S100A7 Antibody, Biotin conjugated

What is S100A7 and why is it significant in research?

S100A7 (psoriasin) is a member of the S100 family of proteins containing EF-hand calcium-binding motifs. It plays important roles in inflammatory processes, antimicrobial defense, and certain cancers. S100A7 differs from other S100 proteins in its lack of calcium binding ability in one EF-hand at the N-terminus. The protein is overexpressed in hyperproliferative skin diseases, exhibits antimicrobial activities against bacteria, and induces immunomodulatory activities . Its expression has also been associated with specific stages of breast cancer and squamous cell carcinomas, making it a significant research target for understanding disease pathology .

What are the molecular characteristics of human S100A7?

Human S100A7 is a relatively small protein with the following specifications:

• Amino acid length: 101 amino acids

• Molecular weight: Approximately 11-11.5 kDa

• Gene ID (NCBI): 6278

• Subcellular localization: Cytoplasm and secreted

• Structure: Contains EF-hand calcium-binding motifs

• Common synonyms: Psoriasin, Psoriasin 1, PSOR1, Protein S100-A7

What does biotin conjugation add to S100A7 antibodies?

Biotin conjugation provides significant advantages for S100A7 antibody applications. The biotin-streptavidin system offers one of the strongest non-covalent interactions in biology, enhancing detection sensitivity in assays. In studies of S100A7-RAGE binding, biotinylated S100A7 allows for precise quantification of binding through luminescence measurements. The biotin modification maintains S100A7 aqueous solubility through strategically placed polyethylene glycol spacer arms, preserving the protein's functional properties while enabling detection through secondary streptavidin systems .

What is the optimal methodology for S100A7 antibody-based ELISA?

For S100A7 detection using sandwich ELISA:

• Plate preparation: Pre-adsorb 96-well polystyrene plates with purified murine recombinant soluble RAGE (100 ng/well) overnight, then block with 0.25% casein (I-block).

• Sample application: Apply biotinylated S100A7 at varying concentrations (0-500 ng/mL) in blocking buffer containing 1mM calcium or other divalent cations as needed.

• Detection system: After washing, incubate with streptavidin alkaline phosphatase (1:1500 dilution) in I-block at room temperature for 60 minutes.

• Signal development: Wash with PBST, distilled water, and Tris buffer (20 mM Tris, 1 mM MgCl₂, pH 9.5), then add CDP-Star with Emerald II enhancer, incubating at room temperature for 20 minutes followed by overnight at a 4°C.

• Measurement: Quantify luminescence using a microplate luminometer, with luminescence units (LU) directly proportional to S100A7 binding .

For competitive or direct ELISA formats, the biotin-conjugated S100A7 antibody is typically used at dilutions of 1:500-1:1000 .

How can immunohistochemistry protocols be optimized for S100A7 detection?

Optimized IHC protocols for S100A7 detection include:

• Tissue preparation: Formalin-fixed, paraffin-embedded sections (5 μm thickness).

• Antigen retrieval: Use TE buffer (pH 9.0) as primary option; citrate buffer (pH 6.0) can be used as an alternative .

• Antibody application: For biotin-conjugated S100A7 antibody, use at dilutions of 1:100-1:500 for IHC-F applications .

• Detection system: For immunofluorescence, use donkey anti-rabbit cy3 (1:250) or donkey anti-mouse FITC (1:250) as secondary antibodies.

• Co-staining method: When performing co-staining, mix monoclonal mouse anti-S100A7 or smooth muscle actin (1:25) with the primary S100A7 or S100A15 antibody.

• Nuclear counterstaining: Stain all sections with DAPI before mounting .

• Controls: Include recombinant S100A7 protein (50 ng/lane) and human keratinocyte lysate (20 μg/lane) as positive controls for validation .

What storage conditions maintain optimal activity of biotin-conjugated S100A7 antibodies?

For optimal activity preservation:

Note: For reconstitution of lyophilized antibodies, follow manufacturer-specific instructions regarding diluent volume and composition .

How is S100A7 involved in inflammatory responses?

S100A7 demonstrates significant inflammatory activities through several mechanisms:

• Chemotactic activity: S100A7 functions as a chemoattractant for multiple leukocyte populations. In dual chamber assay studies, S100A7 attracted granulocytes, monocytes, and lymphocytes at similar concentrations (showing peak activity at concentrations below 100 ng/mL and diminishing at higher concentrations, creating a bell-shaped response curve typical of chemokines) .

• Receptor interaction: S100A7's chemotactic activity is mediated by RAGE (receptor of advanced glycated end products). Unlike its homolog S100A15 (which functions through a Gi protein-coupled receptor), S100A7-RAGE binding is not affected by pertussis toxin treatment .

• Zinc dependency: The binding of S100A7 to RAGE, subsequent signaling, and chemotactic activity are zinc-dependent in vitro. This corresponds with structural studies showing zinc-mediated changes in S100A7 dimer configuration .

• Synergistic inflammation: When combined with S100A15, S100A7 demonstrates potentiated inflammatory responses in vivo, suggesting that their similar but distinct mechanisms may work cooperatively in disease states .

• Disease association: Elevated levels of S100A7 have been documented in inflammatory conditions including psoriasis and oral submucous fibrosis (OSF), with significant positive correlation between S100A7 levels and clinical parameters such as duration of exposure to inflammatory triggers .

How can S100A7 and S100A15 be differentiated in experimental settings?

Despite their high homology, S100A7 and S100A15 can be differentiated using the following experimental approaches:

• Specific antibodies: Development of monospecific antisera using unique N-terminal sequences. For S100A15, synthetic peptides corresponding to its unique N-terminal sequence can be used to generate and affinity-purify specific antibodies .

• Immunoblotting validation: Validated antibodies show distinct recognition patterns:

  • Specific anti-S100A15 antibodies detect a single monomer band of recombinant S100A15 without cross-reacting with S100A7

  • Monoclonal anti-S100A7 antibodies (from manufacturers like Abcam, Imgenex) specifically detect S100A7 monomers and uncleaved recombinant proteins

• Cross-reactivity testing: Comprehensive testing against related S100 family proteins (S100A8, S100A10) is essential for confirming specificity .

• Functional discrimination: Different responses to pertussis toxin (S100A15 chemotaxis is inhibited, S100A7 is not) can be used as a functional discriminator .

• Tissue expression patterns: Differential expression in tissues (e.g., S100A15 present in myoepithelial cells where S100A7 is absent) can help distinguish the proteins in histological studies .

Note: Previous studies using non-specific antibodies or primers should be interpreted with caution as they may not have accurately distinguished between these highly similar proteins .

What is the significance of S100A7 in breast cancer research?

S100A7 has emerged as an important marker in breast cancer with several notable characteristics:

How can specificity of S100A7 antibodies be validated?

Comprehensive validation protocols for S100A7 antibodies should include:

• Recombinant protein testing: Test against recombinant S100A7, S100A15, and other S100 family members (S100A8, S100A10) at standard concentrations (50 ng/lane) .

• Native protein detection: Confirm detection of native S100A7 in human keratinocyte lysates (20 μg/lane) .

• Preabsorption studies: Perform preabsorption with corresponding proteins to block appropriate S100 antibody staining .

• Monomer detection: Verify that antibodies detect the expected 11 kDa monomer band of S100A7 .

• Cross-reactivity assessment: Test for cross-reactivity with S100A15, which shows high sequence homology to S100A7. Many commercial and custom-developed antibodies detect both proteins .

• Western blot gradient testing: Test antibody specificity across a concentration gradient to determine optimal dilution for specific detection (typically 1:500-1:1000 for Western blot applications) .

What factors can interfere with S100A7-RAGE binding assays?

Several factors can impact S100A7-RAGE binding assays:

• Divalent cation concentration: S100A7-RAGE binding is zinc-dependent, and alterations in zinc concentration can significantly affect binding efficiency and signal strength .

• pH sensitivity: Buffer pH variations can alter binding; most assays are optimized at pH 7.2-7.4 .

• Blocking reagent choice: 0.25% casein (I-block) is recommended; other blocking agents may introduce variability .

• Biotin-conjugation efficiency: Variations in biotin-conjugation can affect detection sensitivity; using standardized biotin-S100A7 with verified conjugation efficiency is critical .

• Wash stringency: Rapid washing is necessary to remove unbound ligand while preserving specific interactions .

• Incubation temperature: Temperature fluctuations during incubation periods can affect binding kinetics; room temperature standardization is important .

• Detection system linearity: Luminescence measurements should be verified to be within the linear range of the detection system .

How can researchers distinguish between technical artifacts and true S100A7 signal?

To distinguish genuine S100A7 signal from artifacts:

• Inclusion of multiple controls:

  • Positive controls: Recombinant S100A7 protein and lysates from tissues known to express S100A7 (e.g., psoriatic skin, MCF-7 cells, MDA-MB-468 cells)

  • Negative controls: Samples from tissues not expressing S100A7

  • Isotype controls: To identify non-specific binding of primary antibodies

• Antibody validation:

  • Compare results from multiple antibody clones targeting different epitopes

  • Include preabsorption controls with recombinant S100A7 protein

• Signal verification techniques:

  • Confirm protein size by Western blot (11 kDa)

  • Verify cellular localization pattern (cytoplasmic and secreted) is consistent with known S100A7 distribution

  • For co-staining experiments, use sequential staining protocols to eliminate cross-reactivity concerns

• Technical considerations:

  • For ELISA, include standard curves with recombinant protein

  • For IHC, test multiple antigen retrieval methods (TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Validate signal across multiple dilutions to ensure specificity is maintained

How do S100A7 and S100A15 contribute to differential inflammatory responses?

Despite their high sequence homology, S100A7 and S100A15 exhibit distinct inflammatory mechanisms:

• Receptor specificity: S100A7 functions through RAGE (receptor of advanced glycated end products), while S100A15 signals through a Gi protein-coupled receptor (GiPCR). This is evidenced by the differential response to pertussis toxin, which inhibits S100A15-mediated chemotaxis but has no effect on S100A7-induced neutrophil migration .

• Leukocyte subset targeting: S100A15 shows selective chemotactic activity for granulocytes and monocytes, while S100A7 exhibits broader activity, attracting lymphocytes as well. This differential targeting suggests complementary roles in orchestrating immune responses .

• Zinc dependency: S100A7-RAGE binding, signaling, and chemotaxis demonstrate zinc dependency in vitro, reflecting zinc-mediated conformational changes in the S100A7 dimer structure. This requirement may provide a regulatory mechanism absent in S100A15 function .

• Synergistic inflammation: When present together, S100A7 and S100A15 potentiate inflammation in vivo beyond what either protein achieves independently. This synergism may explain the aggressive inflammatory phenotype in conditions where both proteins are upregulated .

• Reactive oxygen species (ROS) relationship: S100A7 increases intracellular ROS levels in keratinocytes, creating a feedback loop where S100A7 is both stimulated by and intensifies hypoxic conditions. This relationship with oxidative stress may represent a unique contribution to inflammatory pathology .

What methodological approaches can improve detection of S100A7 in complex biological samples?

For enhanced detection of S100A7 in complex samples:

• Sample preparation optimization:

  • For salivary samples: Centrifugation at 2600g for 15 minutes at 4°C to remove cellular debris before analysis

  • For tissue samples: Optimization of protein extraction buffers to maintain S100A7 solubility and native conformation

• Enhanced ELISA techniques:

  • Sandwich ELISA utilizing capture antibodies directed against RAGE and detection with biotin-conjugated S100A7-specific antibodies

  • Signal amplification using horseradish peroxidase (HRP) Streptavidin systems with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrates for improved visualization

  • Triplicate sampling to improve statistical reliability of measurements

• Multiplex detection systems:

  • Simultaneous detection of S100A7 and S100A15 using differentially labeled antibodies

  • Flow cytometry-based detection for cellular expression analysis

  • Multiplex bead-based assays for detection in biological fluids

• Advanced imaging techniques:

  • Super-resolution microscopy for subcellular localization studies

  • Proximity ligation assay (PLA) for detecting S100A7-RAGE interactions in situ

  • Co-immunoprecipitation with biotin-conjugated antibodies for protein-protein interaction studies

• Genetic approaches:

  • RNA interference studies to validate antibody specificity

  • S100A7 reporter systems for live-cell imaging

What are the emerging therapeutic implications of targeting S100A7-RAGE interactions?

Recent research highlights several therapeutic implications of targeting S100A7-RAGE interactions:

• Anti-inflammatory potential: The identification of S100A7-RAGE binding as a mediator of inflammatory chemotaxis suggests this interaction could be targeted to reduce inflammatory cell recruitment in conditions like psoriasis .

• Zinc-dependent modulation: The zinc-dependency of S100A7-RAGE binding offers a potential mechanism for modulating this interaction through zinc chelation or supplementation therapies .

• Receptor antagonism: Development of RAGE antagonists specifically designed to block S100A7 binding could provide selective inhibition of this inflammatory pathway while preserving other RAGE functions .

• Synergistic targeting: Since S100A7 and S100A15 demonstrate inflammatory synergism, dual targeting strategies might be necessary for optimal therapeutic efficacy in conditions where both proteins are elevated .

• Cancer implications: Given S100A7's differential expression in breast cancer subtypes, particularly its elevation in ER/PR negative cases, targeting S100A7-RAGE interactions could have implications for cancer therapy, potentially reducing inflammatory tumor microenvironments .

• Biomarker utilization: The identification of salivary S100A7 as significantly elevated in conditions like oral submucous fibrosis provides opportunities for non-invasive monitoring of disease progression and treatment response .

• Combination approaches: Given the multiple pathways involved in S100A7-mediated inflammation, combination therapies targeting both S100A7-RAGE and associated signaling pathways may provide synergistic therapeutic benefits .

What are the optimal approaches for generating specific S100A7 antibodies?

Generating highly specific S100A7 antibodies requires careful consideration of several factors:

• Antigen selection:

  • For monoclonal antibodies: Use full-length recombinant S100A7 expressed in Escherichia coli BL21 (DE3) with polyhistidine tags that can be removed using thrombin

  • For polyclonal antibodies: Use KLH-conjugated synthetic peptides derived from unique regions of human S100A7 to avoid cross-reactivity with S100A15

• Immunization protocol:

  • Rabbits are commonly used hosts for polyclonal antibody production

  • Immunization schedule: Three immunizations over 2 months (1 mg per animal per immunization)

  • For monoclonal antibodies, mice are typically immunized following standard hybridoma technology protocols

• Antibody purification:

  • Isolation of IgG fraction using ammonium sulfate precipitation (50%)

  • Further purification using protein A affinity chromatography

  • For peptide-specific antibodies, affinity purification using synthetic peptide-coupled to Affigel-15

• Conjugation methods:

  • For biotin conjugation, NHS-PEO4-biotin containing polyethylene glycol spacer arms is recommended to maintain S100A7 aqueous solubility

  • Typical conjugation: Mix S100A7 with 2 molar excess of NHS-PEO4-biotin and incubate on ice for 2 hours

  • Separation of biotinylated-S100A7 from unreacted NHS-PEO4-biotin using gel filtration (e.g., Econo-Pac 10 DG column)

• Validation strategy:

  • Cross-reactivity testing against recombinant S100A15 and other S100 family members (S100A8, S100A10)

  • Testing against native protein in human keratinocyte lysates

  • Determination of optimal concentration through serial dilution testing