S100A11 Antibody

What is S100A11 and why is it important in cancer research?

S100A11 (also known as calgizzarin or S100C) is a 10-12 kDa member of the S100 family of calcium-binding proteins containing two EF-hand motifs. It has emerged as a significant research target due to its dual roles in cellular growth regulation and its upregulation in various metastatic cancers. The protein plays a crucial role in plasma membrane repair, cell migration, and invasion - processes that are essential for cancer progression.

Human S100A11 consists of 105 amino acids with two EF-hand motifs (amino acids 13-49 and 55-90) and one high-affinity calcium-binding site (amino acids 68-79) . Intracellularly, S100A11 can suppress growth, while extracellularly, it exists as monomers or homodimers, binds to RAGE (Receptor for Advanced Glycation End products), induces EGF, and promotes cell growth .

Research has shown S100A11's involvement in malignant pleural mesothelioma and its potential as a therapeutic target, highlighting its importance in oncology research .

How does S100A11 function in normal and cancer cells?

S100A11 exhibits context-dependent functions in both normal and cancer cells:

In Normal Cells:

• Facilitates differentiation and cornification of keratinocytes

• Plays essential roles in efficient plasma membrane repair

• Responds to calcium signaling during cellular injury

• Forms complexes with annexin proteins (particularly Annexin A2) to facilitate membrane resealing

In Cancer Cells:

• Overexpressed in many human cancers

• Contributes to increased plasma membrane dynamics needed for cancer cell migration through dense stroma

• Helps metastatic cancer cells cope with physical stress during invasion

• Acts via RAGE-NF-κB-Akt pathway to trigger production of EGF family proteins, stimulating growth

S100A11 exhibits a "dual mediator" role - it can suppress growth following DNA damage or TGF-β exposure by transferring to nuclei and inducing p21WAF1, but it can also be actively secreted to act as an extracellular growth stimulator .

What are the key considerations when selecting an S100A11 antibody for research?

When selecting an S100A11 antibody, researchers should consider:

Antibody Format:

• Monoclonal vs. Polyclonal: Monoclonal antibodies (e.g., EPR11171(B), EPR11172, Clone 672816) offer higher specificity but recognize single epitopes, while polyclonal antibodies provide signal amplification by recognizing multiple epitopes

• Host Species: Available options include rabbit, goat, mouse, and sheep-derived antibodies

• Applications: Ensure the antibody is validated for your specific application (WB, IHC, ICC, Flow Cytometry, IP, or ELISA)

Validation Parameters:

• Verified reactivity with target species (human, mouse, rat)

• Confirmed specificity (ability to distinguish S100A11 from other S100 family proteins)

• Documented molecular weight detection (~10-12 kDa band on Western blots)

• Validated working dilutions for specific applications

Experimental Requirements:

• Recognition of specific forms (monomer, homodimer, or heterodimer with S100B)

• Ability to detect S100A11 in subcellular compartments of interest (cytoplasmic, nuclear, or extracellular)

• Neutralizing capability if functional studies are planned

How can I validate the specificity of an S100A11 antibody?

Comprehensive validation of S100A11 antibody specificity should include:

Western Blot Validation:

• Confirm detection of the expected ~10-12 kDa band in positive control lysates (HeLa, JEG-3, SKBR3, BxPC-3, PC-3, HACAT cells)

• Include negative controls (tissues/cells known to express low S100A11 levels)

• Test antibody on recombinant S100A11 protein

• Consider using S100A11 knockout/knockdown samples as negative controls

Cross-Reactivity Assessment:

• Test against other S100 family members, particularly those with high sequence homology

• Evaluate specificity across species (human S100A11 shares 78% amino acid identity with mouse and 82% with porcine S100A11)

Immunostaining Validation:

• Compare staining patterns with documented S100A11 localization (typically cytoplasmic and/or nuclear in various cell types)

• Include appropriate negative controls (primary antibody omission, isotype controls)

• Consider dual-staining with alternative S100A11 antibodies targeting different epitopes

Functional Validation:

• For neutralizing antibodies, confirm their ability to block S100A11-mediated functions (e.g., RAGE binding, plasma membrane repair)

What are the optimal conditions for using S100A11 antibodies in Western blotting?

Optimal Western blotting conditions for S100A11 detection include:

Sample Preparation:

• Efficient lysis of cells (HeLa, JEG-3, SKBR3, BxPC-3, PC-3, or HACAT recommended as positive controls)

• Reducing conditions are typically used

• Loading 10-20 μg of total protein per lane is generally sufficient

Recommended Protocol:

• Separate proteins using 12-15% SDS-PAGE gels (optimal for detecting the ~10-12 kDa S100A11 protein)

• Transfer to PVDF membrane (preferred over nitrocellulose for small proteins)

• Block using appropriate buffer (typically 5% non-fat milk or BSA)

• Incubate with primary antibody:

  • Monoclonal antibodies: 1-2 μg/mL (e.g., MAB4874)

  • Polyclonal antibodies: 1:100-1:5000 dilution (e.g., CAB21929)

• Incubate with HRP-conjugated secondary antibody (1:2000-1:5000)

• Develop using enhanced chemiluminescence

• Expected result: Distinct band at approximately 10-12 kDa

Special Considerations:

• For subcellular localization studies, perform fractionation prior to Western blotting

• If detecting oligomeric forms, consider non-reducing conditions

• Some antibodies may require specific immunoblot buffer groups (e.g., Buffer Group 1 or 8)

How can I optimize S100A11 antibody use for immunohistochemistry and immunocytochemistry?

For optimal IHC/ICC results with S100A11 antibodies:

Sample Preparation:

• IHC-P: Formalin-fixed, paraffin-embedded tissues with antigen retrieval (heat-mediated in EDTA buffer is recommended)

• ICC: Cells fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100

Staining Protocol:

• Antigen Retrieval: Critical for formalin-fixed tissues

• Blocking: Use 5-10% normal serum from the same species as the secondary antibody

• Primary Antibody Incubation:

  • IHC-P: Dilutions range from 1:50-1:3000 depending on antibody

  • ICC: Typically 1:50-1:250 dilution

• Detection System:

  • For brightfield: HRP-conjugated secondary followed by DAB

  • For fluorescence: Fluorophore-conjugated secondary antibodies

• Counterstaining: DAPI for nuclear visualization in fluorescence applications

Expected Results:

• S100A11 typically shows cytoplasmic and/or nuclear staining patterns

• Strong expression in epithelial cells, particularly in esophagus

• Variable expression in different cancer types

Controls and Validation:

• Positive control tissues: Esophagus, thyroid carcinoma

• Negative controls: PBS in place of primary antibody, tissues with low S100A11 expression (e.g., cerebral cortex)

• Consider using antibodies that have been validated by the Human Protein Atlas

What are common issues when using S100A11 antibodies and how can they be resolved?

Special Considerations for S100A11:

• S100A11 exhibits calcium-dependent functions; consider the use of calcium chelators in functional studies

• For detecting extracellular S100A11, analyze culture media or secreted fractions

• When studying S100A11 in membrane repair, ensure experimental conditions preserve calcium-dependent protein interactions

How can I effectively use S100A11 antibodies in co-immunoprecipitation and interaction studies?

For effective co-immunoprecipitation (Co-IP) of S100A11 and its binding partners:

Optimized Protocol:

• Lysate Preparation:

  • Use mild lysis buffers to preserve protein-protein interactions

  • Include protease inhibitors and phosphatase inhibitors

  • For calcium-dependent interactions, maintain physiological calcium levels (or use calcium chelators as negative controls)

• Immunoprecipitation Strategy:

  • Direct approach: Use anti-S100A11 antibody for pull-down

  • Reverse approach: Use antibodies against known binding partners (e.g., Annexin A2)

  • For tagged proteins: Consider using anti-tag antibodies (e.g., anti-RFP for S100A11-RFP fusions)

• Interaction Verification:

  • Western blot analysis using specific antibodies against suspected binding partners

  • Include appropriate controls (IgG control, lysate input)

  • Consider cross-linking for transient or weak interactions

Studying S100A11-Annexin Interactions:
S100A11 forms complexes with Annexin A2 during plasma membrane repair. This interaction can be studied by:

• Co-expressing S100A11-GFP and ANXA2-RFP followed by immunoprecipitation with anti-RFP antibody

• Testing various S100A11 mutants (ΔC, ΔN, ΔCa) to identify interaction domains

• Using calcium chelators to determine calcium-dependency of interactions

Membrane Repair Assay Applications:
For studying S100A11's role in membrane repair:

• Induce plasma membrane injury (laser wounding or mechanical scraping)

• Co-IP S100A11 with Annexin A2 before and after injury

• Analyze recruitment of these proteins to injury sites using live-cell imaging

How can S100A11 antibodies be utilized in cancer research beyond basic expression studies?

S100A11 antibodies enable various advanced cancer research applications:

Functional Studies in Cancer Progression:

• Neutralizing antibodies can block extracellular S100A11 functions, helping determine its role in tumor microenvironment signaling

• Antibodies detecting phosphorylated or modified forms can reveal activation states in cancer cells

• Proximity ligation assays using S100A11 antibodies can visualize protein-protein interactions in situ

Therapeutic Target Validation:

• Malignant pleural mesothelioma research has identified S100A11 as a potential therapeutic target

• Antibodies can help validate S100A11 as a druggable target by disrupting its interactions with partners like RAGE

Biomarker Development:

• Immunohistochemical studies using well-validated S100A11 antibodies help establish its utility as a diagnostic/prognostic biomarker

• Correlating S100A11 expression patterns with clinical outcomes helps stratify patients

Resistance Mechanisms:

• Studying S100A11's role in plasma membrane repair provides insights into how cancer cells survive physical stress during metastasis

• Antibodies can track changes in S100A11 expression/localization following treatment with chemotherapeutic agents

Combined Methodologies:

• ChIP-seq using S100A11 antibodies can identify genes directly regulated by nuclear S100A11

• Mass spectrometry following S100A11 immunoprecipitation can identify novel binding partners in cancer contexts

What are the latest methodological advances in using S100A11 antibodies for mechanistic studies?

Recent methodological advances utilizing S100A11 antibodies include:

Live Cell Imaging Applications:

• Combining S100A11 antibody fragments with cell-penetrating peptides allows real-time tracking of endogenous S100A11

• Laser injury models with fluorescently-tagged S100A11 and binding partners (ANXA1, ANXA2) enable visualization of membrane repair dynamics

• FRET-based approaches using antibody-based biosensors can detect S100A11 conformational changes upon calcium binding

Single-Cell Analysis:

• S100A11 antibodies optimized for flow cytometry allow correlation of expression with other markers at single-cell resolution

• Mass cytometry (CyTOF) with metal-conjugated S100A11 antibodies enables multi-parameter analysis of tumor heterogeneity

Spatial Transcriptomics Integration:

• Combining S100A11 immunohistochemistry with spatial transcriptomics reveals relationships between S100A11 protein expression and local gene expression profiles

• This approach correlates protein expression with RNA-seq data in the same tissue regions

Extracellular Vesicle Analysis:

• S100A11 antibodies help characterize extracellular vesicle cargo, revealing mechanisms of intercellular communication in cancer

• Surface plasmon resonance using immobilized S100A11 antibodies can quantify S100A11 in biological fluids

CRISPR-Based Functional Screening:

• CRISPR-edited cells with tagged endogenous S100A11 can be probed with antibodies to study natural expression levels

• Anti-S100A11 antibodies validate knockout/knockdown efficiency in CRISPR screens targeting S100A11 function

These advanced methodologies represent the cutting edge of S100A11 research, enabling precise mechanistic studies of its functions in various biological contexts, particularly in cancer and cellular repair mechanisms.