S100A3 Antibody

What is S100A3 and why is it significant in research?

S100A3 is a member of the S100 family of calcium-binding proteins involved in numerous cellular processes including cell differentiation, proliferation, and migration. Its significance stems from its potential role in multiple diseases, including cancer, inflammatory disorders, and neurodegenerative conditions . As a calcium-binding protein, S100A3 participates in signaling pathways that regulate critical cellular functions, making it an important target for researchers investigating these fundamental biological processes .

What applications are validated for S100A3 antibodies?

S100A3 antibodies have been validated for several key applications in molecular and cellular biology research. Western Blotting (WB) is the most commonly validated application across different antibody products . Additional validated applications include immunohistochemistry (IHC), immunocytochemistry (ICC), immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA) . The specific validation depends on the particular antibody product, with some antibodies demonstrating broader application profiles than others. For comprehensive studies, researchers should verify the specific applications validated for their chosen antibody product.

What species reactivity can be expected with S100A3 antibodies?

The species reactivity of S100A3 antibodies varies by product but commonly includes human samples as the primary target . Many antibodies show cross-reactivity with mouse and rat S100A3 proteins, making them suitable for comparative studies across these common laboratory models . Some antibody products may also demonstrate reactivity with bovine (cow) samples . When designing experiments involving multiple species, researchers should carefully verify the cross-reactivity profile of their specific antibody to ensure consistent detection across experimental models.

What is the difference between polyclonal and monoclonal S100A3 antibodies?

Polyclonal antibodies are generated by immunizing animals (typically rabbits) with recombinant S100A3 protein or peptides, resulting in antibodies that recognize multiple epitopes. This provides broader detection capabilities but potentially more background. Monoclonal antibodies are produced from single B-cell clones, offering higher specificity for a single epitope but potentially more limited detection capabilities .

What are the optimal conditions for using S100A3 antibodies in Western blotting?

For optimal Western blotting results with S100A3 antibodies, researchers should follow these methodological guidelines:

• Sample preparation: Use RIPA or NP-40 based lysis buffers with protease inhibitors to preserve protein integrity.

• Recommended dilution: Most S100A3 antibodies perform optimally at dilutions between 1:500 and 1:2000 for Western blotting .

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Overnight at 4°C with gentle agitation.

• Detection system: HRP-conjugated secondary antibodies with appropriate species specificity.

• Positive controls: SKOV3 and HepG2 cell lysates have been verified as positive controls .

• Expected molecular weight: Approximately 11-12 kDa for the S100A3 protein.

Optimization may be required for specific experimental conditions and antibody products.

How can I confirm the specificity of my S100A3 antibody results?

Confirming antibody specificity is crucial for reliable research findings. For S100A3 antibodies, implement these validation strategies:

• Positive controls: Include lysates from cells known to express S100A3, such as SKOV3 or HepG2 cell lines .

• Negative controls: Use tissues or cells with confirmed absence of S100A3 expression.

• Knockdown/knockout validation: Compare results between wild-type samples and those with S100A3 knockdown or knockout, as demonstrated in studies examining S100A3 function in cancer cells .

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to block specific binding.

• Multiple antibodies: Use antibodies targeting different epitopes of S100A3 to cross-validate findings.

• Molecular weight verification: Confirm the detected band matches the expected size of S100A3 (approximately 11-12 kDa).

• Immunoprecipitation followed by mass spectrometry: For ultimate confirmation of specificity in complex samples.

This comprehensive approach ensures confident interpretation of experimental results.

What subcellular localization pattern should be expected when detecting S100A3?

S100A3 demonstrates a distinctive subcellular distribution pattern that researchers should be aware of when conducting immunofluorescence or subcellular fractionation experiments:

• Dual localization: S100A3 is evenly distributed between the cytosol and nucleus in many cell types .

• Nuclear interaction: While S100A3 is present in both compartments, its functionally significant interactions with proteins like RARα occur predominantly in the nucleus .

• Visualization pattern: In immunofluorescence experiments, S100A3 typically displays a punctate pattern within the nucleus when co-localized with nuclear binding partners .

• Cell type variations: The distribution pattern may vary depending on cell type and experimental conditions.

• Response to stimuli: Upon treatment with agents like all-trans retinoic acid (ATRA), the interaction pattern and potentially the localization of S100A3 may change .

When performing subcellular localization studies, proper fractionation controls (nuclear and cytoplasmic markers) should be included to confirm compartment separation.

How does S100A3 interact with retinoic acid receptors and what are the implications?

The interaction between S100A3 and retinoic acid receptors (RARs) represents a significant area of research with implications for cancer biology and cellular differentiation:

• Binding specificity: S100A3 shows strong interactions with RARα and RARγ, but not with RARβ or RXRα, as demonstrated through co-immunoprecipitation and GST pull-down assays .

• Interaction domain: The binding occurs at the RARα ligand-binding domain, with the I396 residue playing a crucial role in this interaction .

• Regulatory function: S100A3 controls the constitutive and ATRA-dependent degradation of RARα and PML-RARα fusion proteins .

• Context-dependent effects:

  • In breast and lung cancer cells, S100A3 knockdown decreases RARα levels, inducing resistance to ATRA-dependent anti-proliferative and differentiating effects .

  • In acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells, S100A3 knockdown reduces RARα/PML-RARα levels and increases both basal and ATRA-induced differentiation .

• Ligand dependency: S100A3 interacts predominantly with unliganded RARα, and this interaction is reduced by ATRA treatment .

These findings suggest that S100A3 plays a critical role in modulating retinoic acid signaling pathways, with potential therapeutic implications for cancers treated with retinoids.

What methodological approaches can be used to study S100A3 protein-protein interactions?

To investigate S100A3 protein-protein interactions, researchers can employ these advanced methodological approaches:

• Co-immunoprecipitation: Use anti-S100A3 antibodies to precipitate protein complexes from cell lysates, followed by Western blotting for potential interacting partners. This approach has successfully revealed interactions with RARα in nuclear fractions .

• GST pull-down assays: Employ GST-tagged S100A3 to pull down interacting proteins from cell lysates, as demonstrated in studies with retinoid receptors .

• Proximity ligation assay (PLA): Visualize protein-protein interactions in situ by detecting proteins in close proximity (<40 nm).

• Subcellular fractionation: Separate nuclear and cytoplasmic fractions before immunoprecipitation to identify compartment-specific interactions, as shown with the nuclear-specific interaction between S100A3 and RARα .

• Immunofluorescence co-localization: Visualize potential interactions through co-localization studies, with quantitative analysis of overlapping signals .

• Deletion and point mutation analysis: Create mutants of S100A3 or its potential binding partners to map interaction domains and critical residues.

• Functional validation: Confirm biological relevance of interactions through knockdown/overexpression studies examining effects on protein stability, localization, or function .

These complementary approaches provide robust evidence for protein-protein interactions and their biological significance.

How can S100A3 expression and function be modulated in experimental systems?

Modulating S100A3 expression and function is essential for investigating its biological roles. Researchers can employ these methodological strategies:

• RNA interference:

  • siRNA transfection for transient knockdown

  • shRNA for stable knockdown via lentiviral transduction

  • These approaches have been successfully used to demonstrate S100A3's role in regulating RARα and PML-RARα stability

• CRISPR/Cas9 gene editing:

  • Complete knockout of S100A3

  • Introduction of specific mutations to study structure-function relationships

• Overexpression systems:

  • Transient transfection of S100A3 expression vectors

  • Stable cell lines with inducible S100A3 expression

  • Tagged versions (GFP, FLAG, etc.) for tracking and purification

• Calcium modulation:

  • Calcium chelators to disrupt S100A3 calcium-binding

  • Calcium ionophores to increase intracellular calcium

• Protein-protein interaction inhibitors:

  • Small molecules targeting specific interaction interfaces

  • Competing peptides derived from interaction domains

• Post-translational modification analysis:

  • Phosphorylation site mutations

  • Investigation of other modifications affecting function

These experimental approaches allow for comprehensive investigation of S100A3's multifaceted roles in cellular processes and disease mechanisms.

What is known about S100A3's role in pathological conditions and its potential as a therapeutic target?

S100A3's involvement in pathological conditions and its therapeutic potential are emerging areas of research:

• Cancer biology:

  • S100A3 regulates RARα stability and activity in breast and lung cancer cells, affecting ATRA-dependent anti-proliferative and differentiating effects

  • Knockdown of S100A3 in these contexts induces resistance to ATRA, suggesting complex context-dependent roles

• Hematological malignancies:

  • In acute promyelocytic leukemia (APL) and acute myeloid leukemia (AML), S100A3 regulates PML-RARα and RARα levels

  • S100A3 knockdown increases both basal and ATRA-induced differentiation in these blood cancers

  • This presents a potential therapeutic strategy for enhancing ATRA efficacy in leukemia treatment

• Therapeutic targeting strategies:

  • Modulation of S100A3-RARα interaction could potentiate ATRA activity in appropriate cancer contexts

  • S100A3 represents a novel target for rational drug combinations aimed at enhancing retinoid-based therapies

  • Context-specific approaches may be necessary due to opposing effects in different cancer types

• Biomarker potential:

  • Expression patterns of S100A3 may serve as biomarkers for predicting response to retinoid-based therapies

  • Correlation between S100A3 levels and disease progression or prognosis requires further investigation

Further research into these aspects could lead to novel therapeutic strategies targeting S100A3 or its interactions in various disease contexts.

What are common problems when using S100A3 antibodies and how can they be resolved?

These troubleshooting strategies address the most common technical challenges encountered when working with S100A3 antibodies and can help researchers obtain reliable, reproducible results.

How should S100A3 antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of S100A3 antibodies is critical for maintaining their performance across experiments:

• Storage temperature: Store antibody aliquots at -20°C for long-term storage. Avoid repeated freeze-thaw cycles by preparing small working aliquots.

• Working dilutions: For frequently used antibodies, working dilutions can be stored at 4°C with preservatives (0.02% sodium azide) for up to 1 month.

• Shipping and transport: When receiving new antibodies, ensure immediate proper storage. If temporarily transported, maintain cold chain with ice packs.

• Aliquoting: Upon first thaw, divide concentrated antibody stocks into single-use aliquots to prevent degradation from repeated freeze-thaw cycles.

• Handling precautions:

  • Avoid contamination by using clean pipette tips

  • Centrifuge vials briefly before opening to collect all liquid

  • Handle at appropriate temperatures (ice for concentrated stocks)

  • Avoid vortexing; mix by gentle pipetting or flicking

• Documentation: Maintain records of antibody source, lot number, aliquoting dates, and experimental performance to track potential variability.

• Stability indicators: Monitor for signs of degradation such as precipitates, color changes, or declining performance, which may indicate the need for a fresh antibody lot.

Following these storage and handling recommendations will help ensure consistent antibody performance across experiments and extend the useful life of valuable research reagents.

What controls should be included when designing experiments with S100A3 antibodies?

A robust experimental design incorporating appropriate controls is essential for generating reliable data with S100A3 antibodies:

• Positive controls:

  • Cell lines with confirmed S100A3 expression (SKOV3, HepG2)

  • Recombinant S100A3 protein (for Western blot ladder verification)

  • Tissues known to express S100A3 (for immunohistochemistry)

• Negative controls:

  • Primary antibody omission (to detect non-specific secondary antibody binding)

  • Isotype control antibodies (matching IgG at equivalent concentration)

  • S100A3 knockout or knockdown samples (genetic negative controls)

  • Tissues or cells known not to express S100A3

• Specificity controls:

  • Peptide competition/blocking experiments with the immunizing peptide

  • Multiple antibodies targeting different S100A3 epitopes

  • Western blot to confirm correct molecular weight (11-12 kDa)

• Technical controls:

  • Loading controls for Western blots (β-actin, GAPDH)

  • Nuclear/cytoplasmic fractionation markers when studying subcellular localization

  • Treatment controls (e.g., ATRA treatment in studies of RARα interaction)

• Quantification controls:

  • Standard curves with recombinant protein (for quantitative applications)

  • Technical replicates to assess methodological variation

  • Biological replicates to assess biological variation

Incorporating these controls ensures experimental rigor and facilitates troubleshooting if unexpected results are obtained.

How does calcium binding affect S100A3 function and antibody recognition?

As a member of the S100 family of calcium-binding proteins, S100A3's function is intimately connected to calcium binding, though this aspect remains incompletely characterized:

• Structural changes: Calcium binding likely induces conformational changes in S100A3, potentially exposing or masking interaction surfaces relevant to protein partners like RARα.

• Antibody epitope accessibility: Calcium-dependent conformational changes may affect antibody recognition, particularly for conformation-specific antibodies. Different calcium concentrations in buffers could therefore influence immunodetection results.

• Functional implications: The calcium-bound state of S100A3 may determine its interaction with partners like RARα. Studies examining whether calcium chelation affects the S100A3-RARα interaction could provide mechanistic insights .

• Experimental considerations:

  • Buffer calcium concentrations should be standardized in binding studies

  • Calcium chelators (EGTA, EDTA) or calcium ionophores could be used to manipulate S100A3's calcium-binding state

  • Comparing antibody recognition under varying calcium conditions may reveal conformational epitopes

• Research opportunities: Investigating how calcium binding influences S100A3's regulatory effects on protein stability (particularly RARα and PML-RARα) represents an important direction for future mechanistic studies .

Understanding these calcium-dependent mechanisms could provide insights into both basic S100A3 biology and potential strategies for therapeutic intervention.

What are the latest advances in understanding the S100A3 interactome?

Recent research has expanded our understanding of the S100A3 interactome, with significant implications for its biological functions:

• Novel interactions with nuclear receptors:

  • Strong interactions with RARα and RARγ, but not with RARβ or RXRα

  • Binding to PML-RARα fusion protein in acute promyelocytic leukemia cells

  • These interactions occur predominantly in the nuclear compartment

• Co-interacting proteins:

  • S100A3, FABP5, and HSPB1/HSP27 have been identified as co-immunoprecipitating with RARα

  • These interactions are stronger with unliganded RARα and reduced by ATRA treatment

• Functional implications:

  • S100A3 binding controls constitutive and ATRA-dependent degradation of RARα and PML-RARα

  • This regulatory mechanism has context-dependent effects on cellular responses to ATRA

• Emerging methodologies:

  • Proximity-dependent biotinylation (BioID, TurboID)

  • Mass spectrometry-based interactome mapping

  • High-throughput yeast two-hybrid screening

  • These approaches may reveal additional S100A3 interaction partners

• Research opportunities:

  • Investigation of cell type-specific interactomes

  • Dynamic changes in interactions under various cellular conditions

  • Structural studies of interaction interfaces

These advances provide a foundation for further exploration of S100A3's diverse cellular functions through its protein interaction network.