TNIP1 Antibody, FITC conjugated

What is TNIP1 and why is it important in immunological research?

TNIP1, also known as ABIN-1 (A20-binding inhibitor of NF-kappa-B activation 1), is a critical regulator of inflammatory pathways. It functions by interacting with TNFAIP3 (A20) to inhibit NF-κB activation . TNIP1 represses RARs (Retinoic Acid Receptors) in the presence of RA, placing it in a small category of corepressors of agonist-bound nuclear receptors . Recent research has identified TNIP1 variants (particularly Q346P) associated with systemic autoimmune diseases featuring antinuclear antibodies with IgG4 elevation . TNIP1's importance stems from its role in regulating multiple immune signaling pathways, including TLR7 signaling and autophagy-related processes, making it a valuable target for understanding autoimmune disease mechanisms .

What applications is the TNIP1 Antibody, FITC conjugated suitable for?

The FITC-conjugated TNIP1 antibody is primarily validated for ELISA applications with a recommended dilution range of 1:100-1:500 . While the FITC-conjugated version has specific applications, non-conjugated versions of TNIP1 antibodies have broader application ranges including:

It's important to note that optimal dilutions may be sample-dependent and should be determined empirically for each experimental system .

How should TNIP1 Antibody, FITC conjugated be stored for optimal stability?

For optimal stability, store the TNIP1 antibody, FITC conjugated at -20°C. Based on storage protocols for similar antibodies, it's likely stable for one year after shipment when properly stored. The antibody is typically provided in PBS buffer with 0.02% sodium azide and 50% glycerol at pH 7.3 . For small volume products (e.g., 20μl), they may contain 0.1% BSA as a stabilizer. Aliquoting may be unnecessary for -20°C storage, but is recommended for antibodies that will undergo multiple freeze-thaw cycles to preserve activity .

What is the molecular weight of TNIP1 and what protein bands should researchers expect?

The calculated molecular weight of human TNIP1 is 72 kDa, which corresponds with the observed molecular weight in Western blot applications . Researchers should expect to see a band at approximately 72 kDa when using this antibody for protein detection. Multiple isoforms or post-translational modifications may result in additional bands, but the primary band should appear at 72 kDa .

How does the TNIP1 Q346P variant affect autoimmune disease pathogenesis, and how can FITC-conjugated antibodies help investigate this?

The TNIP1 Q346P variant (Q333P in humans) drives autoimmune pathology through distinct mechanisms different from previously described TNIP1 variants. Unlike the D485N variant that impairs NF-κB signaling, Q346P specifically dysregulates interferon-β (IFNβ) production through two key mechanisms:

• Impaired MyD88/IRAK1 recruitment to autophagosomes: The Q346P variant shows reduced colocalization with MyD88 in autophagosomes compared to wild-type TNIP1, resulting in sustained TLR7 signaling . This is evidenced by diminished interaction between TNIP1 Q333P and MyD88 in co-immunoprecipitation experiments.

• Defective mitophagy: The variant impairs TNIP1 localization to damaged mitochondria and disrupts mitophagosome formation, leading to accumulated damaged mitochondria in salivary epithelial cells of Q333P Tnip1 mice .

FITC-conjugated TNIP1 antibodies can help investigate these mechanisms through immunofluorescence microscopy to visualize:

• TNIP1 localization to autophagosomes (using co-staining with autophagosome markers)

• Colocalization with MyD88 and IRAK1

• Association with mitochondrial markers

• Differences in localization patterns between wild-type and variant TNIP1

These experiments would allow quantitative assessment of colocalization coefficients and provide visual evidence of the altered protein interactions in disease states .

What controls and validation steps should be included when using TNIP1 Antibody, FITC conjugated in flow cytometry or imaging applications?

When using FITC-conjugated TNIP1 antibody for flow cytometry or imaging applications, the following controls and validation steps are essential:

• Antibody specificity controls:

  • Isotype control: Rabbit IgG-FITC at the same concentration to assess non-specific binding

  • Blocking peptide competition: Pre-incubate antibody with immunogen peptide (recombinant Human TNFAIP3-interacting protein 1 protein, amino acids 526-636) to confirm specificity

  • Genetic validation: Compare staining in TNIP1 knockout/knockdown cells versus wild-type cells

• Technical controls:

  • Unstained cells: To establish autofluorescence baseline

  • Single-color controls: For compensation when using multiple fluorophores

  • Fixation/permeabilization validation: Compare different protocols as TNIP1 localization includes both cytoplasmic and nuclear compartments

• Application-specific validation:

  • For flow cytometry: Titration experiments (1:50, 1:100, 1:200, 1:500) to determine optimal signal-to-noise ratio

  • For imaging: Co-localization with known TNIP1 interaction partners (TNFAIP3, MyD88, LC3) to validate functional localization

  • Secondary antibody-only controls: To assess background from secondary reagents

Validation should include Western blot confirmation of protein expression in the same samples used for flow cytometry or imaging to correlate protein levels with fluorescence intensity .

How can researchers optimize protocols to investigate TNIP1's role in selective autophagy using FITC-conjugated antibodies?

Investigating TNIP1's role in selective autophagy requires careful protocol optimization:

• Autophagy induction and blocking:

  • Establish baseline autophagy with starvation (EBSS medium for 2-4 hours)

  • Block autophagosome-lysosome fusion with Bafilomycin A1 (100 nM for 2-4 hours) to accumulate autophagosomes

  • Compare TLR7 stimulation (R848, 1 μg/ml) versus unstimulated conditions to observe TNIP1 recruitment to autophagosomes

• Immunofluorescence optimization:

  • Fixation method: 4% paraformaldehyde (10 minutes) preserves autophagosome structures

  • Permeabilization: 0.1% Triton X-100 (5 minutes) for optimal antibody penetration

  • Blocking: 5% BSA in PBS (1 hour) to minimize non-specific binding

  • TNIP1 antibody dilution: Start with 1:100 and optimize based on signal-to-noise ratio

• Co-localization analysis:

  • Counter-stain with LC3B antibody (autophagosome marker)

  • Include MyD88 staining to assess recruitment to autophagosomes

  • Quantify co-localization using Pearson's correlation coefficient or Manders' overlap coefficient

• Advanced approaches:

  • Live-cell imaging: Monitor dynamic recruitment of TNIP1 to autophagosomes using genetically-encoded fluorescent tags combined with immunostaining

  • Super-resolution microscopy: Resolve sub-autophagosomal structures (STED or STORM microscopy)

  • FRET analysis: Assess direct protein-protein interactions between TNIP1 and autophagy proteins

When analyzing results, compare wild-type TNIP1 with the Q333P variant, which shows reduced colocalization with MyD88 in autophagosomes as demonstrated in previous studies .

What approaches can resolve contradictory data when TNIP1 antibody signals differ between applications or experimental conditions?

When faced with contradictory TNIP1 antibody results across different applications, researchers should implement a systematic troubleshooting approach:

• Antibody validation across multiple techniques:

  • Cross-validate with multiple TNIP1 antibodies targeting different epitopes

  • Confirm antibody specificity using TNIP1 knockout/knockdown controls in each application

  • Verify antibody lot consistency through standardized positive controls

• Protein expression and modification analysis:

  • Evaluate TNIP1 post-translational modifications that might mask epitopes under certain conditions

  • Consider that activated TNIP1 appears as a higher molecular weight band after TLR stimulation (specifically with R848 and CpG-B)

  • Assess protein expression levels across different cell types; TNIP1 has been validated in multiple cell lines including A549, HEK-293, HeLa, and U2OS cells

• Technical optimization strategies:

  • For Western blotting: Test different lysis buffers, as TNIP1 interacts with ubiquitin and autophagy machinery

  • For immunofluorescence: Compare different fixation methods; paraformaldehyde might preserve certain epitopes better than methanol

  • For flow cytometry: Optimize permeabilization conditions to ensure antibody access to all cellular compartments

• Experimental condition variables:

  • Document cell activation state; TLR stimulation triggers TNIP1 activation visible as a higher molecular weight band

  • Control for autophagy status; treatment with Bafilomycin A1 increases TNIP1 protein levels

  • Consider cell confluency and passage number effects on TNIP1 expression

• Data integration approach:

  • Create a comprehensive data matrix comparing results across all techniques

  • Weight evidence based on validation controls included in each experiment

  • Consider orthogonal approaches such as mass spectrometry to resolve discrepancies in protein identification

In published studies, TNIP1 antibodies have successfully detected the protein in various applications (WB, IP, IHC, IF/ICC) with consistent results when properly optimized .

How can TNIP1 Antibody, FITC conjugated be utilized to investigate the relationship between TNIP1 and TLR7 signaling in autoimmune disorders?

The relationship between TNIP1, TLR7 signaling, and autoimmunity can be investigated using FITC-conjugated TNIP1 antibodies through several methodological approaches:

• Characterization of patient samples:

  • Compare TNIP1 expression and localization in peripheral blood mononuclear cells (PBMCs) from patients with suspected TNIP1-mediated autoimmunity versus healthy controls

  • Flow cytometry with FITC-TNIP1 antibody (1:100 dilution) can quantify expression levels across immune cell subsets

  • Correlate TNIP1 expression with serum IgG4 levels and antinuclear antibody titers

• Functional assessment of TLR7 signaling:

  • Stimulate cells with TLR7 agonist R848 (1 μg/ml) and assess TNIP1 recruitment to signaling complexes

  • Use immunofluorescence microscopy to visualize TNIP1-FITC colocalization with MyD88 and autophagosome markers before and after stimulation

  • Quantify TNIP1-MyD88 interaction using proximity ligation assay in different genetic backgrounds

• Genetic rescue experiments:

  • In cells expressing TNIP1 Q333P variant, evaluate if overexpression of wild-type TNIP1 rescues:

    • IFNβ production (measured by ELISA)

    • MyD88 localization to autophagosomes

    • Mitophagy efficiency

  • TLR7 knockout/inhibition experiments to determine if TLR7 blockade can rescue phenotypes in Q333P expressing cells

• Multi-parameter analysis:

  • Design panels for spectral flow cytometry combining TNIP1-FITC with:

    • TLR7 expression

    • Phosphorylated TBK1 (marker of pathway activation)

    • Interferon-stimulated gene products

    • B cell activation markers

This approach has strong clinical relevance as research has demonstrated that TNIP1-mediated autoimmunity may respond to TLR7-targeted therapeutics, providing a pathway-specific treatment strategy for patients with this genetic variant .

What are the optimal fixation and permeabilization protocols for intracellular staining with TNIP1 Antibody, FITC conjugated?

Optimal fixation and permeabilization for intracellular TNIP1 staining requires careful protocol selection based on experimental goals:

• Standard protocol for most applications:

  • Fixation: 4% paraformaldehyde in PBS for 10-15 minutes at room temperature

  • Wash: 3× with PBS

  • Permeabilization: 0.1-0.3% Triton X-100 in PBS for 5-10 minutes

  • Blocking: 5% normal serum (match secondary antibody host) or 3% BSA in PBS for 30-60 minutes

  • TNIP1-FITC antibody incubation: 1:100 dilution in 1% BSA/PBS overnight at 4°C

• Flow cytometry-specific protocol:

  • Fixation: BD Cytofix (contains 4% paraformaldehyde) for 15 minutes at room temperature

  • Permeabilization: BD Perm Buffer III (for phospho-epitopes) or 0.1% saponin (for cytoplasmic proteins)

  • TNIP1-FITC antibody: 1:100 dilution in permeabilization buffer for 30-60 minutes

• Preserving autophagosome structures:

  • Fixation: 2% paraformaldehyde in PBS (milder to preserve delicate structures)

  • Permeabilization: 0.1% saponin (gentler than Triton X-100)

  • This approach is especially important when studying TNIP1 colocalization with autophagosomes

• Special considerations:

  • For detecting nuclear TNIP1: Include antigen retrieval with TE buffer pH 9.0 before blocking (similar to IHC protocols)

  • For dual staining with phospho-proteins: Fix first, then permeabilize with methanol at -20°C for 10 minutes

  • For mitochondrial colocalization studies: Use 0.002% digitonin for selective plasma membrane permeabilization while preserving organelle integrity

Optimization is critical as TNIP1 has multiple cellular localizations including cytoplasmic, nuclear, and autophagosomal compartments, with localization patterns changing upon cellular stimulation .

How can researchers quantitatively analyze TNIP1 expression and localization data from fluorescence microscopy experiments?

Quantitative analysis of TNIP1 expression and localization from fluorescence microscopy requires rigorous image acquisition and analytical approaches:

• Image acquisition parameters:

  • Use identical exposure settings across all experimental conditions

  • Acquire Z-stacks (0.3-0.5 μm steps) to capture the full cellular volume

  • Include fluorescence calibration standards for absolute intensity measurements

  • Minimize photobleaching by using low laser power/LED intensity

• Basic quantification methods:

  • Mean fluorescence intensity (MFI): Measure TNIP1-FITC signal intensity within defined cellular regions

  • Nuclear/cytoplasmic ratio: Quantify TNIP1 distribution between compartments using nuclear counterstain (DAPI) for segmentation

  • Threshold-based analysis: Apply consistent thresholds to identify TNIP1-positive structures

• Colocalization analysis approaches:

  • Pearson's correlation coefficient: Measures linear correlation between TNIP1 and markers like LC3 (autophagosomes) or MyD88

  • Manders' overlap coefficient: Quantifies fraction of TNIP1 overlapping with a second marker

  • Object-based colocalization: Count percentage of TNIP1-positive puncta that also contain autophagosome markers

• Advanced analytical techniques:

  • Single-molecule localization microscopy: For nanoscale distribution analysis of TNIP1

  • Fluorescence intensity distribution analysis: Plot TNIP1 intensity across linear regions of interest (line scans)

  • Morphological analysis of TNIP1-positive structures: Size, shape, and density of puncta

  • Machine learning classification: Train algorithms to identify distinct TNIP1 localization patterns

• Recommended software:

  • ImageJ/FIJI with JACoP plugin for colocalization analysis

  • CellProfiler for automated high-content analysis

  • Imaris for 3D reconstruction and analysis

  • MATLAB for custom analytical workflows

Statistical analysis should include multiple cells (>50) from at least three independent experiments, with appropriate statistical tests (ANOVA with post-hoc comparisons) to evaluate differences between experimental conditions .

What strategies can optimize multiplexed detection of TNIP1 alongside other markers in flow cytometry?

Optimizing multiplexed detection of TNIP1-FITC with other markers in flow cytometry requires careful panel design and protocol optimization:

• Panel design considerations:

  • Strategic fluorophore selection: Reserve bright fluorophores (PE, APC) for lower-abundance targets

  • Position FITC-conjugated TNIP1 in appropriate detector: FITC emission overlaps with PE, requiring compensation

  • Complementary markers to include:

    • TLR7 (key interaction partner)

    • MyD88 (signaling adaptor)

    • LC3 (autophagosome marker)

    • Cell lineage markers (CD19, CD3, CD14 for B cells, T cells, monocytes)

• Protocol optimization:

  • Sequential staining: Surface markers first, followed by fixation/permeabilization, then intracellular TNIP1-FITC

  • Buffer selection: Use buffers with protein carrier (1% BSA) to reduce non-specific binding

  • Blocking strategy: Include 10% serum and Fc receptor blocking reagents

  • Titrate TNIP1-FITC antibody (start with 1:100 dilution) to determine optimal signal-to-noise ratio

• Technical validation:

  • Single-color controls: Essential for accurate compensation

  • Fluorescence-minus-one (FMO) controls: To set accurate gates, especially important for TNIP1-FITC

  • Biological controls: Compare known TNIP1-high (stimulated B cells) and TNIP1-low populations

• Advanced approaches:

  • Multispectral flow cytometry: For panels with >10 markers, enabling separation of FITC from spectrally similar fluorophores

  • Phospho-flow analysis: Combine TNIP1-FITC with phospho-TBK1 to assess signaling activity

  • Imaging flow cytometry: Visualize TNIP1 localization patterns while obtaining quantitative data

• Data analysis recommendations:

  • Hierarchical gating strategy starting with viable single cells

  • Consider dimensionality reduction techniques (tSNE, UMAP) for high-parameter data

  • Back-gating to verify population identification

  • Biexponential display scales for optimal visualization of TNIP1-FITC signal distribution

These approaches enable simultaneous assessment of TNIP1 expression, cell phenotype, and functional status in complex samples such as peripheral blood mononuclear cells from autoimmune disease patients .