescence data, several patterns are significant:

• Punctate cytoplasmic TNIP1 staining following TLR stimulation indicates recruitment to autophagosomes. This should be validated by co-localization with LC3B and ATG7 . The degree of this punctate redistribution correlates with autophagy activation.

• Decreased nuclear TNIP1 following inflammatory stimulation suggests mobilization to cytoplasmic signaling complexes.

• Co-localization with MyD88 and IRAK1 in punctate structures indicates TNIP1's involvement in regulating TLR signaling via autophagy mechanisms .

Disease-associated TNIP1 variants, such as Q333P, show defective localization to autophagosomes and impaired co-localization with MyD88 . When interpreting such data, quantify co-localization using established metrics (Pearson's correlation coefficient, Manders' overlap coefficient) across multiple cells.

Additionally, temporal dynamics are crucial - TNIP1 localization changes follow specific kinetics after stimulation, with early recruitment to signaling complexes followed by later autophagosome association. This sequential pattern helps distinguish TNIP1's direct signaling roles from its autophagy-related functions. For comprehensive interpretation, combine localization data with functional readouts like cytokine production or activation of downstream signaling molecules.

What criteria should be used to validate TNIP1 antibody specificity in autoimmune disease models?

Validating TNIP1 antibody specificity in autoimmune disease models requires a multi-layered approach to ensure reliable data interpretation, particularly given TNIP1's involvement in multiple autoimmune conditions including lupus, scleroderma, and psoriasis . Establish the following validation criteria:

First, perform genetic validation using parallel testing in wild-type and TNIP1-deficient or TNIP1-mutant models. Recent studies with Q346P Tnip1 mice (vikala model) provide an excellent genetic control system . The antibody should demonstrate appropriate signal differences between these models across multiple applications (Western blot, immunohistochemistry, flow cytometry).

Second, apply peptide competition assays where the antibody is pre-incubated with excess TNIP1 recombinant protein or immunizing peptide before application. Signal abolishment confirms specificity. For biotin-conjugated antibodies, include additional controls with unrelated biotinylated proteins to rule out non-specific biotin interactions.

Third, validate across multiple detection methods. Results from biotin-conjugated antibody experiments should align with those using unconjugated primary antibodies detected via standard secondary methods. This cross-validation is particularly important when studying newly identified TNIP1 variants in disease models.

Fourth, demonstrate appropriate subcellular localization patterns. TNIP1 should show both nuclear and cytoplasmic distribution , with stimulus-dependent changes in localization. In autoimmune models, TNIP1 variants may show altered localization patterns, as seen with the Q333P variant's impaired recruitment to autophagosomes .

Finally, confirm physiological relevance by demonstrating expected changes in TNIP1 expression or localization following disease-relevant stimulation (e.g., TLR ligands) and correlation with disease phenotypes (e.g., interferon production, autoantibody levels).

How can I differentiate between direct and indirect effects of TNIP1 on TLR signaling pathways?

Differentiating between direct and indirect effects of TNIP1 on TLR signaling pathways requires sophisticated experimental approaches that isolate specific interaction mechanisms. Recent findings have revealed that TNIP1 regulates TLR signaling through multiple mechanisms, including direct protein-protein interactions and autophagy-mediated regulation .

To dissect these mechanisms:

• Utilize domain-specific TNIP1 mutants in reconstitution experiments. The Q333P mutation in the AHD3 domain specifically impacts type I interferon signaling while preserving NF-κB inhibition , allowing you to separate these functions. Similarly, mutations in the ubiquitin-binding domain versus the LIR motif can help distinguish ubiquitin-dependent from autophagy-dependent effects.

• Employ protein interaction studies with temporal resolution. Use co-immunoprecipitation at different time points after TLR stimulation to track the kinetics of TNIP1's association with key signaling molecules (MyD88, IRAK1, TBK1). Direct effects typically manifest as immediate protein-protein interactions, while indirect effects emerge later through secondary mechanisms.

• Use autophagy inhibition versus direct signaling inhibition. Compare the effects of autophagy inhibitors (bafilomycin A1) with direct TLR signaling inhibitors. If TNIP1's effects are primarily autophagy-dependent, they should be mimicked by autophagy inhibition .

• Apply proximity labeling techniques (BioID, APEX) with TNIP1 as the bait to identify direct versus indirect interactors in living cells following TLR stimulation.

• Conduct in vitro reconstitution assays with purified components. Direct effects should be reproducible with purified proteins, while indirect effects requiring additional cellular machinery will not be observed.

By integrating these approaches, you can create an interaction map distinguishing TNIP1's direct binding partners from those affected through secondary mechanisms like altered protein degradation, sequestration, or conformational changes in signaling complexes.

How might biotin-conjugated TNIP1 antibodies be utilized to study the newly identified Q333P variant in autoimmune disorders?

Biotin-conjugated TNIP1 antibodies offer powerful tools for investigating the newly identified Q333P variant associated with systemic autoimmune disorders featuring antinuclear antibodies and IgG4 elevation . These conjugated antibodies can be deployed in multiple sophisticated applications to elucidate how this variant alters TNIP1 function.

First, for subcellular localization studies, biotin-conjugated antibodies enable high-sensitivity multi-color imaging to track the Q333P variant's impaired localization to autophagosomes and damaged mitochondria . Using streptavidin-conjugated quantum dots or fluorophores allows for multiplexed imaging with markers for autophagosomes (LC3B, ATG7), mitochondria (TOMM20), and signaling molecules (MyD88, IRAK1) to visualize the variant's defective recruitment patterns in patient-derived cells or mouse models.

Second, for protein-protein interaction analysis, proximity ligation assays (PLA) using biotin-conjugated TNIP1 antibodies combined with antibodies against putative interaction partners can quantitatively assess how the Q333P variant affects TNIP1's interactions with MyD88, IRAK1, and TBK1 in situ . This approach provides spatial information about where interactions occur (or fail to occur) within cells.

Third, for mechanistic studies, combine biotin-conjugated TNIP1 antibodies with FRET-based sensors for TLR7 activity and type I interferon signaling to directly visualize how the Q333P variant alters signaling dynamics in living cells. Time-lapse imaging following TLR7 stimulation can reveal how wild-type versus mutant TNIP1 differentially regulates these pathways.

Finally, for translational applications, develop tissue-specific TNIP1 expression atlases comparing healthy controls with autoimmune patients carrying the Q333P variant, focusing on affected tissues like salivary glands and lymphoid organs. Biotin-conjugated antibodies enable signal amplification that improves detection sensitivity in tissue microarrays, facilitating correlation of TNIP1 expression patterns with clinical parameters.

What methodological approaches can determine if TNIP1's regulation of autophagy versus inflammatory signaling can be therapeutically targeted separately?

Determining whether TNIP1's dual functions in autophagy regulation and inflammatory signaling can be therapeutically targeted separately requires sophisticated methodological approaches that isolate these functions mechanistically. Recent research has revealed that different TNIP1 domains mediate distinct functions, suggesting the possibility of selective targeting .

First, employ domain-specific functional mapping using CRISPR-Cas9 to generate cell lines expressing TNIP1 with mutations in specific functional domains: the ubiquitin-binding domain (critical for NF-κB regulation), the LC3-interacting region (essential for autophagy functions), and the AHD3 domain (important for type I interferon regulation) . Characterize these domain-specific mutants using biotin-conjugated wild-type TNIP1 antibodies combined with domain-specific antibodies to compare localization patterns and protein interactions.

Second, develop quantitative multiplex assays that simultaneously monitor autophagy flux (LC3-II/LC3-I ratio, p62 degradation) and inflammatory signaling (NF-κB activation, type I interferon production) in the same cells. This approach allows direct comparison of how domain-specific mutations or candidate therapeutic compounds differentially affect these pathways. High-content imaging using biotin-conjugated TNIP1 antibodies with autophagosome markers and signaling molecules enables single-cell correlation analysis.

Third, employ chemical genetics approaches by screening compound libraries for molecules that selectively modulate TNIP1's autophagy functions without affecting inflammatory signaling, or vice versa. For each candidate compound, assess:

• Effects on TNIP1 localization to autophagosomes versus inflammatory signaling complexes

• Impact on protein-protein interactions with autophagy proteins versus signaling molecules

• Differential effects on autophagy flux versus inflammatory cytokine production

Finally, validate the most promising approach in disease-relevant models, such as the Q346P Tnip1 mouse model , tracking whether selective targeting of one pathway versus the other differentially affects autoimmune phenotypes. This comprehensive methodology would determine whether TNIP1's functions can be therapeutically uncoupled, potentially enabling pathway-specific interventions for autoimmune disorders.

How can biotin-conjugated TNIP1 antibodies be integrated into multi-omics approaches to understand TNIP1's role in coordinating autophagy and immunity?

Biotin-conjugated TNIP1 antibodies provide unique advantages for integration into multi-omics frameworks to comprehensively understand TNIP1's coordination of autophagy and immunity. These conjugated antibodies enable specific isolation of TNIP1-associated complexes that can be analyzed across multiple molecular dimensions.

For spatially-resolved proteomics, implement proximity-dependent biotinylation (BioID or TurboID) by fusing these enzymes to TNIP1, then use streptavidin pulldown followed by mass spectrometry to identify the complete TNIP1 proximal proteome in different subcellular compartments (autophagosomes, mitochondria, signaling complexes). Compare wild-type TNIP1 with disease-associated variants like Q333P under basal and stimulated conditions to map context-specific interaction networks.

For integrated epigenome and transcriptome analysis, combine CUT&RUN or CUT&Tag using biotin-conjugated TNIP1 antibodies with RNA-seq to correlate TNIP1's nuclear localization patterns with transcriptional outputs. Since TNIP1 functions as a corepressor for retinoic acid receptors and other nuclear receptors , this approach can reveal how its nuclear functions cooperate with cytoplasmic activities in coordinating immune responses.

For single-cell multi-modal analysis, employ CITE-seq or ASAP-seq with biotin-conjugated TNIP1 antibodies alongside markers for autophagy activity and immune cell activation states. This approach can identify cell populations where TNIP1 expression correlates with specific functional states across heterogeneous immune populations in health and autoimmune conditions.

For metabolic profiling integrated with TNIP1 function, use immunocapture of TNIP1-bound mitochondria (given TNIP1's role in mitophagy) followed by metabolomic analysis to understand how TNIP1 variants affect mitochondrial metabolism and subsequent immune activation.

Finally, develop computational frameworks that integrate these multi-omic datasets to model the dynamic interplay between TNIP1-regulated autophagy and immune signaling networks. Such integrated approaches will reveal how perturbations in TNIP1 function propagate across cellular systems to drive autoimmune pathology, potentially identifying network vulnerabilities for therapeutic targeting.

What is the tissue expression profile of TNIP1 across human and mouse samples?

TNIP1 expression shows remarkable conservation across human and mouse tissues, with highest expression in immune cells and epithelial barriers. The protein exhibits both nuclear and cytoplasmic localization, reflecting its dual functions in transcriptional regulation and cytoplasmic signaling. Expression patterns are dynamically regulated during inflammatory responses, with notable upregulation in activated immune cells and inflamed tissues . This expression profile underlies TNIP1's critical role in multiple autoimmune conditions affecting various organ systems.

How do different TNIP1 variants affect protein function across multiple signaling pathways?

This comprehensive analysis reveals that different TNIP1 variants selectively impact specific signaling pathways and functions. The Q333P variant, recently identified in human autoimmune disease, specifically impacts type I interferon regulation while preserving NF-κB inhibitory function . This selective effect occurs because the mutation resides in the AHD3 domain critical for interactions with TAX1BP1 and regulation of mitophagy, but not in the ubiquitin-binding region required for NF-κB regulation . By contrast, the D472N variant primarily affects NF-κB regulation. These distinct functional consequences highlight the domain-specific nature of TNIP1's regulatory activities and suggest that different TNIP1 variants may predispose to distinct autoimmune phenotypes, potentially guiding precision medicine approaches to TNIP1-related disorders.

What is the reactivity and application performance of biotin-conjugated TNIP1 antibodies compared to other conjugates?

Performance rating scale: + (Poor) to ++++ (Excellent); N/A: Not applicable

Biotin-conjugated TNIP1 antibodies show superior performance in applications requiring signal amplification or multi-label detection. The strong biotin-streptavidin interaction (Kd ≈ 10^-15 M) provides exceptional sensitivity for detecting low-abundance TNIP1 in complex samples. This is particularly valuable for tissue staining applications and for detecting TNIP1 in specific subcellular compartments like autophagosomes or mitochondria .

For microscopy applications, biotin-conjugated antibodies offer greater flexibility than direct fluorophore conjugates, as they can be paired with different streptavidin-fluorophore conjugates depending on the experimental design. They are ideal for multiplexed imaging studies investigating TNIP1's co-localization with autophagy proteins (LC3B, ATG7) and signaling molecules (MyD88, IRAK1) .

The main limitation of biotin-conjugated antibodies is potential interference from endogenous biotin in certain tissues, requiring appropriate blocking steps. Additionally, they require a two-step detection process compared to directly conjugated fluorophore antibodies. Nevertheless, their versatility and signal amplification capabilities make them the preferred choice for advanced TNIP1 research applications, particularly when investigating the complex interplay between TNIP1's functions in autophagy and immune regulation.