UBXN2A Antibody, FITC conjugated

What is UBXN2A and why is it important in scientific research?

UBXN2A is a member of the UBX domain-containing protein family that functions as adaptors for VCP (Valosin-Containing Protein), a critical AAA+ ATPase involved in numerous cellular processes. UBXN2A contains a UBX domain that adopts a ubiquitin fold and associates with the N-terminus of VCP . Research indicates that UBXN2A participates in the VCP-adaptor network linked to various biological processes including protein quality control, membrane fusion, and cellular signaling . Understanding UBXN2A's interactions provides insight into fundamental cellular mechanisms, making it a significant target for scientific investigation.

What applications are most suitable for FITC-conjugated UBXN2A antibodies?

FITC-conjugated UBXN2A antibodies are particularly well-suited for applications requiring direct fluorescent detection without secondary antibodies. Based on analysis of available UBXN2A antibodies, FITC-conjugated versions are most commonly used for:

• Flow cytometry (FACS) with recommended dilutions of 1:10-1:50

• Immunofluorescence microscopy for subcellular localization studies

• Live cell imaging where cell permeabilization would disrupt native protein localization

• Multi-color immunofluorescence studies where primary antibody host species constraints exist

The direct fluorescent conjugation eliminates potential cross-reactivity issues that can occur with secondary antibodies and simplifies experimental protocols.

What is the epitope specificity of commonly available UBXN2A antibodies?

Most commercially available UBXN2A antibodies, including those with FITC conjugation, are generated against specific amino acid sequences. The predominant epitope targets include:

When selecting a FITC-conjugated UBXN2A antibody, researchers should consider whether the epitope region might be masked in their experimental context, such as in protein complexes or due to post-translational modifications.

How should sample preparation be optimized for FITC-conjugated UBXN2A antibody use in immunofluorescence?

For optimal results with FITC-conjugated UBXN2A antibodies in immunofluorescence applications:

• Fixation: Paraformaldehyde (4%) fixation for 10-15 minutes at room temperature is recommended to preserve cellular architecture while maintaining epitope accessibility .

• Permeabilization: Use Triton X-100 (0.1-0.2%) for intracellular staining as recommended for related UBXN2A antibodies .

• Blocking: Employ 3-5% BSA or normal serum from a species unrelated to the antibody host (typically not rabbit for UBXN2A antibodies) to reduce background.

• Antibody concentration: Start with dilutions of 1:50 for immunofluorescence microscopy and optimize based on signal-to-noise ratio .

• Incubation conditions: Incubate with antibody for 1-2 hours at room temperature or overnight at 4°C in a humidified chamber to prevent sample drying.

• Counterstains: When using DAPI for nuclear counterstaining, ensure filter sets are appropriately chosen to avoid spectral overlap with FITC.

Researchers should note that subcellular localization studies have shown UBXN2A exhibits both cytoplasmic and centrosomal localization in certain cell types .

What protocol modifications are needed for flow cytometry applications with FITC-conjugated UBXN2A antibodies?

For flow cytometry applications, consider these methodological adjustments:

• Cell preparation: Single-cell suspensions must be properly prepared with gentle dissociation methods to preserve cellular integrity.

• Fixation/permeabilization: Since UBXN2A is primarily intracellular, use a compatible fixation/permeabilization solution (e.g., methanol or commercial kits designed for intracellular antigens).

• Antibody dilution: Begin with 1:10-1:50 dilution as recommended for UBXN2A antibodies in flow cytometry .

• Controls: Include:

  • Unstained cells for autofluorescence assessment

  • Isotype control (FITC-conjugated rabbit IgG) at the same concentration

  • Single-color controls if performing multicolor analysis

• Compensation: Properly compensate for FITC spillover when using multiple fluorophores.

• Data analysis: Gate strategy should account for cell size, granularity, and viability before analyzing FITC signal.

The relatively high concentration required (1:10-1:50) reflects the typically low abundance of UBXN2A in many cell types.

How can specificity of FITC-conjugated UBXN2A antibodies be validated?

Rigorous validation of FITC-conjugated UBXN2A antibodies should include multiple approaches:

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (amino acids 166-195 for C-terminal antibodies) before staining to confirm signal specificity .

• Knockout/knockdown controls: Compare staining in UBXN2A knockout or siRNA-treated cells versus wild-type cells.

• Orthogonal detection methods: Confirm UBXN2A localization using an alternative method (e.g., unconjugated antibody from a different host species or epitope).

• Western blot correlation: Confirm the antibody recognizes a band of appropriate molecular weight (approximately 29.3 kDa) in lysates from the same cells used for imaging or flow cytometry .

• Cross-reactivity assessment: Test the antibody on cells from species outside the stated reactivity range (human and mouse) to confirm specificity .

How does UBXN2A protein interaction with other UBXD family members affect antibody epitope accessibility?

Recent proteomics research has revealed that UBXN2A interacts with multiple other UBXD proteins in the VCP-adaptor network . This has important implications for antibody-based detection:

• Interaction complexes: UBXN2A associates with at least 6 distinct UBXD proteins and with UFD1L/NPLOC4 components , potentially masking epitopes.

• Epitope occlusion: The C-terminal region (amino acids 166-195) used to generate many UBXN2A antibodies may be partially obstructed in certain protein complexes.

• Experimental disruption: Detergent conditions during sample preparation may alter these protein-protein interactions, affecting epitope accessibility.

• Research strategy: For studies focusing on UBXN2A in complex with specific binding partners, researchers should test multiple antibodies targeting different epitopes or employ proximity ligation assays instead of direct immunofluorescence.

Understanding these interaction dynamics can help researchers interpret unexpected immunofluorescence patterns when investigating UBXN2A's biological roles.

How can researchers distinguish between specific and non-specific signals when using FITC-conjugated UBXN2A antibodies?

Discriminating true signal from background is particularly important with direct FITC conjugates due to the absence of signal amplification:

• Signal characteristics to evaluate:

  • Subcellular localization pattern: Authentic UBXN2A staining should correspond to its known distribution patterns including cytoplasmic localization and centrosomal association in certain cell types .

  • Signal intensity correlation: Signal strength should correlate with expected UBXN2A expression levels across different cell types or experimental conditions.

  • Phenotype concordance: Staining patterns should align with expected biological effects when UBXN2A function is perturbed.

• Technical controls to implement:

  • Concentration-matched FITC-conjugated isotype control antibodies

  • Secondary-only controls when comparing to indirect detection methods

  • Autofluorescence controls, particularly critical with FITC due to its spectral overlap with common autofluorescence sources

  • Blocking peptide competition assays using the specific immunogen peptide (AA 166-195)

• Analytical approaches:

  • Signal-to-background quantification across multiple fields

  • Colocalization analysis with known UBXN2A binding partners

  • Correlation of fluorescence intensity with protein levels determined by orthogonal methods

What are the considerations for studying UBXN2A localization changes during cellular stress responses?

UBXN2A participates in cellular stress response pathways through its role in the VCP-adaptor network. When designing experiments to monitor dynamic changes in UBXN2A localization:

• Temporal dynamics: Design time-course experiments with appropriate intervals (5 min, 15 min, 30 min, 1 hr, 3 hr) after stress induction.

• Stress specificity: Different stressors may affect UBXN2A differently:

  • Proteotoxic stress (proteasome inhibitors like MG132)

  • ER stress (tunicamycin, thapsigargin)

  • DNA damage (UV radiation, cisplatin)

  • Mitochondrial stress (antimycin A, oligomycin)

• Compartmentalization changes: UBXN2A may relocalize between:

  • Cytoplasmic distribution

  • Centrosomal association

  • Potential mitochondrial association (suggested by FAF2 association data)

• Fixation timing: Rapid fixation is critical for capturing transient relocalization events.

• Co-visualization strategies: Co-stain for VCP or other known UBXN2A interacting partners to track complex formation or dissolution.

What is known about UBXN2A's role in the VCP-adaptor network and how can FITC-conjugated antibodies help elucidate this?

UBXN2A functions within the complex VCP-adaptor network that regulates diverse cellular processes. FITC-conjugated UBXN2A antibodies can help illuminate these relationships:

• Network composition: UBXN2A interacts with multiple protein complexes including:

  • Other UBXD family adaptors

  • The UFD1L-NPLOC4 complex essential for some VCP functions

  • A phosphatase complex shared with UBXN2B

• Visualization approaches using FITC-conjugated antibodies:

  • Co-immunofluorescence with VCP and other adaptors

  • Proximity ligation assays to confirm direct interactions

  • FRET-based approaches for dynamic interaction studies

  • Live-cell imaging to track complex formation and dissolution

• Research applications:

  • Mapping UBXN2A interaction domains through mutational analysis

  • Identifying stimuli that alter UBXN2A complex formation

  • Determining cell-type specific differences in UBXN2A network composition

The direct fluorescence detection provided by FITC conjugation is particularly valuable for multi-protein complex visualization where secondary antibody cross-reactivity might be problematic.

How can researchers use FITC-conjugated UBXN2A antibodies to investigate its potential role in cellular pathologies?

UBXN2A's involvement in the VCP-adaptor network suggests potential roles in disease processes. FITC-conjugated antibodies facilitate several investigative approaches:

• Tissue expression studies:

  • Immunohistochemical analysis shows strong cytoplasmic and membranous positivity in renal tubules

  • Expression pattern changes in disease states can be quantified by comparing staining intensity and localization

• Cellular pathology models:

  • Neurodegenerative disease models (VCP mutations cause IBMPFD syndrome)

  • Cancer cell lines with altered proteostasis

  • Renal pathology models given strong UBXN2A expression in kidney tubules

• Quantitative approaches:

  • Flow cytometry to measure UBXN2A levels across patient-derived samples

  • High-content imaging to correlate UBXN2A expression/localization with disease markers

  • Tissue microarray analysis for large-scale patient cohort studies

• Mechanistic investigations:

  • Colocalization with disease-associated proteins

  • Changes in UBXN2A-VCP interaction dynamics in disease models

  • Correlation between UBXN2A levels and cellular stress markers

What are the methodological considerations for multiplex staining involving FITC-conjugated UBXN2A antibodies?

For researchers conducting multiplex immunofluorescence studies:

• Spectral compatibility:

  • FITC emission spectrum (peak ~520 nm) must be considered when selecting additional fluorophores

  • Recommended compatible partners: Texas Red, Cy5, APC (minimal spectral overlap)

  • Partners to avoid: PE, GFP (significant spectral overlap with FITC)

• Sequential staining protocol:

  • Begin with lowest abundance target (often UBXN2A)

  • Implement effective blocking between sequential stainings

  • Consider tyramide signal amplification for low-abundance targets when using multiplexed approach

• Controls for multiplexing:

  • Single-color controls for compensation/spectral unmixing

  • Antibody order controls to detect potential interference

  • Matched isotype controls for each antibody in the panel

• Analysis considerations:

  • Use spectral unmixing algorithms for closely overlapping fluorophores

  • Employ colocalization analysis tools (Pearson's correlation, Manders' coefficients)

  • Consider automated cell classification based on staining patterns

How should researchers address weak or inconsistent signals when using FITC-conjugated UBXN2A antibodies?

When encountering signal problems with FITC-conjugated UBXN2A antibodies:

• Signal enhancement strategies:

  • Increase antibody concentration (starting with 1:10 dilution for flow cytometry)

  • Extend incubation time (overnight at 4°C)

  • Optimize fixation protocol (test both 4% PFA and methanol fixation)

  • Try alternative epitope unmasking methods for tissue sections

  • Consider tyramide signal amplification systems compatible with FITC

• Common technical issues and solutions:

  • Photobleaching: Use anti-fade mounting media, minimize exposure time, add n-propyl gallate

  • Low target abundance: Verify UBXN2A expression in your cell type by Western blot first

  • Epitope masking: Test alternative fixation protocols or antibodies targeting different epitopes

  • pH sensitivity: Ensure buffers are maintained at pH 7.2-7.4, as FITC fluorescence is pH-sensitive

• Biological factors affecting detection:

  • UBXN2A expression levels vary by cell type

  • Protein interactions may mask epitopes

  • Post-translational modifications might affect antibody binding

  • Cell cycle-dependent localization patterns

How can researchers properly interpret UBXN2A subcellular localization patterns?

Accurate interpretation of UBXN2A localization requires understanding its biological context:

• Expected localization patterns:

  • Primarily cytoplasmic distribution in most cell types

  • Centrosomal localization observed in some cells

  • Potential association with membranes and specific organelles

  • Dynamic relocalization during cellular stress responses

• Validation approaches:

  • Confirm patterns with antibodies against different UBXN2A epitopes

  • Co-stain with markers for specific subcellular compartments

  • Use GFP-tagged UBXN2A constructs to confirm antibody-detected patterns

  • Perform subcellular fractionation followed by Western blotting

• Biological significance assessment:

  • Compare localization patterns before and after relevant stimuli

  • Correlate localization changes with functional outcomes

  • Determine if observed patterns match known UBXN2A protein interactions

  • Investigate whether disease states alter normal UBXN2A distribution