SLC11A1 Antibody

What is SLC11A1 and why is it important in research?

SLC11A1 (Solute Carrier Family 11 Member 1) is a 550-amino acid transmembrane protein with a molecular mass of approximately 59.9 kDa in humans . It belongs to the natural resistance-associated macrophage protein (NRAMP) family and is also known by several synonyms including NRAMP1, Leishmaniasis resistance, and LSH . The protein is primarily localized to cell membranes and undergoes post-translational modifications, particularly glycosylation .

SLC11A1 has emerged as a significant research target due to its multifaceted role in macrophage activation, including induction of pro-inflammatory cytokines such as TNF-α, regulation of MHC II expression, and generation of reactive oxygen species (ROS) . Recent studies have identified SLC11A1 as a potential prognostic marker and immunotherapy response indicator in conditions like gliomas and Alzheimer's disease, highlighting its importance in understanding immune response mechanisms in pathological conditions .

How do SLC11A1 antibodies differ from other immunological reagents?

SLC11A1 antibodies are specifically designed for the immunodetection of solute carrier family 11 member 1 protein . Unlike general-purpose antibodies, these reagents target unique epitopes within the SLC11A1 protein structure, enabling precise detection in complex biological samples. When selecting SLC11A1 antibodies for research, it's crucial to consider:

• Epitope specificity: Some antibodies target N-terminal regions while others recognize C-terminal or internal sequences

• Cross-reactivity profile: Many SLC11A1 antibodies cross-react with orthologs from mouse, rat, bovine, frog, chimpanzee, and chicken species, facilitating comparative studies

• Application optimization: While Western Blot and ELISA are widely validated applications, some antibodies are specifically optimized for immunohistochemistry or flow cytometry

These specialized characteristics make SLC11A1 antibodies valuable tools for investigating specific biological questions related to cellular localization, protein expression patterns, and functional roles in immune responses.

What are the validated applications for SLC11A1 antibodies in research settings?

SLC11A1 antibodies have been validated for multiple research applications with varying optimization requirements:

For optimal results in Western Blot applications, researchers should note that SLC11A1 typically appears at higher molecular weights (90-120 kDa) than the predicted 59.9 kDa due to glycosylation and other post-translational modifications . Validation studies have confirmed antibody specificity using transfected cell lines expressing human SLC11A1, with appropriate negative controls using mock transfection or irrelevant transfectants .

How should researchers design validation experiments for new SLC11A1 antibodies?

When validating new SLC11A1 antibodies, a systematic approach incorporating multiple controls is essential:

• Expression system validation:

  • Compare antibody detection in SLC11A1-transfected cells versus mock-transfected controls

  • Use HEK293 human embryonic kidney cell lines for consistent expression systems

  • Include irrelevant transfectants as additional negative controls

• Multi-technique validation approach:

  • Western blot: Confirm specific band detection at expected molecular weight range (90-120 kDa)

  • Flow cytometry: Verify surface expression in transfected cells using appropriate secondary antibodies

  • Immunofluorescence: Assess subcellular localization patterns

• Blocking experiments:

  • Pre-incubate antibody with recombinant SLC11A1 protein before staining

  • Observe elimination of specific signal as confirmation of antibody specificity

Statistical significance should be established through at least three independent experiments, with consistent results across different detection methods providing strong evidence for antibody specificity and functionality .

What are the optimal conditions for detecting SLC11A1 in Western blot experiments?

Successful Western blot detection of SLC11A1 requires careful optimization of several parameters:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors if phosphorylation status is relevant

  • Heat samples at 70°C rather than 95°C to prevent aggregation of membrane proteins

• Gel and transfer conditions:

  • Use 8-10% SDS-PAGE gels to adequately resolve the 90-120 kDa range

  • Transfer to PVDF membranes (preferred over nitrocellulose for glycoproteins)

  • Extend transfer time to 2 hours or use semi-dry transfer systems for efficient transfer of larger proteins

• Blocking and antibody incubation:

  • Use 5% non-fat dry milk in TBST for blocking (1 hour at room temperature)

  • Dilute primary anti-SLC11A1 antibody to approximately 1 μg/mL in blocking buffer

  • Incubate with primary antibody overnight at 4°C for optimal sensitivity

  • Wash extensively (4-5 times) with TBST before and after secondary antibody incubation

• Detection considerations:

  • Use Immunoblot Buffer Group 1 for optimal results under reducing conditions

  • Be prepared to detect bands at significantly higher molecular weight (90-120 kDa) than the theoretical 59.9 kDa due to glycosylation

These conditions have been experimentally validated to produce specific detection of SLC11A1 while minimizing background and non-specific binding.

How can researchers effectively use SLC11A1 antibodies in flow cytometry experiments?

For successful flow cytometry experiments with SLC11A1 antibodies:

• Cell preparation protocol:

  • Harvest adherent cells using enzyme-free dissociation buffer to preserve surface epitopes

  • Fix cells with 2% paraformaldehyde if working with intracellular epitopes

  • Permeabilize with 0.1% saponin buffer for intracellular detection

• Staining strategy:

  • Use 1 μg/mL of anti-SLC11A1 antibody for optimal staining

  • Include fluorophore-conjugated secondary antibodies like allophycocyanin-conjugated anti-mouse IgG

  • Set quadrant markers based on control antibody staining

• Controls and validation:

  • Include isotype controls (e.g., MAB0041) to establish background staining levels

  • Use both positive controls (SLC11A1-transfected cells) and negative controls (irrelevant transfectants)

  • Consider co-staining with GFP in transfection experiments for dual verification

• Analysis considerations:

  • Gate on viable cells using appropriate viability dyes

  • Analyze expression in relevant cell populations (e.g., monocytes, macrophages, microglia)

  • Compare expression levels across different cell types or treatment conditions

These methodological approaches have been validated in transfection experiments with HEK293 cells and can be adapted for primary cells or other cell lines of interest .

How is SLC11A1 expression relevant to glioma research and immunotherapy approaches?

Recent studies have established SLC11A1 as a significant biomarker in glioma with implications for treatment stratification:

• Prognostic significance:

  • SLC11A1 expression increases with glioma progression and predicts unfavorable prognosis

  • Cox analysis reveals a hazard ratio of 2.33 (95% CI: 1.92-2.58, P < 0.001) for SLC11A1 expression

  • Expression levels can stratify glioma patients into subgroups with different immune activation statuses

• Immunotherapy response prediction:

  • Patients with higher SLC11A1 levels show better immunotherapeutic response profiles

  • ImmunCellAI analysis indicates that 79% of patients with high SLC11A1 expression respond to immunotherapy compared to 53% with low expression

  • TIDE assessment confirms similar findings: 72% response rate in high-SLC11A1 versus 35% in low-SLC11A1 groups

• Mechanistic insights:

  • SLC11A1 expression positively correlates with infiltration of various immune cells, particularly macrophages and monocytes

  • Linear regression analysis demonstrates significant relationships between SLC11A1 and immune checkpoint molecules PDCD1 (PD-1) and CTLA4

  • Real-time PCR validation confirms these associations in clinical samples from Shanghai General Hospital

These findings suggest that SLC11A1 assessment could guide personalized treatment decisions, potentially directing patients with high expression toward immunotherapy while those with low expression might benefit more from chemotherapeutic approaches like temozolomide .

What role does SLC11A1 play in neurodegenerative diseases like Alzheimer's?

Emerging research has revealed significant connections between SLC11A1, ferroptosis, and neuroinflammation in Alzheimer's Disease (AD):

• Expression patterns in AD:

  • SLC11A1 is predominantly expressed in microglia within the hippocampus and shows significant upregulation in AD patients

  • Similar upregulation patterns are observed in the middle temporal gyrus, suggesting a broader pathological role across brain regions

  • In peripheral blood, SLC11A1 is primarily expressed in monocytes and shows significant upregulation in AD patients

• Mechanistic connections:

  • SLC11A1 is closely associated with inflammation related to ferroptosis in AD

  • It plays a multifaceted role in macrophage activation, inducing pro-inflammatory cytokines, regulating MHC II expression, and generating reactive oxygen species

  • Expression correlates strongly with severity of both Aβ and Tau pathologies in model mice

• Cellular interactions:

  • Gene Set Enrichment Analysis (GSEA) indicates significant association of SLC11A1 with pathways involved in antigen processing and presentation

  • A ferroptosis-associated subpopulation of M1-type microglia in AD expresses both SLC11A1 and peripheral blood monocyte markers

  • This suggests potential infiltration of peripheral monocytes triggering neuroinflammation through ferroptosis pathways

These findings establish SLC11A1 as a potential therapeutic target in AD, particularly in addressing neuroinflammatory aspects of disease progression. The interaction between peripheral immune cells and central nervous system inflammation highlights the complex immunological dimensions of neurodegenerative processes .

How can SLC11A1 antibodies be used to investigate cell-specific expression in complex tissues?

Investigating cell-specific SLC11A1 expression in heterogeneous tissues requires sophisticated experimental approaches:

• Single-cell transcriptomic integration:

  • Combine bulk RNA-seq with single-cell RNA-seq (scRNA-seq) to identify cell populations expressing SLC11A1

  • In brain tissues, SLC11A1 expression is predominantly localized to microglia

  • In peripheral blood, expression is highest in monocytes

• Immunofluorescence co-labeling strategies:

  • Use SLC11A1 antibodies in conjunction with cell-type-specific markers:

    • For microglia: Co-stain with IBA1 or TMEM119

    • For monocytes: Co-stain with CD14 or CD16

    • For macrophages: Co-stain with CD68 or CD163

• Flow cytometry analysis of tissue-derived cells:

  • Digest tissues to single-cell suspensions using appropriate enzymatic cocktails

  • Stain with fluorophore-conjugated SLC11A1 antibodies alongside lineage markers

  • Gate on specific cell populations to quantify expression levels across cell types

• Spatial transcriptomics correlation:

  • Correlate SLC11A1 immunostaining patterns with spatial transcriptomic datasets

  • Map expression relative to anatomical structures or pathological features

  • Integrate with single-cell data to construct comprehensive expression atlases

These approaches have been successfully employed to demonstrate that SLC11A1 is significantly upregulated in M1-type microglia and peripheral blood monocytes in Alzheimer's Disease, highlighting the interaction between peripheral immune cells and the central nervous system .

What are common challenges in SLC11A1 antibody experiments and how can they be overcome?

Researchers frequently encounter several challenges when working with SLC11A1 antibodies:

• Molecular weight discrepancy:

  • Challenge: SLC11A1 often appears at 90-120 kDa rather than the predicted 59.9 kDa

  • Solution: Use appropriate molecular weight markers spanning 40-150 kDa range and understand that glycosylation significantly affects migration patterns

• Nonspecific binding:

  • Challenge: Multiple bands or high background in Western blots

  • Solution: Increase blocking time/concentration, optimize antibody dilution (starting with 1 μg/mL) , and consider using specialized blocking reagents for membrane proteins

• Isoform detection:

  • Challenge: Two different isoforms have been reported for SLC11A1

  • Solution: Select antibodies that recognize conserved regions if detecting all isoforms is desired, or epitope-specific antibodies if distinguishing between isoforms

• Cross-reactivity concerns:

  • Challenge: Potential cross-reactivity with other NRAMP family members

  • Solution: Validate specificity using SLC11A1 knockout/knockdown controls or comparing with known expression patterns across tissues

• Low signal in fixed tissues:

  • Challenge: Epitope masking during fixation procedures

  • Solution: Optimize antigen retrieval methods (heat-induced or enzymatic), reduce fixation time, or use specialized fixatives that better preserve membrane protein epitopes

Each of these challenges can be systematically addressed through careful experimental design and optimization of protocols specific to the application and sample type being studied.

How should researchers interpret variable SLC11A1 antibody staining patterns across different cell types?

Interpreting variable staining patterns requires consideration of multiple biological and technical factors:

• Cell type-specific expression patterns:

  • SLC11A1 is predominantly expressed in monocytes, macrophages, and microglia

  • Expression levels vary significantly across cell types and activation states

  • M1-type microglia show higher expression compared to other microglial subpopulations

• Subcellular localization variations:

  • SLC11A1 primarily localizes to cell membranes but can redistribute under different conditions

  • Activation state can affect trafficking between membrane and intracellular compartments

  • Consider fixation and permeabilization effects on epitope accessibility

• Quantification approaches:

  • For flow cytometry: Compare mean fluorescence intensity (MFI) values between populations

  • For immunohistochemistry: Use digital image analysis with consistent thresholding

  • For Western blot: Normalize band intensity to appropriate loading controls

• Validation strategies:

  • Correlate protein detection with mRNA expression data

  • Use multiple antibodies targeting different epitopes for confirmation

  • Include positive control cells with known expression levels

When interpreting results, it's important to recognize that SLC11A1 expression is dynamically regulated in response to inflammatory stimuli and disease states, as demonstrated in studies of glioma and Alzheimer's Disease .

What factors affect SLC11A1 detection sensitivity in different experimental contexts?

Multiple factors can influence the sensitivity of SLC11A1 detection:

• Sample preparation impacts:

  • Protein extraction method: Membrane protein extraction buffers containing mild detergents (0.5-1% NP-40 or Triton X-100) improve SLC11A1 solubilization

  • Fixation protocol: Paraformaldehyde concentration and duration significantly affect epitope preservation

  • Storage conditions: Freeze-thaw cycles can degrade membrane proteins like SLC11A1

• Antibody selection considerations:

  • Affinity: Higher affinity antibodies improve detection of low abundance targets

  • Clone type: Monoclonal antibodies provide consistent results but may be sensitive to epitope modifications

  • Recognition region: Antibodies targeting different domains may have varying sensitivities based on protein conformation

• Detection system optimization:

  • Signal amplification: Consider tyramide signal amplification for immunohistochemistry

  • Enhanced chemiluminescence formulations: Use high-sensitivity ECL for Western blots

  • Secondary antibody selection: Match host species and use highly cross-adsorbed secondary antibodies

• Disease state influences:

  • Expression levels increase in inflammatory conditions and certain pathological states

  • Post-translational modifications may vary in disease contexts

  • Protein-protein interactions can mask epitopes

Optimizing these factors is particularly important when studying SLC11A1 in complex tissues like brain samples from Alzheimer's patients or in comparing expression between different cell populations in glioma research .

How might SLC11A1 antibodies contribute to developing new immunotherapy stratification approaches?

The emerging role of SLC11A1 as an immunotherapy response indicator presents several promising research directions:

• Companion diagnostic development:

  • SLC11A1 antibody-based immunohistochemistry assays could stratify patients likely to respond to immune checkpoint inhibitors

  • Standardized scoring systems correlating expression levels with clinical outcomes

  • Integration with existing biomarkers like PD-L1 expression and tumor mutational burden

• Monitoring approaches:

  • Longitudinal assessment of SLC11A1 expression in liquid biopsies during immunotherapy

  • Correlation of expression changes with treatment response or resistance development

  • Development of flow cytometry panels incorporating SLC11A1 for immune monitoring

• Mechanistic investigations:

  • The positive correlation between SLC11A1 and immune checkpoint molecules (PDCD1/PD-1 and CTLA4) suggests interconnected regulatory mechanisms

  • Research could explore how SLC11A1 influences T cell activation and exhaustion

  • Investigation of SLC11A1's role in modulating tumor microenvironment immune landscape

• Therapeutic targeting potential:

  • Development of agents modulating SLC11A1 activity to enhance immunotherapy efficacy

  • Exploration of SLC11A1 as a direct therapeutic target in combination with checkpoint inhibitors

  • Investigation of cell-specific targeting approaches in diseases like glioma

The observation that glioma patients with high SLC11A1 expression show significantly better responses to anti-PD-1 and anti-CTLA4 treatment (79% vs 53% response rates) provides a strong foundation for these research directions .

What novel techniques might enhance SLC11A1 detection in complex biological samples?

Advancing SLC11A1 detection methodologies holds significant potential for improved research outcomes:

• Proximity ligation assays (PLA):

  • Enable detection of protein-protein interactions involving SLC11A1

  • Investigate signaling complexes in different cellular compartments

  • Study co-localization with immune checkpoint receptors or inflammatory mediators

• Mass cytometry (CyTOF) applications:

  • Develop metal-conjugated SLC11A1 antibodies for high-dimensional analysis

  • Simultaneously assess dozens of markers alongside SLC11A1

  • Create comprehensive immune cell phenotyping panels incorporating SLC11A1

• Spatial transcriptomics integration:

  • Correlate SLC11A1 protein expression with transcriptomic profiles in spatial context

  • Map expression relative to tissue architecture and pathological features

  • Develop multiplexed imaging approaches combining RNA and protein detection

• Single-cell proteomics approaches:

  • Apply advanced techniques like SCoPE-MS (Single-Cell ProtEomics by Mass Spectrometry)

  • Quantify SLC11A1 protein levels in individual cells alongside the broader proteome

  • Correlate with single-cell transcriptomics data for multi-omics integration

These advanced techniques would build upon current findings regarding SLC11A1's expression in specific cell populations like M1-type microglia and peripheral blood monocytes, potentially revealing new insights into its functional roles in diseases like Alzheimer's and glioma .

How might SLC11A1 antibodies contribute to understanding the ferroptosis-inflammation axis in neurological disorders?

The newly discovered connection between SLC11A1, ferroptosis, and neuroinflammation opens several research avenues:

• Microglial phenotyping in disease progression:

  • Develop antibody panels combining SLC11A1 with ferroptosis markers (e.g., GPX4, ACSL4)

  • Track microglial subpopulation dynamics during disease progression

  • Correlate with clinical parameters and disease severity

• Blood-brain barrier interactions:

  • Investigate how peripheral monocytes expressing SLC11A1 infiltrate the CNS

  • Study the role of SLC11A1 in monocyte-to-microglia transition

  • Explore potential as a therapeutic target to limit peripheral immune cell infiltration

• Therapeutic modulation approaches:

  • Test whether ferroptosis inhibitors affect SLC11A1 expression and associated inflammation

  • Develop targeted therapies modulating SLC11A1 in specific cell populations

  • Explore combination approaches targeting both ferroptosis and inflammatory pathways

• Biomarker development for neurological diseases:

  • Assess SLC11A1 expression in peripheral blood monocytes as a potential biomarker

  • Correlate with disease progression in conditions beyond Alzheimer's (e.g., Parkinson's, ALS)

  • Develop minimally invasive monitoring approaches based on SLC11A1 detection

The identification of a ferroptosis-associated subpopulation of microglia in AD that expresses SLC11A1 and displays characteristics of peripheral blood monocytes provides a foundation for these investigations . This could ultimately lead to new therapeutic strategies targeting the intersection of ferroptosis and neuroinflammation in neurodegenerative diseases.