TNFAIP3 Antibody, FITC conjugated

What is TNFAIP3 and why is it significant in immunological research?

TNFAIP3 (A20) is a ubiquitin-editing enzyme with dual functionality, possessing both ubiquitin ligase and deubiquitinase activities. It plays a crucial role in regulating immune and inflammatory responses triggered by cytokines like TNF-alpha and IL-1 beta, primarily by terminating NF-kappa-B activation . TNFAIP3 is a key component of a ubiquitin-editing protein complex that includes RNF11, ITCH, and TAX1BP1, which collectively ensures inflammatory signaling pathways remain transient rather than chronic . Located on chromosome band 6q23, TNFAIP3 functions as a tumor suppressor gene in Hodgkin lymphoma and several non-Hodgkin lymphoma subtypes . Its significance in research stems from its essential role in preventing excessive inflammation, as demonstrated by the severe inflammatory phenotypes observed in TNFAIP3-deficient models .

What are the technical advantages of using FITC-conjugated TNFAIP3 antibodies?

FITC-conjugated TNFAIP3 antibodies offer several methodological advantages for researchers. The direct fluorescent conjugation eliminates the need for secondary antibody incubation steps, reducing experiment time and potential sources of non-specific binding . FITC's excitation maximum at approximately 495nm and emission maximum around 520nm makes it compatible with most flow cytometers and fluorescence microscopes without requiring specialized filters . Additionally, FITC-conjugated antibodies allow direct visualization of TNFAIP3 in intact cells for localization studies and can be effectively used in multicolor flow cytometry panels, as FITC occupies the green channel while leaving other fluorescence channels available for additional markers .

How do I validate the specificity of a TNFAIP3-FITC antibody?

Validating antibody specificity is crucial for generating reliable research data. For TNFAIP3-FITC antibodies, a multi-step validation approach is recommended:

• Knockout/knockdown controls: Use TNFAIP3 knockout cell lines (such as the Human TNFAIP3 knockout HeLa cell line) as negative controls to confirm absence of signal .

• Stimulation tests: Analyze cells before and after stimulation with TNF-alpha, as TNFAIP3 is rapidly and transiently induced by TNF stimulation .

• Western blot correlation: Perform parallel Western blot analysis using the same antibody in its unconjugated form to confirm the correct molecular weight band (approximately 80-89 kDa) .

• Blocking peptide competition: Pre-incubate the antibody with the immunizing peptide prior to staining to demonstrate signal specificity .

• Isotype controls: Include appropriate isotype controls at the same concentration as the TNFAIP3-FITC antibody to identify any non-specific binding .

How should I design multicolor flow cytometry panels incorporating TNFAIP3-FITC antibody?

When designing multicolor flow cytometry panels with TNFAIP3-FITC antibody, consider these methodological principles:

• Spectral compatibility: FITC (emission ~520nm) has potential spectral overlap with PE (emission ~575nm). Apply proper compensation using single-stained controls .

• Panel design strategy:

  • Reserve brighter fluorochromes (APC, PE) for lower-expressed targets

  • Pair TNFAIP3-FITC with markers in non-overlapping channels (APC, APC-Cy7, Pacific Blue)

  • Include viability dyes in channels distant from FITC (e.g., far red channels)

• Surface and intracellular staining sequence: Perform surface marker staining first, followed by fixation/permeabilization and intracellular staining for TNFAIP3 .

• Relevant markers to consider:

  • Include NF-κB pathway components (p65, IκBα)

  • Add cell-type specific markers depending on your research focus

  • Consider adding functional markers relevant to inflammation status

• Control tubes: Always include FMO (Fluorescence Minus One) controls for accurate gating, especially for intracellular markers with variable expression .

What fixation and permeabilization protocols work best for intracellular TNFAIP3 staining?

For optimal intracellular detection of TNFAIP3 using FITC-conjugated antibodies, consider these methodological approaches:

• Recommended fixation protocols:

  • 4% paraformaldehyde for 10-15 minutes at room temperature

  • Methanol fixation (100% cold methanol for 10 minutes at -20°C) may provide better detection of certain epitopes

• Permeabilization options:

  • Saponin-based buffers (0.1-0.5%) for reversible membrane permeabilization

  • Triton X-100 (0.1-0.5%) for more thorough permeabilization when higher signal intensity is needed

  • Commercial kits specifically designed for nuclear antigens may improve TNFAIP3 detection

• Optimization steps:

  • Test different fixation times (10-20 minutes) and permeabilization reagents

  • For tissue sections, consider antigen retrieval by boiling in 10mM sodium citrate buffer (pH 6.0) for 10-20 minutes followed by cooling at room temperature for 20 minutes

  • Perform titration of antibody concentration in each fixation/permeabilization condition

• Buffer considerations:

  • Include protein (1% BSA) in all buffers to reduce non-specific binding

  • Maintain permeabilization agent in all wash buffers until final wash step

  • Consider adding RNase inhibitors if performing RNA-protein co-detection

How can I use TNFAIP3-FITC antibody to study NF-κB signaling pathways?

TNFAIP3-FITC antibodies can be effectively employed to study NF-κB signaling through these methodological approaches:

• Time-course experiments:

  • Stimulate cells with TNF-α, IL-1β, or LPS at different time points

  • Use flow cytometry with TNFAIP3-FITC to track dynamic changes in TNFAIP3 expression

  • Correlate with NF-κB activation markers such as phospho-p65 or nuclear translocation of p65

• Co-localization studies:

  • Perform immunofluorescence with TNFAIP3-FITC and antibodies against NF-κB pathway components

  • Track subcellular localization changes upon stimulation

  • Quantify co-localization coefficients between TNFAIP3 and its binding partners (TRAF6, RIPK1)

• Functional correlation analysis:

  • Measure TNFAIP3 expression levels in conjunction with NF-κB target gene expression

  • Compare TNFAIP3 levels with ubiquitination status of key signaling intermediates

  • Design experiments to analyze both early (0-2 hours) and late (2-24 hours) phases of NF-κB activation

• Cell-specific analyses:

  • In mixed cell populations, use lineage markers together with TNFAIP3-FITC

  • Compare NF-κB activation patterns in TNFAIP3-high versus TNFAIP3-low expressing cells

  • Examine the correlation between TNFAIP3 expression and cell survival after TNF-α treatment

How can I use TNFAIP3-FITC antibody to investigate its role in inflammatory bowel disease models?

Research on TNFAIP3's role in inflammatory bowel disease (IBD) can be enhanced using FITC-conjugated antibodies through these approaches:

• Single-cell analysis of intestinal tissue:

  • Isolate intestinal epithelial cells (IECs) and lamina propria mononuclear cells from IBD models

  • Use TNFAIP3-FITC with flow cytometry to quantify expression levels across cell populations

  • Correlate TNFAIP3 expression with inflammatory status and disease severity

• Barrier function correlation studies:

  • Study TNFAIP3 expression in relation to tight junction proteins (occludin, claudins)

  • Analyze how TNFAIP3 expression correlates with epithelial permeability measurements

  • Investigate TNFAIP3's role in preserving barrier function through co-staining experiments

• Antimicrobial peptide (AMP) expression analysis:

  • Design experiments to examine the relationship between TNFAIP3 expression and AMP production

  • Use TNFAIP3-FITC flow cytometry in parallel with qPCR for AMPs like Reg3 and Ang4

  • Investigate how TNFAIP3 alters AMP expression that allows microbial colonization of the inner mucus layer

• Experimental methodology for IBD models:

  Technique	Application with TNFAIP3-FITC	Data Analysis Approach
  Flow cytometry	Quantify TNFAIP3+ cells in DSS or IL-10-/- colitis models	Compare % positive cells and MFI between inflamed/non-inflamed tissues
  Imaging cytometry	Visualize TNFAIP3 together with bacterial penetration	Spatial correlation analysis of TNFAIP3 levels with microbial proximity
  Ex vivo intestinal organoids	Monitor TNFAIP3 expression after inflammatory stimuli	Time-course analysis of expression changes
  FACS sorting	Isolate TNFAIP3high vs TNFAIP3low epithelial cells	Transcriptome comparison between populations

What are the technical considerations when studying the dual ubiquitin-editing functions of TNFAIP3?

TNFAIP3 possesses both deubiquitinase (DUB) and E3 ligase activities, making it a complex enzyme to study. When investigating these dual functions:

• Experimental design for functional studies:

  • Combine TNFAIP3-FITC staining with ubiquitin chain-specific antibodies (K48 vs K63 linkages)

  • Design pulse-chase experiments to track TNFAIP3 activity kinetics following stimulation

  • Use proteasome inhibitors to distinguish between effects on ubiquitination versus degradation

• Target protein analysis methodology:

  • Study TNFAIP3's interaction with known substrates (RIPK1, TRAF6, NEMO/IKBKG)

  • Design co-immunoprecipitation experiments followed by ubiquitin linkage-specific Western blots

  • Correlate TNFAIP3 expression levels with ubiquitination status of target proteins

• Domain-specific functional analysis:

  • Use domain-specific antibodies alongside TNFAIP3-FITC to investigate structure-function relationships

  • Employ site-directed mutagenesis to create variants with selective loss of DUB or E3 ligase functions

  • Analyze how mutations affect TNFAIP3's ability to terminate NF-κB signaling

• Technical workflow for ubiquitin editing studies:

  Step	Technique	Purpose
  1	Cell stimulation with TNF-α	Induce TNFAIP3 expression and NF-κB activation
  2	TNFAIP3-FITC flow cytometry	Quantify expression level and timing
  3	Cell lysis and IP of target proteins	Isolate TNFAIP3 substrates
  4	Ubiquitin chain analysis	Determine K48 vs K63 linkage modifications
  5	Correlation analysis	Relate TNFAIP3 levels to ubiquitination patterns

Why might I see decreased TNFAIP3-FITC signal after certain stimulation conditions?

Several technical and biological factors can cause reduced TNFAIP3-FITC signals in stimulated samples:

• Biological regulation issues:

  • TNFAIP3 expression is rapidly and transiently induced by TNF-α, with peak expression typically occurring 1-2 hours post-stimulation followed by downregulation

  • Proteasomal degradation may reduce TNFAIP3 levels in later time points

  • Certain stimuli might simultaneously activate and repress TNFAIP3 expression through different pathways

• Technical considerations:

  • Epitope masking due to protein-protein interactions or post-translational modifications

  • Inadequate fixation/permeabilization for accessing intracellular TNFAIP3

  • FITC photobleaching during long protocols or extended exposure to light

  • Suboptimal stimulation timing that misses peak expression windows

• Methodological solutions:

  • Perform detailed time-course experiments (0, 0.5, 1, 2, 4, 8, 24 hours) to identify optimal detection windows

  • Test multiple fixation/permeabilization protocols to optimize epitope accessibility

  • Include proteasome inhibitors (MG132) in later time points to prevent TNFAIP3 degradation

  • Minimize light exposure during all protocol steps and use anti-fade mounting media for imaging

How can I distinguish between TNFAIP3 isoforms using antibody-based detection methods?

TNFAIP3 exists in at least two isoforms, presenting technical challenges for specific detection:

• Isoform-specific detection strategy:

  • Review the epitope location of your TNFAIP3-FITC antibody relative to known isoform differences

  • Design PCR primers to quantify isoform-specific mRNA expression alongside protein detection

  • Consider using isoform-specific antibodies in parallel with your FITC-conjugated antibody

• Technical approach for distinguishing isoforms:

  • Combine flow cytometry with Western blotting to correlate fluorescence intensity with specific bands

  • Predetermine the expected molecular weights: full-length TNFAIP3 is typically observed at 80-89 kDa

  • Use isoform-specific positive controls (recombinant proteins or cells with known isoform expression)

• Analytical considerations:

  • Design gating strategies that can distinguish populations expressing different isoforms

  • Consider fluorescence intensity distributions rather than simple positive/negative gating

  • When possible, correlate antibody-based detection with genetic analysis of isoform expression

• Validation approach for isoform specificity:

  Validation Method	Implementation	Expected Outcome
  Western blot correlation	Run parallel WB and flow cytometry	Confirm antibody detects expected isoform bands
  Genetic knockdown	Selectively knockdown specific isoforms	Demonstrate selective loss of signal
  Mass spectrometry	Identify proteins in antibody-precipitated samples	Confirm isoform specificity
  Recombinant standards	Test antibody against purified isoforms	Establish detection limits for each isoform

What approaches can I take if my TNFAIP3-FITC antibody shows cross-reactivity with unexpected proteins?

Cross-reactivity can significantly impact experimental interpretation. Here's a systematic approach to address this issue:

• Verification and characterization of cross-reactivity:

  • Perform Western blot analysis to identify unexpected bands using TNFAIP3 knockout cells as controls

  • Compare staining patterns between multiple anti-TNFAIP3 antibodies targeting different epitopes

  • Check for binding to unrelated cell types that should not express TNFAIP3

• Technical optimization strategies:

  • Titrate antibody concentration to find an optimal signal-to-noise ratio

  • Modify blocking conditions (try different blocking agents like BSA, normal serum, or commercial blockers)

  • Adjust incubation times and temperatures to enhance specific binding

  • Increase wash steps duration and volume to reduce non-specific interactions

• Analytical approaches to minimize cross-reactivity impact:

  • Use multiple antibodies in parallel experiments for verification

  • Include appropriate knockout controls in all experiments

  • Develop gating strategies that account for background fluorescence

  • Pre-absorb antibody with recombinant proteins that show cross-reactivity

• Advanced troubleshooting for persistent cross-reactivity:

  • Consider antibody purification through affinity chromatography

  • Use competitive binding assays with the immunizing peptide

  • Implement machine learning algorithms to distinguish true signal from cross-reactivity patterns

  • Consider developing new antibodies with higher specificity if available options are insufficient

How should I interpret differences in TNFAIP3 expression between healthy and disease models?

Interpreting TNFAIP3 expression changes requires comprehensive analysis and careful consideration:

• Quantitative analysis framework:

  • Compare both percentage of positive cells and mean fluorescence intensity (MFI)

  • Establish normal ranges of TNFAIP3 expression in relevant cell types

  • Consider using relative expression (fold change) rather than absolute values when comparing across experiments

• Biological context considerations:

  • Elevated TNFAIP3 may indicate active inflammatory signaling being regulated

  • Decreased expression could suggest compromised regulatory capacity

  • TNFAIP3 functions as a tumor suppressor in lymphomas, so reduced expression may correlate with malignancy

• Correlation with functional outcomes:

  • Analyze relationship between TNFAIP3 levels and NF-κB activation markers

  • Correlate expression with inflammatory cytokine production

  • Examine connection between TNFAIP3 expression and antimicrobial peptide production in epithelial cells

• Interpretation framework for disease models:

  Disease Context	Typical TNFAIP3 Pattern	Potential Interpretation
  IBD models	Altered expression in epithelial cells	May affect barrier function and microbial interactions
  Lymphoma	Reduced expression	Consistent with tumor suppressor role
  Acute inflammation	Transient upregulation	Normal regulatory response to inflammatory stimuli
  Chronic inflammation	Sustained expression changes	Potential dysregulation of inflammatory resolution

How can I correlate TNFAIP3 expression with other inflammatory biomarkers?

Establishing correlations between TNFAIP3 and other inflammatory markers requires integrated analytical approaches:

• Multi-parameter analytical methods:

  • Design multicolor flow cytometry panels that include TNFAIP3-FITC alongside other inflammatory markers

  • Perform correlation analysis between TNFAIP3 MFI and expression levels of other proteins

  • Consider dimensionality reduction techniques (tSNE, UMAP) for visualizing complex relationships

• Temporal analysis considerations:

  • TNFAIP3 is rapidly induced but may show different kinetics than other inflammatory markers

  • Design time-course experiments to capture differential regulation patterns

  • Analyze both early (0-4h) and late phase (4-24h) responses to inflammatory stimuli

• Cell-specific correlation analysis:

  • Different cell types may show distinct TNFAIP3 expression patterns in response to the same stimulus

  • Parse data by cell type before performing correlation analyses

  • Consider how TNFAIP3's role varies between epithelial cells, immune cells, and other tissue types

• Specific inflammatory marker relationships:

  Inflammatory Marker	Relationship with TNFAIP3	Analytical Approach
  NF-κB components (p65, IκBα)	Reciprocal regulation	Time-course analysis of activation/expression
  Pro-inflammatory cytokines	TNFAIP3 limits production	Correlation of TNFAIP3 levels with cytokine expression
  Antimicrobial peptides	TNFAIP3 may alter expression	Combined flow cytometry and gene expression analysis
  Apoptosis markers	TNFAIP3 protects against TNF-induced death	Cell fate correlation with TNFAIP3 levels