DMT1 Antibody

What is DMT1 and why is it important in research?

DMT1, also known as solute carrier family 11 member 2 (SLC11A2), is a proton-coupled divalent metal ion transporter that plays a crucial role in the transport of various divalent metal ions. The full protein consists of 568 amino acids with a calculated molecular weight of 62 kDa, though the observed molecular weight in experimental conditions typically ranges from 60-70 kDa . DMT1 is particularly important in research concerning iron metabolism, neurodegenerative disorders such as Parkinson's disease, and metal homeostasis. Its mRNA possesses a stem-loop structure in the 3′-untranslated region, which is involved in post-transcriptional regulation through interactions with iron regulatory proteins (IRPs) .

What applications can DMT1 antibodies be used for?

DMT1 antibodies can be employed in multiple experimental applications as demonstrated in the scientific literature. The major applications include:

These applications have been validated across human, mouse, and rat samples, with some cited reactivity in pig and chicken models as well . When designing experiments, researchers should consider that optimal dilutions may be sample-dependent and should be determined empirically for each specific experimental system.

How do I properly validate a DMT1 antibody for my experimental system?

Validation of DMT1 antibodies should follow a systematic approach to ensure specificity and reliability. Based on published approaches, comprehensive validation includes:

• Positive and negative control samples: Use cell lines with known DMT1 expression (e.g., HEK-293 cells overexpressing DMT1) as positive controls .

• Genetic models: Utilize knockout or knockdown models where possible. Published validations have included experiments with brain-specific DMT1 knockout mice .

• Multiple detection methods: Cross-validate using different techniques (e.g., Western blot complemented by immunofluorescence).

• Molecular weight verification: Confirm detection at the expected molecular weight (60-70 kDa for DMT1) .

• Physiological manipulation: Test antibody under conditions that alter DMT1 expression, such as iron deficiency induced by desferrioxamine (DFO) treatment, which should increase DMT1 levels .

What are the optimal conditions for Western blot detection of DMT1?

For optimal Western blot detection of DMT1, researchers should consider the following methodological details:

• Sample preparation: Extract proteins using standard methods, ensuring complete lysis and denaturation.

• Protein loading: Load 20-50 μg of total protein per lane.

• Antibody selection: Use thoroughly validated antibodies with demonstrated specificity. For example, an antibody against the C-terminal region (amino acids 552-570) has been used to specifically detect DMT1 isoform I .

• Blocking conditions: Block membranes in 5% nonfat milk to reduce background .

• Primary antibody dilution: Use at 1:4000 dilution or according to manufacturer recommendations .

• Secondary antibody: Apply HRP-conjugated anti-rabbit antibody at approximately 1:6000 dilution .

• Loading control: Confirm equal loading using housekeeping proteins or Ponceau S staining .

• Quantification: Use digitizing software such as UN-SCAN-IT for accurate densitometric analysis .

Importantly, the observed molecular weight of DMT1 is typically 60-70 kDa, which may vary slightly depending on post-translational modifications and the specific isoform being detected .

How do I optimize immunofluorescence experiments with DMT1 antibodies?

For successful immunofluorescence detection of DMT1, consider these methodological recommendations:

• Sample preparation:

  • For paraffin-embedded tissues: Perform antigen retrieval with TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0 .

  • For cultured cells: Fix with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100.

• Antibody dilution:

  • For paraffin sections (IF-P): Use DMT1 antibody at 1:400-1:1600 dilution .

  • For cell cultures (IF/ICC): Use DMT1 antibody at 1:200-1:800 dilution .

• Expected pattern: DMT1 typically shows punctate cytoplasmic staining, as observed in Z310 cells derived from rat choroid plexus epithelia .

• Controls:

  • Negative control: Omit primary antibody while maintaining all other steps of the protocol .

  • Positive control: Use tissues or cells with confirmed DMT1 expression (e.g., mouse brain tissue, small intestine tissue, or HepG2 cells) .

How can DMT1 antibodies be used to study metal transport mechanisms?

DMT1 antibodies can be instrumental in investigating metal transport mechanisms through several advanced experimental approaches:

• Inducible expression systems: Utilize Dmt1-overexpression cell culture systems with doxycycline (DOX) induction to manipulate expression levels and study consequent changes in metal transport .

• Protein-mRNA interaction studies: Combine DMT1 protein detection with techniques like Electrophoretic Mobility Shift Assay (EMSA) to study interactions between Iron Regulatory Proteins (IRPs) and DMT1 mRNA, which regulate DMT1 expression .

• Metal-dependent regulation studies: Expose experimental systems to:

  • Iron chelators like desferrioxamine (DFO) at 100 μM to induce iron deficiency and upregulate DMT1 .

  • Ferric ammonium citrate to increase iron availability .

  • Manganese at 100-200 μM to study cross-regulation between different metals .

• Transport assays: Combine DMT1 antibody detection with functional assays such as the everted gut sleeve technique and colorimetric copper quantification to correlate DMT1 expression with actual metal transport capacity .

How do I analyze DMT1 expression changes in response to experimental treatments?

For rigorous analysis of DMT1 expression changes:

• Parallel analysis of mRNA and protein: Combine real-time RT-PCR for mRNA quantification with Western blot for protein expression to distinguish between transcriptional and post-transcriptional regulation .

• Time course experiments: In published studies, significant increases in DMT1 mRNA have been observed at 24 hours (45.4% increase) and 48 hours (78.1% increase) after manganese exposure (100 μM) .

• Dose-response relationships: A concentration-dependent relationship between manganese exposure (100-200 μM) and DMT1 mRNA levels has been established (r = 0.870, P < 0.01) .

• Heterogeneous nuclear RNA (hnRNA) analysis: Measure DMT1 hnRNA levels to distinguish between effects on transcription versus mRNA stability. For instance, research has shown that manganese exposure increased mature DMT1 mRNA without affecting hnRNA levels, suggesting post-transcriptional regulation .

What considerations should be made when studying DMT1 across different species or model systems?

When working with DMT1 across different species or models:

• Cross-species antibody reactivity: Consider sequence homology. For example, B. malayi DMT1 shares 61% identity and 74% similarity with human NRAMP 2 isoform 3, allowing human-targeted antibodies to recognize the parasite protein .

• Region-specific recognition: Some commercial antibodies target specific regions, such as the polyclonal antibody raised against amino acids 261-291 of human DMT1, which has 71% identity with the corresponding B. malayi protein region .

• Expression profiling across developmental stages: When studying model organisms, assess DMT1 expression across different life stages. This can be done using qPCR with appropriate normalization to housekeeping genes such as GAPDH .

• Genetic models: Consider using genetic variants like the Belgrade (b/b) rat, which expresses mutant DMT1, to study functional aspects without needing to create knockout models .

What are common challenges in DMT1 antibody experiments and how can they be addressed?

Researchers commonly encounter several challenges when working with DMT1 antibodies:

• Multiple isoforms: DMT1 exists in different isoforms that may show variable molecular weights. Use antibodies that target specific regions to distinguish between isoforms. For example, antibodies against the C-terminal region (amino acids 552-570) have been used to specifically detect DMT1 isoform I .

• Batch-to-batch variability: Validate each new lot of antibody against known positive controls. For polyclonal antibodies, variations between lots can be significant.

• Background signals: Optimize blocking conditions (5% nonfat milk has been effective) and antibody dilutions. Perform careful negative controls by omitting primary antibody while maintaining all other steps .

• Cross-reactivity concerns: When using antibodies across species, first confirm sequence homology in the epitope region. For cross-species applications, validation is crucial (e.g., the 71% identity in a specific region between human and B. malayi DMT1) .

How do I properly quantify DMT1 expression in Western blot and immunofluorescence experiments?

For accurate quantification of DMT1 expression:

• Western blot quantification:

  • Use digitizing software such as UN-SCAN-IT for densitometric analysis .

  • Normalize to appropriate loading controls.

  • Include a concentration gradient of samples to ensure measurements fall within the linear range of detection.

  • Perform at least three independent biological replicates for statistical validity.

• Immunofluorescence quantification:

  • Use consistent exposure settings across all samples.

  • Quantify signal intensity using image analysis software.

  • Analyze multiple fields (at least 5-10) per sample.

  • Include positive and negative controls in each experiment to establish baseline signal levels.

• Statistical analysis:

  • Apply appropriate statistical tests (e.g., t-test for comparisons between two groups, ANOVA for multiple groups).

  • Report results with proper statistical notation (e.g., P < 0.05, P < 0.01) .

How can DMT1 antibodies contribute to understanding neurodegenerative diseases?

DMT1 antibodies are increasingly important tools in neurodegenerative disease research:

• Parkinson's disease studies: Research has shown that manganese exposure can alter DMT1 expression at the blood-CSF barrier, potentially contributing to manganese-induced Parkinsonism . DMT1 antibodies can help track these expression changes in specific brain regions.

• Blood-brain barrier research: DMT1 has been identified in choroidal epithelial cells, suggesting its role in metal transport at the blood-CSF barrier. Antibody-based imaging can help map DMT1 distribution in these specialized cellular interfaces .

• Metal dysregulation hypotheses: DMT1 antibodies can help test hypotheses about how altered metal homeostasis contributes to neurodegeneration by enabling the visualization and quantification of DMT1 in brain tissues under different pathological conditions.

What are the considerations for designing multiplex experiments involving DMT1 antibodies?

For multiplex experiments combining DMT1 with other markers:

• Antibody compatibility: Ensure primary antibodies are from different host species to avoid cross-reactivity of secondary antibodies.

• Fluorophore selection: Choose fluorophores with minimal spectral overlap when designing multi-color immunofluorescence experiments.

• Sequential staining protocols: For challenging combinations, consider sequential rather than simultaneous staining, with complete washing between rounds.

• Controls for each marker: Include single-stained controls to verify specificity of each antibody and to set appropriate imaging parameters.

• Co-localization analysis: Apply rigorous quantitative co-localization analysis using specialized software rather than relying on visual assessment alone.