S100A12 Antibody

What is S100A12 and why is it important in inflammatory research?

S100A12 (also known as EN-RAGE, Calgranulin C, CGRP) is a calcium-binding protein primarily expressed by neutrophils, monocytes, and activated macrophages. It serves as a critical damage-associated molecular pattern (DAMP) molecule and plays a significant role in inflammatory signaling pathways . The importance of S100A12 in research stems from its established role as a reliable biomarker of systemic inflammation, particularly in vasculitis and other inflammatory disorders. Elevated serum concentrations of S100A12 (normal < 75 ng/ml) have been documented in various inflammatory conditions including rheumatoid arthritis, psoriatic arthritis, Crohn's disease, ulcerative colitis, and Kawasaki disease .

What are the molecular characteristics of human S100A12?

Human S100A12 is a 92-amino acid protein with a molecular weight of approximately 10-11 kDa . Its sequence (MTKLEEHLEGIVNIFHQYSVRKGHFDTLSKGELKQLLTKELANTIKNIKDKAVIDEIFQGLDANQDEQVDFQEFISLVAIALKAAHYHTHKE) contains calcium-binding EF-hand motifs characteristic of the S100 family proteins . The protein is encoded by the S100A12 gene (UniProt ID: P80511) and belongs to the S100 family of calcium-binding proteins with important roles in inflammatory processes .

How does S100A12 differ from other S100 family proteins in expression pattern and function?

Unlike some other S100 family members that have broader tissue expression, S100A12 shows a relatively restricted expression pattern, being predominantly produced by neutrophils, monocytes, and activated macrophages . S100A12 is specifically expressed by eosinophils and macrophages in asthmatic airways, particularly in regions where mast cells accumulate . Functionally, S100A12 serves as a ligand for RAGE (Receptor for Advanced Glycation End products) and plays a distinct role as an inflammatory mediator, with particularly strong associations with neutrophil-mediated inflammatory conditions .

What criteria should be considered when selecting an S100A12 antibody for research?

When selecting an S100A12 antibody, researchers should consider:

• Antibody type and host species: Options include rabbit polyclonal antibodies (e.g., Affinity Biosciences DF7277) or goat anti-human polyclonal antibodies (e.g., R&D Systems AF1052)

• Validated applications: Confirm that the antibody has been validated for your specific application (WB, IHC, Flow Cytometry, etc.)

• Species reactivity: Verify reactivity to your species of interest. Available antibodies show reactivity to human and mouse S100A12, with predicted reactivity to bovine, horse, rabbit, and dog

• Epitope specificity: For targeted studies, consider antibodies directed against defined epitopes, such as sandwich ELISA methods using monoclonal antibodies against specific S100A12 epitopes

• Validation data: Review available validation data including Western blot images, IHC staining patterns, and flow cytometry results

What validation experiments should be performed to ensure S100A12 antibody specificity?

To ensure antibody specificity:

• Western blot analysis: Validate using human granulocyte lysates, which should show a specific band at approximately 10-11 kDa under reducing conditions

• Immunohistochemistry cross-validation: Compare staining patterns in tissues known to express S100A12, such as tonsil, with established patterns in literature

• Flow cytometry validation: Test antibody performance in peripheral blood monocytes with appropriate isotype controls

• Positive and negative control tissues: Include tissues with known high expression (e.g., inflamed tissues, neutrophil-rich samples) and low/no expression

• Peptide blocking: Perform competition experiments with recombinant S100A12 protein to confirm binding specificity

• Cross-reactivity assessment: Test against related S100 family proteins to ensure specificity within this closely related protein family

What are the optimal conditions for using S100A12 antibodies in Western blot applications?

For optimal Western blot results with S100A12 antibodies:

• Sample preparation: Use neutrophil or granulocyte lysates as positive controls

• Reducing conditions: Perform Western blots under reducing conditions, as demonstrated in validation data from manufacturers

• Protein loading: Given S100A12's relatively low molecular weight (10-11 kDa), use appropriate percentage gels (15-20% polyacrylamide) for optimal separation

• Primary antibody concentration: Start with 1 μg/mL for goat anti-human S100A12 antibodies or manufacturer-recommended dilutions for other antibodies

• Detection system: Use appropriate HRP-conjugated secondary antibodies (e.g., anti-goat IgG or anti-rabbit IgG depending on primary antibody host)

• Buffer system: Consider using Immunoblot Buffer Group 1 or similar buffer systems as recommended in validation studies

How can I optimize immunohistochemistry protocols for S100A12 detection in different tissue types?

For effective IHC staining:

• Fixation and embedding: Use immersion fixed paraffin-embedded sections for consistent results

• Antibody concentration: Start with 15 μg/mL for goat anti-human antibodies in IHC applications or follow manufacturer-specific recommendations

• Incubation conditions: Incubate primary antibody overnight at 4°C for optimal staining

• Detection system: Use appropriate detection systems such as HRP-DAB for chromogenic detection

• Counterstaining: Employ hematoxylin for nuclear counterstaining to provide cellular context

• Positive control tissues: Include human tonsil sections as positive controls, which demonstrate characteristic S100A12 staining patterns

• Antigen retrieval: Optimize antigen retrieval methods based on tissue type and fixation conditions

What are the recommended protocols for measuring S100A12 levels in serum samples using ELISA?

For S100A12 quantification in serum:

• Assay selection: Use a sandwich ELISA with monoclonal antibodies directed against defined S100A12 epitopes for highest specificity

• Sample handling: Process serum samples promptly and store at -80°C to preserve protein integrity

• Reference ranges: Establish that normal serum concentrations of S100A12 are < 75 ng/ml in healthy children and adolescents

• Quality controls: Include appropriate positive controls (e.g., samples from inflammatory disease patients) and negative controls

• Assay validation: Determine assay sensitivity, specificity, and reproducibility for your specific experimental conditions

• Calibration curve: Prepare a standard curve using recombinant human S100A12 protein with appropriate range (covering expected pathological levels up to 250-300 ng/mL based on vasculitis study data)

How does S100A12 correlate with disease activity in inflammatory disorders?

Research demonstrates significant correlations between S100A12 levels and inflammatory disease activity:

• Chronic primary systemic vasculitidies (CPV): S100A12 levels are dramatically elevated at disease onset, with mean values of 247 ng/mL compared to normal values of <75 ng/mL

• Treatment response: S100A12 levels normalize in many patients following 3-6 months of induction therapy, with more substantial normalization at 12 months post-diagnosis

• Individual patient monitoring: Studies show that S100A12 level changes mirror disease activity changes in approximately 70% of individual patients, making it a valuable biomarker for monitoring treatment response

• Comparative performance: S100A12 shows better correlation with disease activity compared to some traditional inflammatory markers, with changes in S100A12 levels (70% of cases) more frequently mirroring disease activity than ESR (42%) or CRP (46%)

How do S100A12 levels compare with other inflammatory markers in monitoring disease progression?

When compared with traditional inflammatory markers:

This comparison demonstrates that S100A12, along with hemoglobin levels, provides more consistent correlation with disease activity than traditional inflammatory markers like CRP and ESR in certain inflammatory conditions .

What is the role of S100A12 in the AGE-RAGE signaling pathway, and how can antibodies help elucidate this mechanism?

S100A12 functions as an important ligand for RAGE (Receptor for Advanced Glycation End products), activating pro-inflammatory signaling cascades. Advanced research using specific S100A12 antibodies can:

• Pathway elucidation: Block specific S100A12-RAGE interactions using epitope-specific antibodies to determine contribution to inflammatory signaling

• Structural studies: Investigate the binding domains and conformational changes involved in S100A12-RAGE interaction through co-immunoprecipitation and structural analysis

• Downstream signaling: Map the signaling events following S100A12-RAGE engagement by using antibodies to track activation of downstream mediators

• Therapeutic targeting: Develop and test neutralizing antibodies against S100A12 as potential therapeutic agents for inflammatory conditions

Research has identified S100A12 as an upstream regulator in the AGE-RAGE pathway with implications in TGFβ2-mediated epithelial to mesenchymal transition, particularly in lens epithelial cells, opening potential therapeutic avenues for targeting this interaction .

What approaches can be used to investigate S100A12 secretion mechanisms in neutrophils and monocytes?

To study S100A12 secretion mechanisms:

• Cell activation models: Establish reliable models of neutrophil and monocyte activation that trigger S100A12 release

• Inhibitor studies: Use specific pathway inhibitors to determine the signaling pathways involved in stimulus-induced S100A12 secretion

• Secretome analysis: Compare the release of S100A12 with other secreted proteins to identify co-regulation patterns

• Live-cell imaging: Develop fluorescently tagged S100A12 constructs to track intracellular trafficking and secretion in real-time

• Knockout/knockdown models: Generate S100A12 knockout or knockdown cellular models to study the impact on inflammatory responses and neutrophil function

• Flow cytometry applications: Use flow cytometry with S100A12 antibodies to quantify intracellular versus surface-bound S100A12 in stimulated and unstimulated cells

How can researchers address potential cross-reactivity between S100A12 and other S100 family proteins in experimental design?

To minimize cross-reactivity concerns:

• Epitope mapping: Select antibodies raised against unique epitopes of S100A12 not shared with other S100 family members

• Validation with recombinant proteins: Test antibody reactivity against a panel of recombinant S100 family proteins to quantify potential cross-reactivity

• Negative control tissues: Include tissues known to express other S100 proteins but not S100A12

• Genetic models: Use S100A12-deficient tissues or cells as gold-standard negative controls

• Complementary techniques: Confirm antibody-based findings with orthogonal methods such as mass spectrometry or RNA expression analysis

• Pre-absorption controls: Perform pre-absorption of antibodies with recombinant S100A12 versus other S100 proteins to demonstrate specificity

What are common technical challenges when working with S100A12 antibodies and how can they be addressed?

Common challenges and solutions include:

• High background in immunostaining: Optimize blocking conditions using 5-10% normal serum from the same species as the secondary antibody; consider adding 0.1-0.3% Triton X-100 for improved blocking

• Multiple bands in Western blot: Ensure complete reduction of samples; optimize primary antibody concentration; consider using freshly prepared samples as S100A12 may form oligomers

• Variable ELISA results: Standardize sample collection and storage protocols; use consistent freeze-thaw cycles; consider matrix effects when analyzing different sample types

• Inconsistent flow cytometry staining: Optimize fixation and permeabilization protocols specifically for intracellular S100A12 detection; use appropriate isotype controls

• Tissue-specific optimization: Different tissues may require specific antigen retrieval methods and antibody concentrations; conduct titration experiments for each new tissue type

• Calcium-dependent conformational changes: Consider that S100A12 undergoes conformational changes upon calcium binding, which may affect epitope accessibility in certain experimental conditions

What methodological approaches are recommended for simultaneous detection of S100A12 with other inflammatory markers?

For multiplexed detection approaches:

• Multicolor flow cytometry: Design panels with appropriate fluorophore combinations for simultaneous detection of S100A12 and other markers like CD66b, MPO, or other S100 proteins

• Multiplex immunohistochemistry: Employ sequential immunostaining protocols with careful antibody stripping or spectral unmixing techniques

• Dual immunofluorescence: Use primary antibodies from different host species (e.g., rabbit anti-S100A12 with mouse anti-MPO) and species-specific secondary antibodies

• Multiplex ELISA platforms: Consider bead-based multiplex assays for simultaneous quantification of S100A12 alongside other inflammatory markers

• Combined protein/RNA detection: Implement protocols for sequential detection of S100A12 protein and mRNA expression in the same sample using immunostaining followed by in situ hybridization

• Proximity ligation assays: For studying protein-protein interactions between S100A12 and potential binding partners in situ

By addressing these technical considerations and implementing appropriate methodological approaches, researchers can maximize the utility of S100A12 antibodies in advancing our understanding of inflammatory processes and developing new diagnostic and therapeutic strategies.