SSNA1 Antibody

What is SSNA1 and why are antibodies against it important for research?

SSNA1 (Sjögren's syndrome nuclear autoantigen 1), also known as NA14, is a 119 amino acid nuclear and cytoplasmic protein belonging to the SSNA1 family. It contains an N-terminal dimeric coiled-coil domain and a C-terminal basic domain that associates with microtubule structures . SSNA1 antibodies are critical research tools because:

• They enable visualization of SSNA1's diverse cellular localizations (basal bodies, axonemes, spindle poles, midbody) across different cell types

• They facilitate investigation of SSNA1's role in microtubule dynamics and stability

• They help study SSNA1's involvement in pathological conditions including hepatocellular carcinoma and Sjögren's syndrome

• They allow detection of SSNA1's interactions with other proteins such as spastin

What cellular and tissue distribution patterns does SSNA1 exhibit?

SSNA1 shows a distinct expression profile across tissues and cellular compartments:

When selecting tissues for antibody validation or expression studies, researchers should consider this distribution pattern to maximize detection probability .

Which applications are SSNA1 antibodies validated for?

SSNA1 antibodies have been validated for multiple experimental applications:

When selecting an antibody, researchers should ensure it has been validated for their specific application and target species. Cross-reactivity tests are recommended when working with non-validated species combinations .

How can researchers optimize detection of SSNA1 at microtubule damage sites?

Detecting SSNA1 at microtubule damage sites requires careful experimental design:

• Damage induction methods:

  • Use purified spastin for controlled microtubule severing

  • Apply mechanical forces through micromanipulation techniques

  • Employ photodamage with focused laser light

• Imaging considerations:

  • Implement TIRF microscopy for enhanced signal-to-noise ratio near substrate

  • Use confocal microscopy with high numerical aperture objectives for intracellular damage sites

  • Consider super-resolution techniques (STED, STORM) for precise localization

• Experimental controls:

  • Include undamaged microtubules as negative controls

  • Use known damage site markers as positive controls

  • Employ SSNA1 depletion/knockout samples to confirm antibody specificity

• Technical recommendations:

  • Fix cells rapidly after damage induction to preserve transient SSNA1 recruitment

  • Consider dual-labeling with tubulin antibodies to clearly identify damaged regions

  • Use time-lapse imaging to capture dynamic recruitment processes

Research has shown that SSNA1 is specifically enriched at microtubule damage sites, occurring both naturally and those induced by spastin, making it an excellent marker for studying microtubule damage responses .

What methodological considerations are important when investigating SSNA1's role in hepatocellular carcinoma progression?

Recent findings indicate SSNA1 is upregulated in HCC tissues, with expression increasing proportionally to disease progression and correlating with poor prognosis . Key methodological considerations include:

• Expression analysis approach:

  • Use quantitative immunohistochemistry to assess SSNA1 levels across HCC stages

  • Employ western blotting with SSNA1 antibodies for comparative analysis between normal and tumor tissues

  • Consider multiplex staining to correlate SSNA1 expression with other HCC markers

• Functional investigations:

  • Design SSNA1 knockdown experiments using siRNA/shRNA to assess functional consequences

  • Monitor STAT3/EMT axis components after SSNA1 depletion

  • Implement scratch assays and transwell invasion assays to evaluate migration and invasion capacity

• Mechanistic studies:

  • Evaluate SSNA1's effects on STAT3 phosphorylation status using phospho-specific antibodies

  • Assess EMT marker expression (E-cadherin, N-cadherin, vimentin) in response to SSNA1 modulation

  • Investigate potential co-immunoprecipitation between SSNA1 and STAT3 pathway components

• Clinical correlation:

  • Stratify patient samples by SSNA1 expression levels and correlate with survival data

  • Analyze SSNA1 expression in primary tumors versus metastatic sites

  • Consider SSNA1 as a potential biomarker for early detection or prognosis prediction

These approaches can help elucidate how SSNA1 contributes to HCC progression through STAT3/EMT axis modulation, potentially revealing new therapeutic targets .

How do different fixation and permeabilization methods affect SSNA1 antibody immunoreactivity?

The detection of SSNA1 by antibodies can be significantly influenced by sample preparation techniques:

Optimization strategies:

• Test multiple fixation protocols in parallel on the same sample type

• Consider dual-fixation approaches (brief PFA followed by methanol) for comprehensive detection

• Optimize antibody concentration for each fixation method

• Implement antigen retrieval for formalin-fixed tissues to expose masked epitopes

• For colocalization studies with tubulin, methanol fixation typically provides superior results

When studying SSNA1's association with microtubule dynamics or damage sites, methanol fixation often yields the most accurate representation of its localization patterns .

What approaches can be used to study the interaction between SSNA1 and spastin using antibody-based techniques?

Investigating the SSNA1-spastin interaction is crucial given SSNA1's protective role against spastin-mediated microtubule severing . Recommended methodological approaches include:

• Co-immunoprecipitation (Co-IP):

  • Use SSNA1 antibodies to pull down protein complexes and probe with spastin antibodies

  • Perform reciprocal Co-IP with spastin antibodies and detect SSNA1

  • Include negative controls (IgG pull-downs) and positive controls (known interactors)

  • Consider native vs. crosslinking conditions to capture transient interactions

• Proximity Ligation Assay (PLA):

  • Apply paired antibodies against SSNA1 and spastin

  • Quantify interaction signals across different cellular compartments

  • Compare interaction frequency under normal vs. stressed cellular conditions

  • Use SSNA1 or spastin knockout/knockdown cells as specificity controls

• Immunofluorescence colocalization:

  • Employ high-resolution imaging techniques (SIM, STED)

  • Analyze colocalization coefficients at microtubule damage sites

  • Perform time-lapse imaging to track temporal dynamics of recruitment

  • Use FRET-based approaches with labeled antibodies for direct interaction assessment

• In vitro reconstitution with purified components:

  • Use antibodies to confirm protein purity and identity

  • Implement TIRF microscopy to visualize labeled SSNA1 accumulation at spastin-induced damage sites

  • Quantify spastin severing activity with varying concentrations of SSNA1

  • Design competition assays with SSNA1 fragments to map interaction domains

These methodologies can help delineate how SSNA1 detects and protects against spastin-induced microtubule damage, potentially informing therapeutic strategies for conditions involving microtubule instability .

How can researchers distinguish between monomeric and fibrillar forms of SSNA1 using antibodies?

SSNA1 has been demonstrated to self-assemble into fibril-like structures both in vitro and in vivo , posing unique challenges for antibody-based detection:

• Epitope accessibility considerations:

  • Select antibodies targeting epitopes that remain accessible in fibrils

  • Use multiple antibodies targeting different regions to differentiate assembly states

  • Consider conformation-specific antibodies that selectively recognize fibrillar forms

• Sample preparation optimization:

  • Employ non-denaturing conditions to preserve fibrillar structures

  • Use differential centrifugation to separate monomeric and fibrillar forms

  • Apply size-exclusion chromatography for fractionation before antibody probing

• Specialized detection techniques:

  • Implement super-resolution microscopy to visualize fibrillar structures

  • Apply thioflavin T or Congo red staining in parallel with antibody labeling

  • Use electron microscopy with immunogold labeling for definitive fibril identification

  • Consider native PAGE followed by western blotting to distinguish assembly states

• Quantitative approaches:

  • Develop ELISAs with capture antibodies specific to different conformational states

  • Implement kinetic aggregation assays with labeled antibodies

  • Apply analytical ultracentrifugation in combination with antibody detection

Research indicates that SSNA1's ability to form fibrils may contribute to its immunogenicity in conditions like Sjögren's syndrome, making the discrimination between monomeric and fibrillar forms particularly relevant for autoimmunity research .

What are the optimal protocols for visualizing SSNA1's effects on microtubule dynamics?

SSNA1 has been shown to modulate all parameters of microtubule dynamic instability, making visualization of these effects crucial for understanding its function :

• In vitro reconstitution approach:

  • Prepare GMPCPP-stabilized microtubule seeds as templates

  • Add purified tubulin (3-10 μM) with and without SSNA1 (2.5 μM)

  • Employ TIRF microscopy for direct visualization

  • Track microtubule growth rates, shrinkage rates, catastrophe frequency, and rescue events

  • Quantify SSNA1 intensity at microtubule ends over time

• Live-cell imaging approach:

  • Express fluorescently-tagged SSNA1 (ensuring tag doesn't interfere with function)

  • Co-express fluorescent tubulin markers

  • Use spinning disk confocal microscopy for rapid acquisition

  • Track colocalization dynamics during growth, shrinkage, and at damage sites

  • Compare control cells with SSNA1-depleted or overexpressing cells

• Parameters to measure:

  • Microtubule growth rate (~7.6 ± 0.9 nm/s initially, slowing to ~3.1 ± 0.8 nm/s with SSNA1)

  • Catastrophe frequency (reduced with SSNA1)

  • Rescue frequency (increased with SSNA1)

  • SSNA1 enrichment at microtubule ends (correlates with growth slowdown)

• Perturbation experiments:

  • Remove soluble tubulin to induce catastrophe

  • Add Op18/Stathmin to sequester tubulin

  • Introduce spastin to create microtubule damage sites

  • Monitor SSNA1 recruitment dynamics under these conditions

These methodologies have revealed that SSNA1 acts as both a microtubule stabilizer and a sensor of microtubule damage, providing insight into its cellular functions .

What controls and validation steps are essential when using SSNA1 antibodies in experimental settings?

Proper validation of SSNA1 antibodies is critical for obtaining reliable research results:

• Specificity controls:

  • SSNA1 knockout/knockdown samples as negative controls

  • Preabsorption with purified antigen to confirm specificity

  • Testing multiple SSNA1 antibodies targeting different epitopes

  • Correlation of signal with known SSNA1 expression patterns across tissues

• Application-specific validation:

  • For Western blotting: Confirm band at expected molecular weight (~16 kDa)

  • For IHC/IF: Verify subcellular localization patterns (nuclear/cytoplasmic, centrosomal, microtubule-associated)

  • For co-IP: Include non-specific IgG controls and known interaction partners

  • For ELISA: Generate standard curves with purified SSNA1 protein

• Cross-reactivity assessment:

  • Test antibody against related family members

  • Validate across species when using for evolutionary studies

  • Confirm specificity in tissues with complex protein mixtures

• Technical optimization:

  • Titrate antibody concentrations for each application

  • Test multiple detection methods (direct vs. indirect)

  • Optimize blocking conditions to minimize background

  • Validate lot-to-lot consistency for critical experiments

• Functional validation:

  • Confirm antibody doesn't interfere with SSNA1's microtubule-binding activity in functional assays

  • Verify detection of both native and denatured forms if relevant to the research question

  • Assess detection sensitivity in samples with varying SSNA1 expression levels

Implementing these validation steps ensures confident interpretation of results and reproducibility across experiments and laboratories.

How can SSNA1 antibodies contribute to cancer biomarker research?

Recent findings indicate SSNA1 has potential as a biomarker in hepatocellular carcinoma, warranting further investigation with antibody-based approaches :

• Diagnostic applications:

  • Develop immunohistochemical scoring systems for SSNA1 expression in HCC tissues

  • Correlate SSNA1 levels with established HCC staging systems

  • Investigate SSNA1 detection in liquid biopsies using specific antibodies

  • Create antibody-based diagnostic assays for early HCC detection

• Prognostic value assessment:

  • Analyze SSNA1 expression in patient cohorts with known outcomes

  • Correlate expression patterns with metastatic potential

  • Develop multimarker panels incorporating SSNA1 antibody detection

  • Investigate SSNA1's relationship to treatment response

• Mechanistic insights:

  • Use phospho-specific antibodies to track STAT3 activation in relation to SSNA1 expression

  • Monitor EMT markers in parallel with SSNA1 detection

  • Develop antibodies against specific SSNA1 post-translational modifications

  • Investigate SSNA1's role in other cancer types beyond HCC

• Therapeutic monitoring:

  • Track changes in SSNA1 expression during treatment

  • Correlate with response to microtubule-targeting therapeutics

  • Explore SSNA1 as a potential therapeutic target itself

Research has shown that elevated SSNA1 expression in HCC patients correlates with poor prognosis, suggesting its value as both a biomarker and potential therapeutic target .

What role does SSNA1 play in autoimmune conditions and how can antibodies help study this connection?

SSNA1 was initially identified as an autoantigen in Sjögren's syndrome, and antibodies are crucial for investigating this connection :

• Autoantibody detection strategies:

  • Develop ELISA assays using purified SSNA1 to detect anti-SSNA1 autoantibodies

  • Implement western blotting with recombinant SSNA1 for autoantibody screening

  • Create multiplexed autoantigen arrays including SSNA1

  • Compare autoantibody reactivity against monomeric versus fibrillar SSNA1

• Epitope mapping approaches:

  • Generate fragment-specific antibodies covering different SSNA1 domains

  • Compare autoantibody and research antibody binding profiles

  • Identify immunodominant epitopes in patient populations

  • Correlate epitope recognition patterns with clinical manifestations

• Pathogenic mechanism investigation:

  • Study SSNA1 fibril formation and its relationship to immunogenicity

  • Examine co-localization of SSNA1 with immune complexes in tissue samples

  • Investigate cellular responses to anti-SSNA1 antibody binding

  • Explore cross-reactivity with other autoantigens or microtubule-associated proteins

• Clinical correlation methodologies:

  • Analyze anti-SSNA1 autoantibody levels across disease subtypes

  • Track longitudinal changes in autoantibody titers

  • Correlate with specific clinical features of Sjögren's syndrome

  • Investigate presence in other autoimmune conditions

Research indicates that approximately 14% of PSS cases test positive for autoantibodies against SSNA1, compared to 2% or less of patients with other rheumatoid diseases, highlighting its value as a specific autoantigen in PSS diagnostics .