UBASH3A Antibody, FITC conjugated

What is UBASH3A and what is its role in T-cell function?

UBASH3A (Ubiquitin-associated and SH3 domain-containing protein A) is a protein that functions as a negative regulator of T-cell activity. It interferes with CBL-mediated down-regulation and degradation of receptor-type tyrosine kinases, promoting the accumulation of activated target receptors such as T-cell receptors (TCR), EGFR, and PDGFRB on the cell surface . UBASH3A plays a critical role in regulating the synthesis and dynamics of the T-cell receptor-CD3 complex, affecting both resting T cells and those under stimulation . Mechanistically, UBASH3A attenuates NF-κB signaling by inhibiting the activation of the IκB kinase complex, demonstrating that it can function through phosphatase-independent mechanisms .

What are the key structural domains of UBASH3A and their functions?

UBASH3A contains three primary structural domains that contribute to its diverse cellular functions:

• N-terminal UBA (ubiquitin-associated) domain: Binds to mono-ubiquitin as well as lysine-63- and methionine-1-linked polyubiquitin chains, enabling interaction with ubiquitinated proteins .

• SH3 (Src homology 3) domain: Mediates protein-protein interactions, particularly binding to CBL-B, an E3 ubiquitin ligase that negatively regulates CD28-mediated signaling and T-cell activation . This domain also allows UBASH3A to functionally sequester dynamin, potentially inhibiting dynamin-dependent endocytic pathways .

• C-terminal histidine phosphatase domain (also called phosphoglycerate mutase-like or PGM domain): Despite structural homology to phosphatases, UBASH3A exhibits negligible protein tyrosine phosphatase activity at neutral pH . It may act as a dominant-negative regulator of UBASH3B-dependent dephosphorylation .

Understanding these domains is essential for interpreting antibody binding specificity and experimental results.

How does UBASH3A antibody conjugated with FITC differ from unconjugated variants in experimental applications?

FITC-conjugated UBASH3A antibodies provide direct fluorescent detection without requiring secondary antibodies, which offers several methodological advantages over unconjugated variants. The FITC (fluorescein isothiocyanate) conjugate emits green fluorescence when excited, allowing direct visualization in techniques such as immunofluorescence microscopy and flow cytometry . This direct labeling approach reduces background signal and simplifies multi-color staining protocols by eliminating cross-reactivity concerns associated with secondary antibodies.

What are the optimal conditions for using FITC-conjugated UBASH3A antibody in flow cytometry?

When using FITC-conjugated UBASH3A antibody for flow cytometry, researchers should consider the following methodological approach:

• Sample preparation: For cell surface staining, use approximately 1×10^6 cells per sample. For intracellular staining, cells must be fixed and permeabilized using appropriate buffers compatible with FITC fluorescence preservation.

• Antibody concentration: Start with the manufacturer's recommended dilution (often 1:100 to 1:500) . Optimal concentration should be determined experimentally through titration for each specific application.

• Staining buffer: Use PBS containing 1-2% BSA or FBS with 0.1% sodium azide, pH 7.4. For cells with Fc receptors, include an Fc block to reduce non-specific binding.

• Incubation conditions: Stain for 30 minutes on ice in the dark to protect the FITC fluorophore from photobleaching.

• Washing steps: Perform at least two washes with cold buffer after staining to remove unbound antibody.

• Controls: Always include:

  • Unstained cells for autofluorescence determination

  • Isotype control (FITC-conjugated rabbit IgG) to assess non-specific binding

  • Positive control (cell line known to express UBASH3A)

• Instrument settings: Set proper compensation when multiplexing with other fluorophores, as FITC has spectral overlap with PE.

• Data analysis: Gate appropriately to exclude dead cells and debris, which can contribute to false-positive signals.

How can researchers validate the specificity of UBASH3A antibody in their experimental systems?

Validating antibody specificity is crucial for reliable research outcomes. For UBASH3A antibody validation, implement the following comprehensive strategy:

• Genetic validation:

  • Use UBASH3A knockout or knockdown cells as negative controls

  • Compare staining patterns between wild-type and UBASH3A-deficient samples

  • Rescue experiments by reintroducing UBASH3A expression in knockout cells

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide (aa 287-455 for the FITC-conjugated antibody)

  • Compare staining between blocked and unblocked antibody

• Cross-validation with multiple antibodies:

  • Test multiple antibodies targeting different UBASH3A epitopes

  • Compare staining patterns across techniques (e.g., flow cytometry, Western blot, ICC/IF)

• Correlation with mRNA expression:

  • Analyze UBASH3A protein detection in relation to mRNA levels across cell types

  • Validate expression patterns in tissues known to express UBASH3A (predominantly T cells)

• Mass spectrometry confirmation:

  • Perform immunoprecipitation followed by mass spectrometry

  • Verify that the immunoprecipitated protein is indeed UBASH3A

A comprehensive validation strategy increases confidence in experimental results and addresses concerns about antibody cross-reactivity.

What fixation and permeabilization methods are recommended for intracellular staining of UBASH3A?

For optimal intracellular detection of UBASH3A using FITC-conjugated antibodies, the following fixation and permeabilization protocols have been validated:

• PFA fixation with Triton X-100 permeabilization:

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

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

  • This method has been successfully used for detecting UBASH3A in HEL (human bone marrow erythroleukemia) cells using immunofluorescence

• Methanol fixation/permeabilization:

  • Fix and permeabilize simultaneously with ice-cold 90% methanol for 30 minutes on ice

  • Particularly effective for detecting phospho-epitopes and some intracellular proteins

• Commercial fixation/permeabilization kits:

  • For flow cytometry, specialized buffers may provide better epitope preservation

  • Follow manufacturer's protocols for time and temperature

Each method has distinct advantages depending on the subcellular localization of UBASH3A in your experimental system. The choice of method may affect antibody binding efficiency and should be optimized for each experimental setup.

How can UBASH3A antibodies be used to investigate T-cell receptor dynamics?

UBASH3A antibodies can serve as powerful tools for investigating T-cell receptor dynamics through several sophisticated approaches:

• Dual-color flow cytometry analysis:

  • Co-stain cells with FITC-conjugated UBASH3A antibody and APC-conjugated anti-CD3ε

  • Quantify correlation between UBASH3A expression levels and TCR-CD3 surface expression

  • Track temporal changes following T-cell activation

• Receptor internalization assays:

  • Based on published protocols, cells can be incubated with APC-conjugated anti-CD3ε (2.5 μg/mL) for 30 min on ice, then transferred to 37°C for various time intervals (0-60 minutes)

  • Surface-bound antibodies can be removed using acidic buffer (100 mM glycine, 100 mM NaCl, pH 2.5)

  • Compare receptor internalization rates between cells with different UBASH3A expression levels

• Confocal microscopy with live-cell imaging:

  • Use FITC-conjugated UBASH3A antibody in combination with other fluorescently-labeled TCR components

  • Track co-localization during TCR clustering and internalization following stimulation

  • Analyze spatial and temporal dynamics using high-resolution imaging

This methodological approach enables researchers to directly assess how UBASH3A levels influence TCR-CD3 expression, internalization, and recycling under both basal and stimulated conditions, providing insight into how UBASH3A negatively regulates T-cell activation.

What are the considerations when investigating the interaction between UBASH3A and CBL-B using antibody-based approaches?

Investigating UBASH3A and CBL-B interactions requires careful experimental design due to the complex nature of their relationship in T-cell signaling:

• Co-immunoprecipitation optimization:

  • When using UBASH3A antibodies for pull-down experiments, consider that the SH3 domain mediates binding to CBL-B

  • Select antibodies targeting epitopes outside the SH3 domain to avoid disrupting the interaction

  • For the FITC-conjugated antibody targeting aa 287-455, verify whether this region overlaps with the SH3 domain

• Proximity ligation assay (PLA) considerations:

  • Combine UBASH3A antibody with CBL-B-specific antibody raised in a different host species

  • Include controls for antibody specificity and background signal

  • Analyze interactions in both resting and stimulated T cells, as activation state may affect complex formation

• FRET/FLIM analysis:

  • If using FITC-conjugated UBASH3A antibody, pair with a CBL-B antibody conjugated to a suitable FRET acceptor

  • Account for potential steric hindrance affecting energy transfer efficiency

  • Validate FRET signals using appropriate negative controls

• Domain-specific mutational analysis:

  • Complement antibody-based approaches with studies using UBASH3A constructs containing mutations in the SH3 domain

  • Compare wild-type and mutant interactions to confirm specificity

Understanding this interaction can provide insights into the synergistic inhibition of T-cell function and potential contributions to autoimmune diseases like type 1 diabetes, which has been linked to both genes .

How do genetic variants in UBASH3A affect protein detection using antibodies?

Genetic variants in UBASH3A can significantly impact antibody-based detection through several mechanisms that researchers must consider:

• Epitope alterations affecting antibody binding:

  • Missense variants within the antibody's target epitope (aa 287-455 for the FITC-conjugated antibody) may alter binding affinity

  • Disease-associated variants could potentially modify protein conformation, indirectly affecting epitope accessibility

• Expression level variations:

  • Type 1 diabetes-associated variants increase UBASH3A expression in primary T cells

  • When comparing patient cohorts, standardize detection methods and include appropriate calibration controls to account for genotype-dependent expression differences

• Post-translational modification differences:

  • UBASH3A has four identified ubiquitination sites at lysine residues 15, 202, 309, and 358

  • Genetic variants may alter ubiquitination patterns, potentially masking antibody epitopes

  • Monoubiquitination at Lys 202 causes UBASH3A to adopt a closed conformation, which affects UBA domain accessibility

• Methodological considerations:

  • Include genotyping in experimental design when working with primary T cells

  • Consider denaturation state in Western blots versus native conformation in flow cytometry

  • Validate antibody performance across samples with known genotypes

These considerations are particularly relevant when studying UBASH3A in the context of autoimmune diseases, where genetic variation may contribute to pathogenesis through altered protein function or expression levels.

What are common sources of background when using FITC-conjugated UBASH3A antibodies, and how can they be minimized?

Background signal is a common challenge when working with FITC-conjugated antibodies. Here are specific sources and solutions for UBASH3A detection:

How can researchers optimize FITC-conjugated UBASH3A antibody performance for challenging samples or low-abundance targets?

When working with challenging samples or detecting low-abundance UBASH3A, consider these optimization strategies:

• Signal amplification techniques:

  • Tyramide signal amplification (TSA): Enhances FITC signal through catalyzed reporter deposition

  • Sequential multiple antibody labeling: Layer primary and secondary antibodies in cycles

  • Consider using unconjugated primary with highly-sensitive detection systems for very low abundance targets

• Sample preparation optimization:

  • For T cells, consider activation status, as UBASH3A function changes upon TCR engagement

  • Enrich target cell populations before staining (e.g., magnetic separation for T cells)

  • Optimize fixation protocols based on subcellular localization (UBASH3A functions in both membrane and cytoplasmic compartments)

• Instrument settings for flow cytometry:

  • Increase PMT voltage within linear range

  • Optimize threshold settings to exclude debris

  • Consider alternative laser/filter combinations for better FITC excitation/emission

• Microscopy enhancement:

  • Use deconvolution algorithms for improved signal-to-noise ratio

  • Consider super-resolution techniques for detailed localization studies

  • Implement longer exposure times with anti-photobleaching agents

• Antibody enhancement:

  • Try titrating from 1:500 up to 1:3000 for optimal signal-to-noise ratio

  • Consider using antibodies targeting different epitopes of UBASH3A

  • For FITC-conjugated antibody with aa 287-455 specificity, verify target accessibility in your experimental system

These approaches can be particularly valuable when studying UBASH3A in primary T cells or in samples from patients with autoimmune conditions where expression levels may vary due to genetic factors.

How does protein conformation affect UBASH3A epitope accessibility and antibody binding?

UBASH3A undergoes conformational changes that can significantly impact epitope accessibility and antibody binding efficacy:

• Ubiquitination-induced conformational changes:

  • Monoubiquitination at Lys 202 causes UBASH3A to adopt a closed conformation

  • This closed state can mask epitopes, particularly affecting antibodies targeting regions involved in this conformational change

  • The FITC-conjugated antibody targeting aa 287-455 may have variable accessibility depending on ubiquitination status

• Domain-specific considerations:

  • The UBA domain (N-terminal) binds to ubiquitin and ubiquitin chains

  • The SH3 domain mediates protein-protein interactions, particularly with CBL-B

  • Antibodies targeting regions involved in protein-protein interactions may have reduced accessibility when UBASH3A is engaged with binding partners

• Native versus denatured detection:

  • Flow cytometry and ICC/IF typically detect native conformations

  • Western blotting with SDS-PAGE detects denatured protein

  • Some epitopes may only be accessible in one conformation state

  • The FITC-conjugated antibody has been validated for ICC/IF applications, suggesting efficacy with fixed but relatively native conformations

• Fixation effects on conformation:

  • PFA fixation (as used in validated protocols) preserves native structure but can potentially mask some epitopes

  • Different fixation methods may expose different epitopes by partially denaturing the protein

Understanding these conformational considerations is essential for selecting appropriate antibodies and interpreting results, particularly when comparing data across different experimental techniques or when studying UBASH3A's role in protein-protein interaction networks relevant to T-cell regulation.

How can UBASH3A antibodies be used to investigate its role in autoimmune diseases?

UBASH3A antibodies provide valuable tools for investigating autoimmunity mechanisms through several methodological approaches:

• Expression analysis in patient samples:

  • Compare UBASH3A protein levels in T cells from patients with autoimmune diseases versus healthy controls

  • Correlate with genetic variants, as type 1 diabetes-associated variants increase UBASH3A expression

  • Use FITC-conjugated antibodies for multi-parameter flow cytometry to analyze expression across T-cell subsets

• Functional assays combining antibody detection with readouts:

  • Measure TCR-CD3 downmodulation rates in patient T cells using antibody-based internalization assays

  • Correlate UBASH3A expression with IL-2 production following TCR stimulation

  • Analyze NF-κB signaling in relation to UBASH3A levels

• Protein-protein interaction studies:

  • Investigate UBASH3A/CBL-B interactions in autoimmune conditions

  • Compare interaction strength between disease and control samples

  • Correlate with clinical parameters or disease severity

• Therapeutic target validation:

  • Use antibodies to monitor UBASH3A expression/function following experimental treatments

  • Track changes in downstream pathways affected by UBASH3A modulation

  • Validate target engagement in drug development pipelines

These approaches can provide insights into how UBASH3A contributes to autoimmune pathogenesis and potentially identify new therapeutic strategies for conditions like type 1 diabetes.

What are the best practices for analyzing UBASH3A expression in relation to genetic variants associated with autoimmunity?

When analyzing UBASH3A expression in relation to disease-associated genetic variants, researchers should follow these best practices:

• Experimental design considerations:

  • Genotype subjects for relevant UBASH3A single nucleotide polymorphisms (SNPs)

  • Include sufficient sample sizes for each genotype group

  • Match cases and controls for age, sex, and ethnicity to minimize confounding factors

  • Consider disease duration and treatment status as potential confounders

• Expression analysis methodology:

  • Use flow cytometry with FITC-conjugated UBASH3A antibody for single-cell resolution

  • Complement protein detection with mRNA analysis (RT-qPCR or RNA-seq)

  • Analyze expression in specific T-cell subsets (CD4+, CD8+, memory, naïve)

  • Include appropriate isotype controls and standardization markers

• Functional correlation:

  • Measure TCR-CD3 complex expression and downmodulation dynamics

  • Assess IL-2 production capacity following stimulation

  • Evaluate NF-κB signaling activation

  • Correlate UBASH3A expression with functional readouts

• Data analysis framework:

  • Account for allele dosage effects (heterozygous vs. homozygous for risk alleles)

  • Consider gene-environment interactions

  • Use multivariate models to adjust for confounding factors

  • Perform longitudinal analyses when possible to assess temporal changes

This structured approach enables robust investigation of how UBASH3A genetic variants contribute to autoimmune disease pathogenesis through altered protein expression and function.

How might advanced imaging techniques enhance our understanding of UBASH3A function using fluorescently labeled antibodies?

Advanced imaging approaches with FITC-conjugated UBASH3A antibodies can reveal unprecedented insights into protein function:

• Super-resolution microscopy applications:

  • STORM or PALM imaging can resolve UBASH3A distribution at nanometer resolution

  • Structured illumination microscopy (SIM) can reveal colocalization with TCR components

  • These techniques can elucidate how UBASH3A spatially organizes within immunological synapses during T-cell activation

• Live-cell imaging strategies:

  • Combine membrane-permeable FITC-conjugated antibody fragments with real-time imaging

  • Track UBASH3A dynamics during T-cell activation and TCR internalization

  • Correlate movement with functional outcomes in signaling cascades

• Multi-color imaging approaches:

  • Simultaneously visualize UBASH3A (FITC-labeled) with TCR components and endocytic machinery

  • Track colocalization changes over time following T-cell stimulation

  • Analyze spatial relationships between UBASH3A and CBL-B during signaling events

• Correlative light and electron microscopy (CLEM):

  • Initially identify UBASH3A distribution using FITC-conjugated antibodies

  • Subsequently analyze the same sample at ultrastructural resolution

  • This approach can reveal how UBASH3A associates with specific subcellular structures

These advanced imaging approaches can answer fundamental questions about how UBASH3A physically interfaces with the T-cell signaling machinery, potentially revealing new therapeutic targets for autoimmune disease intervention.

What emerging technologies might enhance UBASH3A detection beyond current FITC-conjugated antibody applications?

Emerging technologies offer promising avenues to enhance UBASH3A detection and functional analysis:

• Proximity-based protein detection systems:

  • CRISPR-based proximity labeling could tag endogenous UBASH3A with engineered peroxidases

  • BioID or APEX approaches could map the UBASH3A interactome with temporal resolution

  • These methods avoid potential artifacts from antibody binding disrupting native interactions

• Single-molecule tracking technologies:

  • Quantum dot-conjugated antibodies provide superior photostability compared to FITC

  • DNA-PAINT techniques offer extremely high resolution without photobleaching limitations

  • These approaches could track individual UBASH3A molecules during T-cell activation events

• Mass cytometry (CyTOF) applications:

  • Metal-tagged anti-UBASH3A antibodies enable highly multiplexed analysis

  • Simultaneous measurement of UBASH3A with dozens of other markers

  • Particularly valuable for analyzing heterogeneous immune cell populations

• In situ protein analysis:

  • Expansion microscopy combined with FITC-conjugated antibodies to visualize nanoscale distribution

  • CODEX multiplexed imaging for iterative antibody staining

  • These methods reveal spatial relationships between UBASH3A and multiple signaling components

• Genetically encoded sensors:

  • CRISPR knock-in of fluorescent proteins to tag endogenous UBASH3A

  • Development of conformational sensors to detect UBASH3A activation states

  • These approaches enable longitudinal studies without antibody addition

These emerging technologies will likely provide unprecedented insights into UBASH3A biology and its role in T-cell regulation and autoimmune disease pathogenesis.