UBE2L3 Antibody, FITC conjugated

What is UBE2L3 and what biological pathways is it involved in?

UBE2L3 (also known as UBCH7, E2-F1, L-UBC, and UbcM4) is a member of the E2 ubiquitin-conjugating enzyme family that plays a critical role in the ubiquitination pathway. This pathway is an important cellular mechanism for targeting abnormal or short-lived proteins for degradation. UBE2L3 specifically participates in the ubiquitination of several key proteins including p53, c-Fos, and the NF-kB precursor p105 in vitro . UBE2L3 is a functional subunit of the linear ubiquitin chain assembly complex (LUBAC), a crucial regulator of the canonical NF-κB signaling pathway . In this complex, UBE2L3 facilitates the formation of linear polyubiquitin chains that are conjugated to the NF-κB essential modulator (NEMO), ultimately leading to activation of NF-κB and transcription of proinflammatory mediators .

How does FITC conjugation affect UBE2L3 antibody performance in different experimental applications?

FITC (Fluorescein isothiocyanate) conjugation provides direct fluorescent labeling of the UBE2L3 antibody, eliminating the need for secondary detection reagents in fluorescence-based applications. The performance impact varies by application:

When designing experiments with FITC-conjugated UBE2L3 antibodies, researchers should consider that conjugation may slightly reduce antibody affinity compared to unconjugated versions, potentially requiring higher concentrations for optimal results.

What are the optimal fixation and permeabilization protocols for UBE2L3 detection in cellular localization studies?

The optimal protocols depend on the cellular compartment being studied, as UBE2L3 has dual localization in both nucleus and cytoplasm :

For comprehensive detection in both compartments:

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 10 minutes

• Block with 3% BSA in PBS for 1 hour

• Incubate with FITC-conjugated UBE2L3 antibody at 1:50-1:200 dilution

• Wash 3× with PBS-T (0.05% Tween-20)

• Counterstain nucleus with DAPI if needed

For nuclear-focused studies:

• Consider adding a methanol permeabilization step (-20°C, 10 minutes) after PFA fixation

• Increase antibody concentration to the higher end of recommended range (1:50)

For cytoplasmic-focused studies:

• Use a gentler permeabilization with 0.05% saponin

• Use shorter fixation time (10 minutes)

The choice of fixation and permeabilization is critical as overfixation can mask epitopes, particularly for nuclear detection, while insufficient permeabilization can reduce cytoplasmic signal.

How can I validate the specificity of FITC-conjugated UBE2L3 antibody in my experimental system?

A comprehensive validation approach should include multiple methods:

• Positive and negative controls:

  • Use cell lines with verified UBE2L3 expression (HeLa, NIH/3T3)

  • Include UBE2L3 knockout or knockdown samples as negative controls

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare staining patterns with and without peptide blocking

• Western blot correlation:

  • Perform parallel western blot with unconjugated antibody

  • Confirm molecular weight matches expected UBE2L3 size (~18 kDa)

• Multi-antibody validation:

  • Compare staining pattern with another UBE2L3 antibody from different host/clone

  • Patterns should be consistent despite different epitope recognition

• Genetic manipulation verification:

  • Overexpress UBE2L3 and confirm increased signal intensity

  • Perform siRNA knockdown and confirm reduced signal

This multi-faceted approach significantly increases confidence in antibody specificity beyond single-method validation.

How can I design experiments to investigate UBE2L3's role in NF-κB signaling and inflammatory responses?

Designing comprehensive experiments to investigate UBE2L3's role in NF-κB signaling requires a multi-level approach:

• Genetic manipulation strategies:

  • CRISPR/Cas9 knockout of UBE2L3 in relevant cell types

  • Inducible expression systems with wildtype and mutant UBE2L3

  • Generation of mouse models with conditional UBE2L3 deletion in specific cell types

• Stimulation paradigms:

  • Canonical NF-κB activators (TNFα, IL-1β, LPS)

  • Time course analysis (15 min, 30 min, 1h, 2h, 4h post-stimulation)

  • Dose-response relationships

• Readout measurements:

  • Phosphorylation status of IκB by western blot

  • Nuclear translocation of p65 using FITC-conjugated UBE2L3 antibody with co-staining

  • NF-κB reporter assays

  • qPCR analysis of NF-κB target genes

• Interaction analysis:

  • Co-immunoprecipitation of UBE2L3 with LUBAC components

  • Proximity ligation assay to detect UBE2L3-NEMO interactions in situ

  • Ubiquitination analysis of key targets including NEMO

• Pathway inhibition:

  • Combine UBE2L3 manipulations with NF-κB inhibitors

  • Test epistatic relationships with upstream and downstream components

Research has demonstrated that UBE2L3 facilitates LUBAC-mediated NF-κB activation and affects proliferation of plasmablasts and plasma cells . Additionally, studies in mice showed that deletion of Ube2l3 reduces pro-IL-1β turnover in macrophages, leading to excessive mature IL-1β production and neutrophilic inflammation following inflammasome activation .

What methodological approaches can determine if UBE2L3 genetic variants affect protein expression and function in patient-derived samples?

To investigate the impact of UBE2L3 genetic variants on protein expression and function, researchers should implement this methodological framework:

• Patient stratification:

  • Genotype patients for known UBE2L3 risk variants (rs140490, rs140491, rs11089620, rs3747093)

  • Group based on homozygous risk, heterozygous, and homozygous non-risk haplotypes

• Expression analysis:

  • Quantify UBE2L3 mRNA levels by qRT-PCR in patient PBMCs

  • Measure UBE2L3 protein expression using flow cytometry with FITC-conjugated antibody

  • Analyze cell-type specific expression patterns in whole blood using spectral flow cytometry

• Functional assessment:

  • Stimulate patient-derived cells with NF-κB activators

  • Measure downstream proinflammatory cytokine production

  • Assess NF-κB signaling kinetics through phospho-flow cytometry

  • Evaluate UBE2L3-mediated ubiquitination activity in cell lysates

• Mechanistic investigation:

  • Perform ChIP-qPCR to assess transcription factor binding at variant sites

  • Use 3C-qPCR to evaluate chromatin interactions between UBE2L3 and YDJC promoters

  • Conduct siRNA knockdown of regulatory factors (e.g., YY1, CTCF)

• Correlation with disease parameters:

  • Associate UBE2L3 expression/function metrics with clinical disease activity

  • Longitudinal analysis during treatment response

Research has shown that variants on the UBE2L3-YDJC autoimmune risk haplotype increase UBE2L3 expression through strengthening a YY1-mediated interaction between the UBE2L3 and YDJC promoters . Risk alleles demonstrated increased binding affinity for YY1 at the expense of CTCF, leading to enhanced long-range genomic interactions .

How can I troubleshoot weak or inconsistent signal when using FITC-conjugated UBE2L3 antibody in immunofluorescence experiments?

When encountering weak or inconsistent signals, consider this systematic troubleshooting approach:

Optimization steps:

• Always include a positive control cell line (HeLa or NIH/3T3)

• Perform titration experiments to determine optimal antibody concentration

• Compare results with an unconjugated UBE2L3 antibody + secondary approach

• For critical experiments, consider alternative fluorophores with greater photostability

What are the appropriate controls and normalization methods for flow cytometry experiments using FITC-conjugated UBE2L3 antibody?

For robust flow cytometry experiments with FITC-conjugated UBE2L3 antibody:

Essential Controls:

• Unstained cells:

  • Establish autofluorescence baseline

  • Set negative population boundaries

• Isotype control:

  • FITC-conjugated rabbit IgG at matching concentration

  • Assess non-specific binding contribution

• FMO (Fluorescence Minus One):

  • Include all fluorophores except FITC

  • Critical for accurate gating in multi-color panels

• Biological controls:

  • Positive: Cell lines with high UBE2L3 expression (HeLa, NIH/3T3)

  • Negative: UBE2L3 knockdown/knockout cells

  • Stimulated/unstimulated pairs for dynamic studies

Normalization Methods:

• Geometric Mean Fluorescence Intensity (gMFI):

  • Most appropriate for log-distributed data

  • Calculate: gMFI(sample) - gMFI(isotype)

• Staining Index:

  • SI = (MFIpos - MFIneg) / (2 × SDneg)

  • Accounts for population spread and separation

• Relative expression:

  • Calculate fold-change relative to control condition

  • Emphasizes biological significance over absolute values

• Standardization with beads:

  • Use calibration beads with known MESF (Molecules of Equivalent Soluble Fluorochrome)

  • Converts arbitrary units to absolute fluorophore numbers

When analyzing UBE2L3 expression in different cell populations, cell type-specific normalization may be necessary due to UBE2L3's differential expression patterns. Documentation of gating strategies and compensation matrices is essential for reproducibility.

How can I differentiate between UBE2L3's roles in normal protein turnover versus pathological processes using imaging and functional assays?

Differentiating UBE2L3's physiological and pathological roles requires parallel investigation of multiple cellular processes:

• Dual-color pulse-chase experiments:

  • Pulse with one color-tagged ubiquitin, chase with another

  • Measure UBE2L3 colocalization with each pool

  • Physiological turnover shows consistent patterns; pathological shows temporal disruptions

• Proteasome inhibition studies:

  • Compare UBE2L3 substrate accumulation patterns with/without MG132

  • Normal turnover: proportional increase in substrates

  • Pathological: non-linear accumulation of specific substrates

• Stress response analysis:

  • Monitor UBE2L3 activity under various cellular stressors

  • Measure relationship between UBE2L3 expression and pro-IL-1β turnover

  • Compare inflammatory response in WT versus UBE2L3-variant cells

• Quantitative co-localization analysis:

  • Calculate Pearson's correlation coefficients between UBE2L3 and:

    • Proteasome markers (normal turnover)

    • Stress granules or aggregates (pathological)

    • LUBAC components (inflammatory signaling)

• Functional readouts:

  • NF-κB activity (luciferase reporters)

  • IL-1β production and secretion

  • Cell survival metrics

Research has shown that UBE2L3, along with TRIP12 and AREL1 E3 ligases, limits inflammation by reducing the cellular pool of pro-IL-1β . In pathological contexts, deletion or dysfunction of UBE2L3 leads to excessive mature IL-1β production, neutrophilic inflammation, and disease following inflammasome activation .

How do I interpret contradictory results between UBE2L3 expression levels and functional outcomes in patient-derived samples?

Resolving contradictions between UBE2L3 expression and function requires a systematic analytical approach:

• Consider post-translational modifications:

  • Phosphorylation status may alter activity independent of expression

  • Ubiquitination of UBE2L3 itself may affect function

  • Methodological approach: Phospho-specific antibodies and ubiquitination assays

• Evaluate genetic background effects:

  • UBE2L3 variants occur on a haplotype with YDJC

  • Gene-gene interactions may modify UBE2L3 function

  • Approach: Stratify analysis by complete haplotype rather than single variants

• Assess regulatory protein availability:

  • Measure expression of interacting partners (TRIP12, AREL1)

  • Quantify YY1 and CTCF binding at UBE2L3 locus

  • Method: Co-immunoprecipitation and ChIP-qPCR

• Cell-type specific effects:

  • Different cell types may show different UBE2L3 function despite similar expression

  • Approach: Isolated cell-type analysis rather than whole PBMC studies

• Disease context and environment:

  • Inflammatory milieu can alter protein function

  • Stage of disease may influence results

  • Method: Longitudinal sampling and ex vivo stimulation experiments

When interpreting contradictory results, consider that genetic variants on the UBE2L3-YDJC autoimmune risk haplotype increase UBE2L3 expression through complex mechanisms involving altered chromatin interactions . Also, UBE2L3's effects may be threshold-dependent rather than linearly correlated with expression.

What methodological approaches can be used to develop therapeutic strategies targeting UBE2L3 pathway in autoimmune diseases?

Developing therapeutic strategies targeting the UBE2L3 pathway requires multiple complementary approaches:

• High-throughput screening platforms:

  • Develop fluorescence-based UBE2L3 activity assays similar to the UBE3A assay

  • Adapt the FITC-ubiquitin detection system to measure UBE2L3-specific activity

  • Screen compound libraries for selective inhibitors

• Structure-based drug design:

  • Determine crystal structure of UBE2L3 in complex with E3 ligases

  • Identify binding pockets at protein-protein interfaces

  • Design small molecules or peptides to disrupt specific interactions

• Genetic modulation strategies:

  • Design antisense oligonucleotides targeting UBE2L3 mRNA

  • Develop CRISPR-based approaches to modify the UBE2L3-YDJC risk haplotype

  • Test AAV-delivered shRNA for tissue-specific UBE2L3 knockdown

• Pathway-specific interventions:

  • Target the YY1-mediated chromatin interactions that drive UBE2L3 overexpression

  • Develop peptide inhibitors of UBE2L3-LUBAC interaction

  • Create decoy substrates that compete with endogenous proteins

• Validation systems:

  • Humanized mouse models carrying UBE2L3 risk variants

  • Patient-derived organoids for ex vivo testing

  • Biomarker development to stratify patients by UBE2L3 pathway activity

Research has established that UBE2L3 risk variants lead to increased expression and enhanced NF-κB signaling , suggesting that normalized expression or activity could have therapeutic benefit in autoimmune conditions.

How can multiparametric analysis combining UBE2L3 studies with other ubiquitination pathway components improve understanding of disease mechanisms?

Integrating UBE2L3 with broader ubiquitination pathway analysis provides deeper mechanistic insights:

• Comprehensive ubiquitinome profiling:

  • Combine UBE2L3 antibody staining with mass spectrometry-based ubiquitinome analysis

  • Map changes in ubiquitination patterns upon UBE2L3 manipulation

  • Identify substrate specificity through K-linkage type analysis

• E3 ligase interaction mapping:

  • Perform systematic analysis of UBE2L3 interaction with HECT-family E3 ligases

  • Include focused analysis of TRIP12 and AREL1, which work with UBE2L3 in pro-IL-1β regulation

  • Use proximity labeling approaches (BioID, APEX) to identify novel interactors

• Pathway cross-talk analysis:

  • Simultaneous monitoring of UBE2L3 activity with other post-translational modifications

  • Investigate relationship between ubiquitination and inflammasome activation

  • Study interaction between UBE2L3-mediated processes and autophagy pathways

• Single-cell multiparametric approaches:

  • Combine FITC-conjugated UBE2L3 antibody with markers for cell state and other pathway components

  • Perform CyTOF or spectral flow cytometry to identify cell populations with distinctive pathway signatures

  • Correlate with single-cell transcriptomics data

• Systems biology integration:

  • Develop computational models incorporating UBE2L3 and interacting components

  • Simulate effects of genetic variation and therapeutic intervention

  • Identify emergent properties and feedback mechanisms

This integrated approach recognizes that UBE2L3 functions within a complex network. Research has shown that UBE2L3 works with specific E3 ligases like TRIP12 and AREL1 to regulate inflammatory processes , suggesting that pathway-level analysis is essential for understanding disease mechanisms.