S100A14 Antibody

What is S100A14 and why is it important in cancer research?

S100A14 is a member of the S100 protein family of calcium-binding proteins with a calculated molecular weight of 12 kDa, though it typically appears at 10-12 kDa on Western blots . It plays significant roles in cell proliferation, differentiation, and metastasis in various cancer types. Its expression patterns are heterogeneous—overexpressed in lung, breast, and uterine cancers, but underexpressed in colon, kidney, and rectal tumors . This differential expression suggests tissue-specific roles in tumorigenesis, making S100A14 a potential biomarker for cancer diagnosis and prognosis. It has been specifically identified as a potential marker for predicting distant metastasis in breast cancer patients and for detecting circulating tumor cells in peripheral blood from patients with colorectal, prostate, and breast cancers .

Which applications are most effective for S100A14 antibody detection?

S100A14 antibodies have been validated for multiple research applications with varying efficacy:

For IHC applications, antigen retrieval is typically performed using TE buffer (pH 9.0) or alternatively with citrate buffer (pH 6.0) . Experimental optimization is recommended for each specific application and sample type.

How should S100A14 antibodies be stored and handled to maintain reactivity?

Proper storage and handling are critical for maintaining antibody functionality:

• Store at -20°C for long-term storage; antibodies are typically stable for one year after shipment

• For antibodies in liquid form with preservatives (e.g., sodium azide and glycerol), aliquoting may be unnecessary for -20°C storage

• Lyophilized antibody formulations should be reconstituted according to manufacturer instructions and can typically be stored at 4°C for one month after reconstitution

• Avoid repeated freeze-thaw cycles which can degrade antibody quality

• Some formulations contain trehalose, NaCl, and Na₂HPO₄ as stabilizers

• Certain conjugated antibodies (e.g., PerCP-conjugated) should be stored at 4°C in the dark to preserve fluorophore activity

How can researchers validate the specificity of their S100A14 antibodies?

Validating antibody specificity is crucial for reliable research results:

• Positive and negative controls: Use cell lines with known S100A14 expression. For example, MCF-7 cells and rat stomach tissue have been verified as positive WB controls . KYSE180 cells show relatively high levels of S100A14, while EC9706 cells have negligible endogenous S100A14 .

• Knockdown/knockout validation: Utilize S100A14 knockdown or knockout models to confirm specificity. Multiple publications have employed this approach .

• Multiple detection methods: Cross-validate results using different techniques (WB, IHC, IF) to ensure consistency.

• Peptide competition assay: Pre-incubate the antibody with purified S100A14 protein before application to verify that the signal disappears.

• Genetic variant analysis: Consider known genetic variants in S100A14 that might affect antibody binding, such as the 461G>A variant which diminishes a P53-binding site .

What are the best positive control tissues/cells for S100A14 antibody validation?

Based on published research, these samples provide reliable positive controls:

What methodological considerations should be taken for IHC applications with S100A14 antibodies?

For optimal immunohistochemistry results:

• Antigen retrieval: Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is commonly used . Alternatively, TE buffer (pH 9.0) or citrate buffer (pH 6.0) can be employed depending on the specific antibody .

• Blocking: 10% goat serum is typically used to reduce non-specific binding .

• Antibody concentration: A concentration of 2 μg/ml has been validated for many applications, but titration is recommended (typical dilution ranges: 1:500-1:2000) .

• Incubation conditions: Overnight incubation at 4°C has shown optimal results .

• Detection systems: Peroxidase-conjugated secondary antibodies with DAB (3,3'-diaminobenzidine) as the chromogen are commonly used. HRP Conjugated Rabbit IgG Super Vision Assay kits have been successfully employed .

• Interpretation: Consider both membranous and cytoplasmic staining patterns, as S100A14 can localize to both compartments depending on cancer type.

How does extracellular versus intracellular S100A14 affect experimental design and interpretation?

S100A14 can function both intracellularly and as a secreted protein, which affects experimental approaches:

Extracellular S100A14:

• At low doses (0.01-20 μg/ml), extracellular S100A14 stimulates cell proliferation in a concentration- and time-dependent manner, with optimal effects at approximately 10 μg/ml

• Extracellular S100A14 binds to RAGE (Receptor for Advanced Glycation End products) and activates RAGE-dependent signaling cascades

• For studying extracellular effects, recombinant S100A14 protein can be added to culture medium

• Purification methods typically involve histidine-tagged fusion proteins expressed in E. coli

• Control experiments should include similarly produced control proteins (e.g., Myo117) to ensure observed effects are specific to S100A14

Intracellular S100A14:

• Functions in calcium signaling pathways

• Interacts with proteins like HER2, affecting receptor phosphorylation and downstream signaling

• For studying intracellular functions, overexpression or knockdown approaches are typically employed

• Influences store-operated Ca²⁺ influx by suppressing Orai1 and STIM1 expression

Experimental design should account for these dual roles when interpreting results.

How do calcium levels affect S100A14 function and antibody binding?

As a calcium-binding protein, S100A14 undergoes conformational changes upon calcium binding:

• Functional changes: S100A14 contains EF-hand calcium-binding domains that mediate calcium-dependent interactions with target proteins. Mutations in calcium-coordinating residues (e.g., E39, E45) can be introduced to study calcium-independent functions .

• Antibody binding considerations:

  • Some antibodies may preferentially bind calcium-bound or calcium-free forms of S100A14

  • Buffer conditions during experiments can affect calcium binding and potentially antibody recognition

  • Site-directed mutagenesis can be used to generate calcium-binding deficient mutants (mS100A14-N) for control experiments

  • For studying calcium-dependent interactions, buffers should be supplemented with appropriate calcium concentrations

• Experimental implications:

  • Include calcium chelators (e.g., EGTA) as controls when studying calcium-dependent functions

  • Consider calcium concentrations in experimental buffers when performing immunoprecipitation or pull-down assays

  • S100A14 blocks store-operated Ca²⁺ influx by suppressing Orai1 and STIM1 expression, which leads to focal adhesion assembly and MMP downregulation

How do genetic variants of S100A14 affect antibody binding and experimental outcomes?

Several genetic variants have been identified in the S100A14 locus that may affect experimental outcomes:

• Known variants: Four single nucleotide polymorphisms (−43A>G, 461G>A, 1493A>G, and 1545A>T) have been identified in the S100A14 locus and are in absolute linkage disequilibrium .

• Functional consequences: The 461G>A variant diminishes a P53-binding site and is associated with decreased expression of S100A14 both in vitro and in vivo . This variant has been associated with susceptibility to esophageal squamous cell carcinoma (ESCC) among smokers .

• Implications for antibody-based detection:

  • Epitope-specific antibodies may have altered binding to variant forms of S100A14

  • N-terminal targeted antibodies (e.g., ABIN6264901) may be affected by variants in this region

  • Expression levels in clinical samples may vary due to these genetic variants, requiring genotyping in certain research contexts

  • For population studies, consider variant frequencies when interpreting expression data

• Experimental recommendations:

  • When possible, sequence or genotype the S100A14 locus in cell lines or tissue samples used for critical experiments

  • Use multiple antibodies targeting different epitopes for validation

  • Include appropriate positive controls with known genotypes

S100A14 in Disease Models and Translational Research

S100A14 influences cancer progression through multiple mechanisms:

• RAGE-dependent signaling:

  • S100A14 binds to RAGE and stimulates RAGE-dependent signaling cascades

  • At low doses, this promotes cell proliferation and survival

  • S100A14-RAGE interactions can be detected via pull-down experiments and co-immunoprecipitation

• p53 pathway interaction:

  • S100A14 functions as a cancer suppressor working in the p53 pathway

  • The 461G>A variant diminishes a p53-binding site and is associated with decreased S100A14 expression

  • S100A14 requires functional p53 for some of its effects on cell behavior

• HER2 modulation:

  • S100A14 binds to HER2 and modulates HER2 phosphorylation

  • This interaction affects HER2-stimulated cell proliferation

  • S100A14 acts as a functional partner of HER2 in breast cancer progression

• Calcium signaling and metastasis inhibition:

  • In gastric cancer, S100A14 blocks store-operated Ca²⁺ influx by suppressing Orai1 and STIM1 expression

  • This leads to FAK activation, focal adhesion assembly, and MMP downregulation

  • The result is inhibition of cell migration and invasion both in vitro and in vivo

• Cell differentiation induction:

  • S100A14 induces differentiation of gastric cancer cells by upregulating E-cadherin and PGII expression

What methodological approaches can resolve contradictory findings about S100A14 function across different experimental systems?

To address contradictory findings about S100A14:

• Context-dependent functions analysis:

  • Explicitly compare extracellular versus intracellular effects

  • Systematically vary S100A14 concentration (low doses promote proliferation, while effects at higher concentrations may differ)

  • Evaluate effects in multiple cell types from the same tissue origin

• Protein interaction profiling:

  • Perform comprehensive interaction studies (IP-MS) in different cell types

  • Map interaction networks to identify cell-type specific binding partners

  • Validate key interactions using multiple methods (co-IP, proximity ligation assay, FRET)

• Genetic background consideration:

  • Assess p53 status in experimental systems, as S100A14 requires functional p53 for some effects

  • Determine S100A14 genetic variants present in cell lines used

  • Use isogenic cell lines differing only in S100A14 expression

• Systematic review methodology:

  • Perform meta-analysis of published data with careful attention to experimental details

  • Stratify results by cancer type, S100A14 concentration, and experimental approach

  • Distinguish between correlation studies and functional interventions

• Improved experimental controls:

  • Use both gain- and loss-of-function approaches in the same experimental system

  • Include appropriate protein controls (e.g., Myo117) when studying effects of recombinant S100A14

  • Validate antibody specificity using knockout/knockdown approaches

By systematically addressing these factors, researchers can better understand the seemingly contradictory roles of S100A14 in different experimental contexts.

How can multiplexed detection systems improve S100A14 research?

Multiplexed detection of S100A14 alongside other biomarkers offers several advantages:

• Co-expression analysis with other S100 family members:

  • Simultaneous detection of S100A14 with S100A4 can provide insights into metastatic potential, as simultaneous S100A14 underexpression and S100A4 overexpression correlates with high colorectal cancer metastatic potential

  • Multiplex IHC/IF can reveal co-localization patterns with other S100 proteins

• Pathway analysis integration:

  • Combined detection of S100A14 with RAGE, p53, and HER2 can elucidate functional relationships

  • Phosphorylation status of downstream signaling molecules can be assessed in relation to S100A14 expression

• Technical approaches:

  • Multiplex immunofluorescence using spectrally distinct fluorophores

  • Sequential chromogenic IHC with antibody stripping between rounds

  • Mass cytometry (CyTOF) for simultaneous detection of multiple proteins

  • Single-cell RNA sequencing combined with protein detection (CITE-seq)

• Clinical applications:

  • Improved prognostic value by combining S100A14 with other markers

  • Better characterization of circulating tumor cells using multiplexed detection systems

  • Patient stratification for personalized treatment approaches

What are the emerging research frontiers for S100A14 antibody applications?

Several cutting-edge applications for S100A14 antibodies are emerging:

• Therapeutic targeting:

  • Development of function-blocking antibodies targeting S100A14-RAGE or S100A14-HER2 interactions

  • Context-specific intervention based on S100A14's dual roles in different cancers

• Liquid biopsy approaches:

  • Detection of S100A14 in circulating tumor cells or extracellular vesicles

  • Correlation with disease progression and treatment response

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize S100A14 subcellular localization

  • Intravital imaging using labeled antibodies to track S100A14 dynamics in vivo

• Biomarker development:

  • Integration with other cancer biomarkers for improved diagnostic accuracy

  • Longitudinal monitoring of S100A14 expression during treatment

• Drug discovery applications:

  • High-throughput screening assays using S100A14 antibodies to identify compounds that modulate its expression or function

  • Development of proximity-based assays to screen for disruptors of S100A14 protein interactions