S100A7 Antibody, HRP conjugated

What is S100A7 and what biological functions does it serve in research?

S100A7, also known as Psoriasin, is a member of the S100 family of proteins containing 2 EF-hand calcium-binding motifs. It is an 11 kDa protein that differs from other S100 proteins in its lack of calcium binding ability in one EF-hand at the N-terminus. S100A7 is primarily localized in the cytoplasm but can also be secreted extracellularly . It plays crucial roles in regulating cellular processes including cell cycle progression and differentiation. The protein is overexpressed in hyperproliferative skin diseases and exhibits both antimicrobial activities against bacteria and immunomodulatory functions .

In cancer research, S100A7 has gained significance as it correlates with worse prognostic outcomes in breast cancers, particularly in ER-positive tumors where it is associated with higher tumor grade . The protein functions through multiple pathways, including binding to the Receptor for Advanced Glycation End Products (RAGE), which can trigger angiogenic responses in vascular endothelial cells .

What are the optimal dilutions for using HRP-conjugated S100A7 antibodies in different applications?

For HRP-conjugated S100A7 antibodies, optimal dilutions vary by application:

Always perform antibody titration experiments with positive and negative controls to determine optimal working dilutions for your specific experimental system.

What sample types have been validated for S100A7 antibody applications?

Based on published research, S100A7 antibodies have been successfully verified in several human tissue and cell line samples:

• Cell Lines: A431 cells have been verified for Western blot applications

• Tissue Samples: Human tonsil and human lung cancer tissues have been validated for immunohistochemistry applications

• Breast Cancer Samples: Both ER-positive (MCF-7, T47D) and ER-negative breast cancer samples

• Vascular Endothelial Cells: Human umbilical vein endothelial cells (HUVECs) for studying S100A7-induced angiogenic effects

When working with new sample types, it is advisable to include known positive controls alongside experimental samples to validate antibody performance.

How should I design experiments to assess S100A7 expression changes in response to growth factors?

When designing experiments to evaluate S100A7 expression changes in response to growth factors such as IGF-1, a multi-level assessment approach is recommended:

• Transcriptional Regulation: Utilize qRT-PCR to measure S100A7 mRNA expression changes. Design primers specific to the S100A7 gene and normalize expression to established housekeeping genes .

• Promoter Activity Assessment: Consider using S100A7 promoter constructs in luciferase reporter assays to directly measure transcriptional activation. This approach was successfully employed to demonstrate that IGF-1 induces transactivation of the S100A7 promoter in breast cancer cell lines .

• Protein Expression Analysis: Employ Western blotting with HRP-conjugated S100A7 antibodies at 1:200-1:500 dilution to detect changes in intracellular protein levels .

• Secreted Protein Measurement: Use ELISA to quantify S100A7 secretion into culture medium. In previous studies, IGF-1 was shown to induce a 3-fold increase in S100A7 release from MCF-7 cells .

• Signaling Pathway Investigation: Include inhibitors of key signaling molecules (e.g., STAT3, ERK1/2, AKT) to determine the mechanistic pathways regulating S100A7 expression. Previous research has demonstrated that IGF-1/IGF-1R signaling engages STAT3 activation and its recruitment to the S100A7 promoter .

Include appropriate time course and dose-response experiments to fully characterize the kinetics and sensitivity of S100A7 expression changes.

What are the optimal conditions for using HRP-conjugated S100A7 antibodies in protein-protein interaction studies?

When using HRP-conjugated S100A7 antibodies for protein-protein interaction studies, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): Use a dilution of 1:500 for detection of pulled-down complexes. Pre-clear lysates to reduce non-specific binding and include appropriate negative controls.

• Dot Blot Binding Assays: HRP-labeled S100A7 has been successfully used in dot blot assays to detect binding to target proteins. Block membranes with either 5% BSA or 5% nonfat dry milk in TBST before probing with the HRP-labeled S100A7 .

• Surface Plasmon Resonance (SPR): While not using HRP-conjugation directly, SPR has been used to characterize S100A7 binding interactions with high affinity (Kd of 41 nM reported for S100A7 binding to TdfJ) . Consider using unconjugated antibodies for immobilization and HRP-conjugated antibodies for detection in SPR-based sandwich assays.

• RAGE Binding Studies: When studying S100A7-RAGE interactions, consider including the RAGE inhibitor FPS-ZM1 as a negative control to confirm specificity, as this approach has previously demonstrated the functional significance of this interaction .

For all interaction studies, ensure that the HRP conjugation does not interfere with the binding epitopes by comparing results with unconjugated antibodies when possible.

How can I troubleshoot weak or absent signals when using HRP-conjugated S100A7 antibodies?

When facing challenges with weak or absent signals using HRP-conjugated S100A7 antibodies, systematically address the following factors:

• Antibody Concentration: Increase antibody concentration if signal is weak, starting with a 2-fold increase from the recommended dilution range of 1:200-1:500 for Western blot or 1:50-1:300 for IHC .

• Sample Preparation: Ensure proper sample preparation, as S100A7 has a low molecular weight (11 kDa) that may require specialized gel compositions for optimal separation . Consider using gradient gels or higher percentage (15-20%) acrylamide gels for better resolution of low molecular weight proteins.

• Antigen Retrieval: For IHC applications, optimize antigen retrieval methods. S100A7 has both cytoplasmic and secreted localizations , so different fixation protocols may affect epitope accessibility.

• Detection System Sensitivity: Enhance signal using more sensitive detection substrates for HRP, such as enhanced chemiluminescence (ECL) systems with signal amplification capabilities.

• Blocking Conditions: Test alternative blocking agents; some researchers find that 5% BSA may be superior to milk for certain applications with S100A7 .

• Cross-Reactivity Assessment: Verify antibody specificity, as the S100 family has at least 13 members with structural similarities . Use recombinant S100A7 protein as a positive control to confirm antibody functionality.

• Storage and Handling: Ensure proper storage at -20°C and avoid freeze/thaw cycles that could compromise antibody activity .

If signal problems persist after addressing these factors, consider alternative detection methods or antibody clones.

What strategies can optimize specific detection of S100A7 in complex tissue samples?

Optimizing S100A7 detection in complex tissue samples requires careful consideration of several experimental parameters:

• Antibody Titration: Perform careful antibody titration experiments starting with the recommended dilution range (1:50-1:300 for IHC) , testing multiple concentrations to determine the optimal signal-to-noise ratio.

• Blocking Optimization: Test different blocking reagents (BSA vs. milk) and concentrations (3-5%) to minimize background while maintaining specific signal .

• Multi-labeling Approach: Consider dual immunofluorescence labeling with antibodies against known S100A7 interaction partners or cellular compartment markers to confirm specificity of localization patterns.

• Tissue-Specific Controls: Include tissue sections known to express high levels of S100A7 (e.g., human tonsil, lung cancer tissues) as positive controls alongside experimental samples.

• Alternative Fixation Protocols: Test multiple fixation protocols as they can significantly affect epitope preservation and accessibility for S100A7 detection.

• Signal Amplification Systems: For tissues with low S100A7 expression, consider using tyramide signal amplification (TSA) or other HRP signal enhancement methods.

• Serial Dilution Validation: Perform serial dilution tests of both primary and secondary antibodies to identify the concentration that provides optimal specific staining while minimizing background.

Implementing these optimization strategies will significantly improve the reliability and specificity of S100A7 detection in complex tissue samples.

How can S100A7 antibodies be utilized to investigate its role in the tumor microenvironment?

S100A7 plays significant roles in the tumor microenvironment, particularly in breast cancer. To investigate these functions using S100A7 antibodies:

• Tumor-Stromal Interactions: Use immunohistochemistry with S100A7 antibodies (1:50-1:300 dilution) on tumor sections to analyze expression patterns at the tumor-stroma interface. This can reveal spatial relationships between S100A7-expressing cells and infiltrating immune or stromal cells.

• Angiogenesis Assessment: Implement dual staining with S100A7 and endothelial markers to investigate the relationship between S100A7 expression and tumor vasculature. Research has shown that S100A7 can activate RAGE signaling in endothelial cells to promote angiogenesis .

• Conditioned Media Experiments: Detect secreted S100A7 in tumor cell conditioned media using S100A7 antibodies in ELISA assays. This approach can be used to investigate paracrine effects, as demonstrated in studies showing that media from IGF-1-stimulated MCF-7 cells containing secreted S100A7 induces proliferation of human vascular endothelial cells .

• RAGE Signaling Investigation: Use S100A7 antibodies in conjunction with RAGE pathway inhibitors (e.g., FPS-ZM1) to assess the specific contribution of S100A7-RAGE signaling to angiogenic phenotypes .

• Chromatin Immunoprecipitation (ChIP): Implement ChIP assays to investigate the transcriptional regulation of S100A7, as demonstrated in studies showing STAT3 recruitment to the S100A7 promoter in response to IGF-1 stimulation .

These methodological approaches can provide comprehensive insights into how S100A7 contributes to the complex cellular interactions within the tumor microenvironment.

What methods are recommended for investigating S100A7's role in antimicrobial activities?

S100A7 exhibits antimicrobial activities against bacteria . To investigate this function using S100A7 antibodies:

• Bacterial Binding Assays: Utilize dot blot assays with HRP-labeled S100A7 to assess binding to bacterial surface proteins. This approach has been successfully used to investigate S100A7 binding to Neisseria gonorrhoeae TdfJ protein .

• Zinc Sequestration Studies: Develop assays to measure S100A7's role in zinc piracy, as S100A7 binding to bacterial receptors can interfere with zinc acquisition. Use zinc-specific fluorescent probes alongside S100A7 antibody detection to correlate protein binding with zinc deprivation .

• Mutagenesis Approaches: When investigating bacterial proteins that interact with S100A7, consider site-directed mutagenesis of key residues suspected to be involved in binding. This approach revealed that mutagenesis of the loop 3 α-helix of N. gonorrhoeae TdfJ reduced S100A7 binding and zinc piracy .

• Surface Plasmon Resonance: Implement SPR to characterize binding affinities between S100A7 and bacterial proteins, which has demonstrated high-affinity interactions (Kd of 41 nM) .

• Functional Antimicrobial Assays: Combine S100A7 antibody-based detection methods with bacterial growth inhibition assays to correlate S100A7 binding/localization with antimicrobial activity.

These methodological approaches provide a comprehensive framework for investigating the molecular mechanisms underlying S100A7's antimicrobial functions.

How can S100A7 antibodies contribute to investigating its role in cancer progression and metastasis?

S100A7 antibodies can be leveraged to explore several emerging areas in cancer progression and metastasis research:

• Prognostic Biomarker Development: Utilize S100A7 antibodies in tissue microarray analyses to correlate expression with clinical outcomes across diverse cancer types. Research has already demonstrated that S100A7 expression correlates with worse prognosis and higher tumor grade in ER-positive breast cancers .

• Epithelial-Mesenchymal Transition (EMT): Implement co-staining approaches with S100A7 antibodies and EMT markers to investigate potential relationships between S100A7 expression and the acquisition of invasive phenotypes.

• Therapeutic Target Validation: Use S100A7 antibodies to monitor protein expression changes in response to targeted therapies, particularly those addressing the IGF-1/IGF-1R axis, which has been shown to regulate S100A7 expression .

• Liquid Biopsy Development: Explore the potential of detecting circulating S100A7 in patient serum or plasma as a non-invasive biomarker, using sensitive immunoassays based on HRP-conjugated S100A7 antibodies.

• Tumor Microenvironment Interactions: Investigate how S100A7-expressing cancer cells influence the recruitment and polarization of immune cells in the tumor microenvironment through dual immunofluorescence labeling.

• Mechanistic Studies of STAT3 Regulation: Build on findings that IGF-1/IGF-1R signaling engages STAT3 activation and recruitment to the S100A7 promoter by investigating how this regulatory mechanism might be targeted therapeutically.

These research directions represent promising avenues for understanding S100A7's complex roles in cancer progression and developing potential therapeutic approaches.

What are the considerations for developing multiplex assays that include S100A7 detection?

When developing multiplex assays that include S100A7 detection alongside other biomarkers, consider these methodological approaches:

• Antibody Compatibility: Carefully select antibodies raised in different host species to avoid cross-reactivity in multiplex immunofluorescence assays. The rabbit-derived polyclonal S100A7 antibodies should be paired with antibodies from other species.

• Sequential Detection Strategies: For IHC-based multiplex assays, implement sequential detection protocols with thorough stripping or blocking steps between each antibody application to prevent signal carryover.

• Spectral Overlap Minimization: When using fluorescently-labeled secondary antibodies, select fluorophores with minimal spectral overlap to clearly distinguish S100A7 signal from other targets.

• Multiplexed ELISA Development: For detecting soluble S100A7 alongside other biomarkers, develop sandwich ELISA systems using HRP-conjugated S100A7 antibodies with carefully optimized capture and detection antibody pairs to minimize cross-reactivity.

• Tissue Microarray Validation: Validate multiplex protocols on tissue microarrays containing samples with known expression patterns of S100A7 and other biomarkers of interest.

• Digital Pathology Integration: Implement digital image analysis algorithms to quantify co-localization or expression relationships between S100A7 and other markers in multiplex-stained specimens.

• Single-Cell Analysis Compatibility: Ensure compatibility of S100A7 antibodies with single-cell analysis platforms to enable high-dimensional characterization of S100A7-expressing cells in heterogeneous populations.

These considerations will facilitate the successful integration of S100A7 detection into multiplex assay systems for comprehensive biomarker profiling.