S100A1 Antibody

What is S100A1 and why is it important in research?

S100A1 is a member of the S100 family of calcium-binding proteins that undergoes significant conformational changes upon calcium binding. It functions both as an intracellular signaling molecule and as a secreted protein . S100A1 is particularly important in research due to its differential expression across various tissues and its altered expression in multiple disease states including cardiomyopathy, neurological diseases, and several cancer types . Its dual functionality as both an intracellular regulator and extracellular signaling molecule makes it a compelling target for understanding complex cellular communication systems.

Which tissues express S100A1 protein under normal physiological conditions?

S100A1 expression varies considerably across tissues:

• High expression: Heart tissue, particularly cardiomyocytes

• Moderate expression: Kidney (particularly in the normal parenchyma)

• Detectable expression: Brain tissues (supporting cells and inner hair cells of the developing cochlea)

• Low expression (under physiological conditions): Lungs, ovaries, and liver

• Specific expression in the urothelium: Exclusively in terminally differentiated superficial cells of both the ureter and bladder, with nucleocytoplasmic localization

How do I select between monoclonal and polyclonal S100A1 antibodies for my experiment?

Selection should be based on your specific experimental needs, tissue of interest, and application. For critical diagnostic applications or when comparing results across multiple experiments, monoclonal antibodies provide better consistency .

What are the optimal conditions for immunohistochemical detection of S100A1?

For optimal IHC detection of S100A1, consider the following evidence-based protocol:

Antigen Retrieval:

• Primary method: TE buffer pH 9.0 (recommended for most tissues)

• Alternative method: Citrate buffer pH 6.0 (may be preferable for certain tissue types)

Antibody Dilutions:

• Monoclonal antibody (67237-1-Ig): 1:16000-1:64000

• Polyclonal antibody (16027-1-AP): 1:50-1:500

Tissue-Specific Considerations:

• For renal tissues: S100A1 is expressed in oncocytomas, clear cell and papillary renal cell carcinomas but not in chromophobe renal cell carcinomas

• For urothelial tissues: Focus on superficial/luminal cells where S100A1 shows nucleocytoplasmic staining

• For liver tissues: Nuclear and cytoplasmic staining can be observed in HCC samples with variable intensity correlating with prognostic factors

Controls:

• Positive tissue controls: Human heart tissue, kidney tissue, tonsillitis tissue, or rat brain tissue

• Negative controls: Omit primary antibody to confirm specificity

For quantitative analysis, image processing software can be used to measure the mean Integrated Optical Density (IOD) of S100A1 staining, which has been successfully used to stratify patients in prognostic studies .

How can I effectively validate S100A1 antibody specificity in my experimental system?

A comprehensive validation approach should include:

• Multiple detection methods:

  • Compare immunohistochemistry/immunofluorescence with RNA in situ hybridization to confirm that protein expression patterns match mRNA expression

  • Perform Western blot analysis to confirm the correct molecular weight (observed: 10 kDa; calculated: 11 kDa)

• Orthogonal validation techniques:

  • Analyze tissues with known S100A1 expression as positive controls (e.g., heart tissue, supporting cells in cochlea)

  • Include S100A1 knockout/knockdown samples when possible

• Cross-reactivity assessment:

  • Test the antibody on different species (human, rat, mouse) to confirm expected reactivity

  • Evaluate potential cross-reactivity with other S100 family members, particularly S100A4 which is known to interact with S100A1

• Technical controls:

  • Use antibodies from different sources or clones targeting different epitopes of S100A1

  • Include isotype controls to rule out non-specific binding

  • For recombinant protein-raised antibodies, test against the immunogen used for antibody production

What is the recommended protocol for using S100A1 antibodies in Western blot applications?

Sample Preparation:

• For tissue samples: Use RIPA buffer with protease inhibitors

• For cell samples: Lyse cells directly in sample buffer supplemented with protease inhibitors

• Expected molecular weight: 10-11 kDa

Protocol Optimization:

• Antibody selection and dilution:

  • Polyclonal antibody 16027-1-AP: Use at 1:500-1:1000 dilution

  • Monoclonal antibodies: Follow manufacturer's recommended dilutions

• Gel separation:

  • Use 15% SDS-PAGE gels for optimal resolution of low molecular weight S100A1 protein

  • Include molecular weight markers covering the 10-15 kDa range

• Transfer conditions:

  • PVDF membrane is recommended for small proteins

  • Use 100V for 60 minutes in cold transfer buffer containing 20% methanol

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBST

  • Incubate with primary antibody overnight at 4°C

  • Wash thoroughly with TBST (3-5 times, 5 minutes each)

  • Incubate with appropriate HRP-conjugated secondary antibody

• Detection:

  • Use enhanced chemiluminescence detection

  • For quantitative analysis, normalize S100A1 signal to appropriate loading controls

Positive Control Samples:

• Human heart tissue lysate shows strong S100A1 expression

• Kidney tissue lysates can also serve as positive controls

How can S100A1 antibodies be used to distinguish between different types of renal cell carcinomas?

S100A1 immunostaining provides a valuable diagnostic tool for differentiating renal cell neoplasms. The distinctive staining patterns are as follows:

Methodological considerations:

• Use appropriate antigen retrieval methods (TE buffer pH 9.0 recommended)

• Include positive controls (normal kidney parenchyma consistently expresses S100A1)

• Use additional markers (such as KRT5, UPK1B) for comprehensive differential diagnosis

• Consider that both immunohistochemistry and RT-PCR analyses show statistically significant differences in S100A1 expression between chromophobe RCC and oncocytomas (P<0.001)

This differential expression pattern makes S100A1 particularly useful for distinguishing chromophobe RCC from oncocytoma, which can be challenging due to overlapping morphological features .

What is the prognostic significance of S100A1 expression in hepatocellular carcinoma (HCC)?

S100A1 has emerged as an independent prognostic factor in HCC. Research indicates:

These findings suggest that S100A1 assessment could be incorporated into prognostic evaluation of HCC patients, and that targeting S100A1 may represent a potential therapeutic strategy for HCC treatment .

How does S100A1 function as an alarmin when released from damaged cardiomyocytes?

S100A1, the S100 isoform with highest abundance in cardiomyocytes, acts as an alarmin (damage-associated molecular pattern/DAMP) when released from damaged heart cells during myocardial infarction:

• Release mechanism:

  • S100A1 is released from necrotic cardiomyocytes during myocardial injury

  • Elevated S100A1 serum levels are detected in patients with acute myocardial infarction and in experimental MI in mice

• Cellular uptake and signaling:

  • S100A1 is selectively internalized by cardiac fibroblasts (CFs) adjacent to damaged cardiomyocytes

  • Endocytosis of S100A1 by CFs leads to Toll-like receptor 4 (TLR4)-dependent activation of:

    • MAP kinases

    • NF-κB signaling pathways

• Phenotypic consequences:

  • CFs exposed to S100A1 adopt an immunomodulatory and anti-fibrotic phenotype

  • This is characterized by enhanced intercellular adhesion molecule-1 (ICAM1) expression

  • Concurrent decrease in collagen production is observed

• In vivo effects:

  • Intracardiac S100A1 injection recapitulates these transcriptional changes in mouse models

  • Antibody-mediated neutralization of S100A1 results in:

    • Enlarged infarct size

    • Worsened left ventricular function post-MI

Methodological approach for studying alarmin function:

• Measure S100A1 serum levels in patients with acute MI using ELISA

• Use S100A1 knockout mice and S100A1 neutralizing antibodies in experimental MI models

• Perform in vitro studies with isolated cardiac fibroblasts exposed to recombinant S100A1

• Analyze downstream signaling pathway activation through phosphorylation-specific antibodies

These findings suggest that extracellular S100A1 may play a potentially beneficial role in MI-related inflammation and repair, highlighting the complexity of S100A1 biology beyond its intracellular functions .

What methodological approaches can be used to study S100A1's dual role as both an intracellular signaling molecule and a secreted protein?

Investigating S100A1's dual functionality requires integrated experimental approaches that distinguish between its intracellular and extracellular roles:

• Intracellular function analysis:

  • Gene manipulation strategies:

    • siRNA knockdown of S100A1 in cell lines to study loss-of-function effects

    • CRISPR/Cas9 genome editing to create S100A1-deficient cell lines

    • Inducible expression systems to control S100A1 levels temporally

  • Protein interaction studies:

    • Co-immunoprecipitation to identify S100A1 binding partners (e.g., LATS1)

    • Proximity labeling techniques (BioID, APEX) to identify context-specific interactions

    • Fluorescence resonance energy transfer (FRET) to study dynamic interactions in living cells

  • Calcium dependency:

    • Calcium imaging coupled with S100A1 activity assays

    • Site-directed mutagenesis of calcium-binding domains to create calcium-insensitive variants

• Extracellular function analysis:

  • Secretion mechanisms:

    • Pulse-chase experiments to track S100A1 secretion

    • Selective inhibitors of unconventional secretion pathways

  • Receptor identification:

    • Receptor binding assays using labeled recombinant S100A1

    • Blocking antibodies against potential receptors (e.g., TLR4)

    • Receptor knockout cell lines to confirm specificity

  • Functional assays:

    • Cell-type specific responses to exogenous S100A1 (e.g., cardiac fibroblasts)

    • Neutralizing antibodies to block extracellular S100A1 in vivo

• Integrated approaches:

  • Compartment-specific S100A1 variants:

    • Creating cell-impermeable S100A1 variants to exclusively study extracellular effects

    • Signal sequence-tagged S100A1 to enhance secretion

    • Membrane-anchored forms to restrict to intracellular compartments

  • Temporal analysis:

    • Time-course experiments to distinguish immediate vs. delayed effects

    • Pulse treatment with extracellular S100A1 followed by washout

  • In vivo models:

    • Tissue-specific S100A1 knockout mice

    • Measuring S100A1 in circulation during disease models (e.g., myocardial infarction)

    • Targeted delivery of S100A1-neutralizing antibodies to specific tissues

These methodological approaches provide a framework for dissecting the complex biology of S100A1 and understanding how its dual functionality contributes to both normal physiology and pathological conditions .

How can contradictory findings about S100A1 expression in different cancer types be reconciled and effectively studied?

S100A1 shows variable expression patterns across cancer types, with apparently contradictory roles as either a tumor promoter or suppressor. A systematic approach to reconciling these findings includes:

• Standardized expression analysis:

  • Multi-omics approach:

    • Integrate transcriptomics, proteomics, and functional data

    • Analyze S100A1 at both mRNA and protein levels in the same samples

    • Include post-translational modification analysis

  • Spatial resolution:

    • Use single-cell techniques to identify cell-specific expression

    • Apply spatial transcriptomics/proteomics to map expression in tumor microenvironment

    • Distinguish between tumor cells and stromal/immune components

• Context-dependent function assessment:

  • Tissue-specific partners:

    • Identify tissue-specific S100A1 interacting proteins

    • Map differential binding partners across cancer types

    • For example, S100A1 interaction with LATS1 in HCC vs. other binding partners in different cancers

  • Calcium-dependency:

    • Analyze calcium levels in different tumor types

    • Determine how calcium concentration affects S100A1 function

    • Examine how calcium dysregulation in cancer affects S100A1 activity

• Methodological standardization:

  • Antibody validation:

    • Use multiple antibodies targeting different epitopes

    • Validate antibody specificity for each tissue type

    • Include appropriate positive and negative controls

  • Expression quantification:

    • Standardize scoring methods (e.g., consistent use of Integrated Optical Density)

    • Apply digital pathology for objective quantification

    • Establish clear cutoffs for "high" vs. "low" expression

• Integrated experimental models:

  • Comparative studies:

    • Side-by-side analysis of S100A1 function in multiple cancer cell lines

    • Use isogenic cell lines differing only in S100A1 expression

    • Compare effects in 2D culture, 3D organoids, and in vivo models

  • Temporal dynamics:

    • Study S100A1 expression throughout cancer progression

    • Analyze expression in paired primary and metastatic samples

    • Examine changes during treatment response

• Clinical correlation standardization:

  • Comprehensive clinical annotation:

    • Include detailed pathological parameters

    • Document treatment history and response

    • Analyze survival data with multivariate approaches

  • Meta-analysis approach:

    • Pool data across studies using standardized reporting

    • Account for methodological differences

    • Stratify by cancer type, stage, and molecular subtype

By implementing these approaches, researchers can better understand why S100A1 shows oncogenic properties in hepatocellular carcinoma while potentially having different roles in other cancer types, leading to more precise diagnostic and therapeutic applications.

What are the potential therapeutic applications of targeting S100A1 or using S100A1 antibodies in disease treatment?

S100A1-targeted therapeutic strategies are emerging across multiple disease areas:

• Cancer therapy:

  • Direct targeting approaches:

    • Small molecule inhibitors of S100A1-target protein interactions

    • Targeted degradation of S100A1 using PROTACs (Proteolysis Targeting Chimeras)

    • siRNA/antisense oligonucleotides for S100A1 knockdown

  • Combinatorial therapies:

    • S100A1 inhibition to sensitize HCC cells to cisplatin treatment

    • Targeting S100A1-LATS1 interaction to modulate Hippo signaling in cancer

    • Biomarker-guided patient stratification for personalized treatment

• Cardiovascular applications:

  • Dual approach in heart failure:

    • Gene therapy to increase intracellular S100A1 in cardiomyocytes

    • Modulation of extracellular S100A1 alarmin function after myocardial injury

  • Post-infarction recovery:

    • Therapeutic regulation of S100A1's effect on cardiac fibroblasts

    • Manipulating the immunomodulatory and anti-fibrotic phenotype induced by S100A1

    • Controlled release systems for localized S100A1 delivery

• Neurological diseases:

  • Neurodegenerative disorders:

    • Targeting S100A1 in Alzheimer's disease and amyotrophic lateral sclerosis

    • Modulating calcium dysregulation through S100A1 pathways

    • Blood-brain barrier penetrating S100A1-modulating compounds

• Diagnostic and theranostic applications:

  • Molecular imaging:

    • Radiolabeled S100A1 antibodies for cancer detection

    • Monitoring therapy response through S100A1 expression

    • Early detection of myocardial injury using S100A1 as biomarker

  • Liquid biopsy development:

    • Detection of circulating S100A1 in serum as a biomarker

    • Multi-biomarker panels including S100A1

• Technical considerations for therapeutic development:

  • Target validation:

    • Confirm S100A1 knockout mice phenotypes in disease models

    • Validate tissue-specific and temporal requirements for inhibition

    • Distinguish between blocking intracellular vs. extracellular S100A1

  • Delivery strategies:

    • Tissue-specific targeting to reduce off-target effects

    • Antibody-drug conjugates for cancer-specific delivery

    • Gene therapy vectors for cardiac-specific expression

  • Safety assessment:

    • Monitor calcium signaling disruption

    • Evaluate effects on normal S100A1-expressing tissues

    • Develop reversible inhibition strategies

The development of S100A1-targeted therapies presents both opportunities and challenges, with the need to carefully distinguish between beneficial and detrimental roles of S100A1 in different disease contexts .

How can advanced imaging techniques be combined with S100A1 antibodies to enhance research and diagnostic applications?

Integrating cutting-edge imaging technologies with S100A1 antibodies offers powerful new approaches for both research and clinical applications:

• Super-resolution microscopy:

  • Techniques applicable with S100A1 antibodies:

    • STORM (Stochastic Optical Reconstruction Microscopy)

    • PALM (Photoactivated Localization Microscopy)

    • SIM (Structured Illumination Microscopy)

  • Research applications:

    • Nanoscale localization of S100A1 in cellular compartments

    • Co-localization with binding partners at molecular resolution

    • Visualization of calcium-dependent conformational changes

  • Implementation strategy:

    • Use directly conjugated primary antibodies for better resolution

    • Apply appropriate fixation to preserve native protein distribution

    • Combine with proximity ligation assays to confirm interactions

• Multiplexed tissue imaging:

  • Advanced techniques:

    • Cyclic immunofluorescence (CycIF)

    • CODEX (CO-Detection by indEXing)

    • Imaging mass cytometry

  • Applications in cancer research:

    • Map S100A1 expression in relation to multiple cell types in tumor microenvironment

    • Correlate with other S100 family proteins and calcium signaling markers

    • Profile expression across entire tumor sections to address heterogeneity

  • Renal tumor diagnostics:

    • Simultaneous detection of S100A1 with other renal tumor markers

    • Digital pathology algorithms for automated classification

    • Integration with histopathological and molecular features

• In vivo imaging:

  • Preclinical approaches:

    • Radiolabeled S100A1 antibodies for PET/SPECT imaging

    • Near-infrared fluorescent (NIRF) labeled antibodies for optical imaging

    • Magnetic resonance imaging with antibody-conjugated nanoparticles

  • Translational applications:

    • Monitoring S100A1 release in acute myocardial infarction models

    • Tracking treatment response in HCC models

    • Early detection of tumors with altered S100A1 expression

• Live-cell imaging:

  • Intracellular S100A1 dynamics:

    • Genetically encoded S100A1 fusion proteins (e.g., S100A1-GFP)

    • Bimolecular fluorescence complementation for interaction studies

    • FRET sensors for calcium-dependent conformational changes

  • Extracellular S100A1 tracking:

    • Fluorescently labeled recombinant S100A1 to track uptake by target cells

    • Monitoring receptor-mediated endocytosis in cardiac fibroblasts

    • Real-time visualization of alarmin responses

• Computational integration:

  • Image analysis algorithms:

    • Machine learning for automated quantification of S100A1 staining

    • Cell-type specific expression analysis in complex tissues

    • 3D reconstruction of S100A1 distribution in tissue microenvironments

  • Multi-omics integration:

    • Spatial transcriptomics overlaid with S100A1 protein visualization

    • Correlation with calcium signaling pathway activity

    • Integration with clinical outcome data