SLC11A2 Antibody, Biotin conjugated

What is SLC11A2 and why is it an important target for biotin-conjugated antibodies?

SLC11A2 (Solute Carrier Family 11 Member 2), also known as DMT1 (Divalent Metal Transporter 1) or NRAMP2, is a critical transmembrane iron transporter involved in cellular iron uptake. This protein plays essential roles in:

Research has demonstrated that SLC11A2 is required for intestinal iron absorption and erythropoiesis, as evidenced by studies showing that SLC11A2 knockout mice develop severe iron-deficiency anemia . Biotin-conjugated antibodies targeting this protein are particularly valuable in research settings because they allow for signal amplification through avidin/streptavidin systems, enabling enhanced sensitivity in detection methods compared to conventional antibodies, especially in tissues where SLC11A2 expression may be relatively low.

What epitope targets are available for biotin-conjugated SLC11A2 antibodies and why does this matter?

Commercially available biotin-conjugated SLC11A2 antibodies target different epitopes of the protein, which significantly impacts their research applications:

The epitope selection is critical because:

• N-terminal epitopes (AA 1-69) may detect specific isoforms or cleaved versions of SLC11A2

• Mid-region epitopes (AA 251-350) often detect a broader range of SLC11A2 variants

• Different epitopes may be more or less accessible depending on protein conformation and experimental conditions

• Epitope selection affects cross-reactivity with SLC11A2 homologs across species

Researchers should select antibodies targeting epitopes appropriate for their specific experimental design, considering factors such as protein folding, post-translational modifications, and species-specific sequence conservation in the target region.

What validated applications exist for biotin-conjugated SLC11A2 antibodies?

Based on product validation data, biotin-conjugated SLC11A2 antibodies have been validated for several applications:

While these are the specifically validated applications for biotin-conjugated antibodies, other SLC11A2 antibodies have demonstrated utility in additional applications such as:

• Immunofluorescence (IF) on both tissue sections and cultured cells

• Flow cytometry

• Immunoprecipitation studies

When adapting biotin-conjugated SLC11A2 antibodies to applications beyond their validated uses, researchers should include appropriate controls and perform preliminary optimization experiments to ensure specificity and sensitivity.

What dilution ranges are optimal for biotin-conjugated SLC11A2 antibodies in different applications?

Optimal dilution ranges for biotin-conjugated SLC11A2 antibodies vary by application and specific antibody:

Important methodological considerations:

• Always perform a dilution series during initial optimization to determine the optimal concentration for your specific sample type

• Higher antibody concentrations may be required for tissues with low SLC11A2 expression

• When using biotin detection systems, remember that endogenous biotin in some tissues (especially liver, kidney, and brain) may contribute to background

• Sample-dependent optimization is critical; the actual working concentration should be determined by the researcher

How can I validate the specificity of biotin-conjugated SLC11A2 antibodies in my experimental system?

Rigorous validation of antibody specificity is crucial for reliable results. The following approaches are recommended:

• Knockout/Knockdown Controls:

  • Use tissue or cells from SLC11A2 knockout mice (Slc11a2−/−) as negative controls

  • Employ siRNA knockdown of SLC11A2 in cell culture systems using validated siRNA sequences (see Table 1 for effective siRNA target sequences) :

  siRNA Target Sequences for mouse Slc11a2
  siRNA1: AGACAGGUGAAUCGGGCCA
  siRNA2: ACAAAUAUGGCUUGCGGAA
  siRNA3: GGACCUUUCUGACGAUGAA
  siRNA4: GGUUUAAAGUGUAUCGAUA

• Conditional Knockout Models:

  • NG2-DMT1 KO or Sox10-DMT1 KO mice with tamoxifen-inducible Cre systems allow for tissue-specific and temporal control of SLC11A2 deletion

  • These models provide valuable negative control tissues for antibody validation

• Peptide Competition Assays:

  • Pre-incubating the antibody with its specific immunogen peptide should abolish specific staining

  • Some manufacturers offer blocking peptides specific to their antibodies

• Multiple Antibody Validation:

  • Compare staining patterns using antibodies targeting different epitopes of SLC11A2

  • Consistent patterns across antibodies increase confidence in specificity

What molecular weight should I expect when detecting SLC11A2 with biotin-conjugated antibodies?

The expected molecular weight for SLC11A2 detection varies slightly between sources and may depend on post-translational modifications:

When interpreting Western blot results:

• Expect the main SLC11A2 band between 60-70 kDa

• Multiple bands may represent different isoforms or post-translational modifications

• Truncated versions of SLC11A2 have been observed in some experimental systems

• Glycosylation patterns may vary across tissue types, potentially affecting apparent molecular weight

• Denaturation conditions can affect observed molecular weight due to the protein's highly hydrophobic nature with multiple transmembrane domains

What sample preparation techniques optimize detection with biotin-conjugated SLC11A2 antibodies?

Optimal sample preparation is critical for successful detection of SLC11A2 using biotin-conjugated antibodies:

• For Western Blotting:

  • Use fresh tissue/cells when possible

  • Include protease inhibitors in lysis buffers to prevent degradation

  • Standard RIPA or NP-40 buffers are typically effective for membrane protein extraction

  • Consider membrane fractionation to enrich for SLC11A2, as it is a transmembrane protein

  • For difficult samples, gentle non-ionic detergents help maintain protein integrity

• For Immunohistochemistry:

  • Paraffin sections: Antigen retrieval is critical; use TE buffer pH 9.0 or alternatively citrate buffer pH 6.0

  • Frozen sections: Fix briefly (10 min) with cold 4% paraformaldehyde

  • Block endogenous biotin using commercial blocking kits to reduce background

  • Quench endogenous peroxidase activity if using HRP-based detection systems

• For Immunofluorescence:

  • Gentle fixation (4% PFA, 10-15 minutes) preserves epitope accessibility

  • Permeabilization with 0.1% Triton X-100 enhances antibody penetration

  • Extended blocking (1-2 hours) with serum matching the secondary antibody host reduces background

• For ELISA:

  • Standardize protein concentration across samples

  • Pre-clear lysates by centrifugation to remove debris

  • Consider detergent compatibility with coating procedures

How can biotin-conjugated SLC11A2 antibodies be used to investigate iron transport mechanisms in disease models?

Biotin-conjugated SLC11A2 antibodies offer powerful tools for studying iron transport in various disease models:

• Iron Deficiency Anemia:

  • Track upregulation of SLC11A2 in duodenal enterocytes during iron deficiency

  • Compare SLC11A2 expression and localization between normal and iron-deficient animals

  • Correlate SLC11A2 protein levels with hematological parameters

• Hemochromatosis Models:

  • Investigate SLC11A2 expression changes in Hfe knockout mice, which model hereditary hemochromatosis

  • Evidence shows that Hfe deficiency can partially rescue the phenotype of SLC11A2-deficient mice, suggesting compensatory mechanisms

  • Study compound mutants (Slc11a2−/−Hfe−/−) to understand transporter interactions

• Neurodegeneration:

  • Examine SLC11A2 expression in brain regions affected by Parkinson's or Alzheimer's disease

  • Use conditional knockout models (such as Sox10-DMT1 KO) to study the role of SLC11A2 in oligodendrocyte function and myelination

  • Investigate iron accumulation patterns in relation to SLC11A2 distribution in neuronal tissues

• Cancer Models:

  • Compare SLC11A2 expression in normal vs. cancer cell lines (HuH-7, COLO 320, Caco-2, SH-SY5Y)

  • Study how altered iron metabolism affects cancer cell proliferation and metastasis

  • Investigate potential correlations between SLC11A2 expression and response to iron chelation therapies

Methodology for such studies typically combines:

• Western blotting to quantify total protein expression

• Immunohistochemistry to determine cellular and subcellular localization

• Co-localization studies with other iron metabolism proteins

• Functional assays of iron uptake correlated with SLC11A2 expression

What are the experimental considerations for studying SLC11A2 in different tissue types?

Different tissues require specific considerations when studying SLC11A2 with biotin-conjugated antibodies:

Methodological recommendations:

• For tissues with high endogenous biotin (liver, kidney, brain), use commercial avidin/biotin blocking kits before applying biotin-conjugated antibodies

• For duodenal sections, orientation is critical to distinguish apical vs. basolateral staining

• For brain sections, use thin sections (5-7μm) and extended antibody incubation times

• For bone marrow, specialized fixatives (such as Bouin's solution) may better preserve morphology

How can I effectively combine biotin-conjugated SLC11A2 antibodies with other iron metabolism markers?

Multiplex analysis combining SLC11A2 with other iron metabolism markers provides comprehensive insights:

• Methodological Approaches:

  • Double immunofluorescence using different detection systems (biotin-streptavidin for SLC11A2 and direct fluorophore conjugates for other markers)

  • Sequential immunostaining with careful antibody stripping between rounds

  • Parallel staining of adjacent sections for markers requiring incompatible protocols

• Recommended Marker Combinations:

  • SLC11A2 + Ferroportin (FPN/SLC40A1): Compare iron importer vs. exporter

  • SLC11A2 + Transferrin Receptor 1 (TfR1): Analyze different iron uptake pathways

  • SLC11A2 + Ferritin: Correlate iron transport with storage

  • SLC11A2 + IRP1/IRP2: Examine post-transcriptional regulation (especially using biotinylated-IRE probe methods)

• Technical Considerations:

  • Choose primary antibodies from different host species to avoid cross-reactivity

  • When using biotin-conjugated SLC11A2 antibodies, other markers should use non-biotin detection systems

  • Control for spectral overlap when using multiple fluorophores

  • Include single-stained controls to verify specificity

• Analysis Approaches:

  • Colocalization quantification using specialized software

  • Correlation of staining intensities across cell populations

  • Subcellular distribution comparison using confocal microscopy

What controls are essential when working with biotin-conjugated SLC11A2 antibodies?

Rigorous experimental controls are crucial for reliable results with biotin-conjugated SLC11A2 antibodies:

• Negative Controls:

  • Primary antibody omission (detection reagents only)

  • Isotype control (non-specific IgG of same isotype and concentration)

  • Tissue from SLC11A2 knockout models (Slc11a2−/−)

  • Cells treated with validated siRNA against SLC11A2

• Positive Controls:

  • Tissues known to express SLC11A2 (duodenum, specific brain regions)

  • Cell lines with verified SLC11A2 expression (HuH-7, COLO 320, Caco-2, SH-SY5Y, HepG2)

  • Overexpression systems with tagged SLC11A2 constructs

• Technical Controls:

  • Endogenous biotin blocking control (especially for liver, kidney, brain samples)

  • Autofluorescence control (unstained sample) for fluorescence applications

  • Absorption controls (pre-incubation with immunizing peptide)

  • Iron manipulation controls (cells/tissues treated with iron chelators or iron loading)

• Validation Approaches:

  • Compare results with multiple antibodies targeting different SLC11A2 epitopes

  • Correlate protein detection with mRNA expression data

  • Verify that manipulations known to alter SLC11A2 (iron deficiency/overload) produce expected changes

How do I troubleshoot common issues with biotin-conjugated SLC11A2 antibodies?

For specific troubleshooting example:

• When detecting SLC11A2 in brain tissue, endogenous biotin often causes high background. Solution: Implement a sequential blocking approach - first block endogenous biotin using an avidin/biotin blocking kit, then perform standard protein blocking, followed by overnight primary antibody incubation at 4°C with extended washing steps.

How can I study SLC11A2 isoforms using biotin-conjugated antibodies?

SLC11A2/DMT1 exists in multiple isoforms that differ in their N-terminal and C-terminal regions. To study these isoforms using biotin-conjugated antibodies:

• Isoform Identification Strategy:

  • Select antibodies targeting different epitopes to distinguish isoforms

  • ABIN7161094 (targeting AA 1-69) may detect N-terminal variants

  • ABIN701067 (targeting AA 251-350) likely detects all major isoforms

  • Combine with RT-PCR to confirm isoform expression at mRNA level

• Technical Approaches:

  • Western blotting with high-resolution gels (8-10% acrylamide) to separate closely sized isoforms

  • 2D electrophoresis to separate isoforms based on both size and charge

  • Immunoprecipitation followed by mass spectrometry for definitive isoform identification

  • Isoform-specific knockdown to confirm antibody specificity

• Functional Studies:

  • Compare isoform expression across tissues with different iron requirements

  • Assess isoform regulation under iron deficiency vs. iron overload conditions

  • Correlate isoform expression with iron regulatory protein (IRP) activity

• Analytical Considerations:

  • Document all observed molecular weights precisely

  • Consider that post-translational modifications may cause shifts in apparent molecular weight

  • Use positive controls with known isoform expression patterns

What are the best practices for storing and handling biotin-conjugated SLC11A2 antibodies?

Proper storage and handling of biotin-conjugated SLC11A2 antibodies are essential for maintaining their activity:

Handling recommendations:

• Centrifuge briefly before opening to collect solution at the bottom of the vial

• Use sterile technique when handling to prevent contamination

• Return to appropriate storage temperature immediately after use

• For diluted working solutions, prepare fresh or store at 4°C for maximum 7 days

• Some preparations contain BSA (0.1%) for additional stability

• Do not heat antibodies during thawing; allow to thaw naturally at 4°C

How can biotin-conjugated SLC11A2 antibodies be used in the study of neurodegenerative diseases?

SLC11A2/DMT1 plays crucial roles in brain iron homeostasis, making it relevant to neurodegenerative disease research:

• Methodological Approaches:

  • Immunohistochemistry to map SLC11A2 distribution in brain regions affected by neurodegeneration

  • Double-labeling with cell type-specific markers (neurons, astrocytes, oligodendrocytes, microglia)

  • Quantitative Western blotting to compare expression levels in disease vs. control samples

  • Correlative studies with iron staining methods (Perls' Prussian blue)

• Specific Applications:

  • Conditional knockout models (NG2-DMT1 KO, Sox10-DMT1 KO) to study oligodendrocyte-specific iron transport

  • Analysis of myelin deficits associated with SLC11A2 deficiency in oligodendrocytes

  • Comparison of SLC11A2 expression in regions with iron accumulation in Parkinson's disease

  • Investigation of blood-brain barrier iron transport mechanisms

• Experimental Design Considerations:

  • Use tamoxifen-inducible Cre systems for temporal control of SLC11A2 deletion

  • Include age-matched controls due to age-related changes in brain iron

  • Consider regional variations in SLC11A2 expression within the CNS

  • Correlate with behavioral or functional measures in animal models

• Technical Recommendations:

  • Extended primary antibody incubation times (24-48h at 4°C) for optimal penetration in brain tissue

  • Thinner sections (5-7μm) for better resolution of subcellular localization

  • Use tyramide signal amplification for detection of low abundance expression

How can I quantify SLC11A2 expression levels using biotin-conjugated antibodies?

Accurate quantification of SLC11A2 expression requires careful methodological considerations:

• Western Blot Quantification:

  • Include recombinant protein standards for absolute quantification

  • Use housekeeping proteins appropriate for the specific tissue type as loading controls

  • Employ digital imaging systems with linear dynamic range

  • Perform densitometric analysis with background subtraction

  • Report results as ratio to housekeeping protein or total protein (Ponceau S staining)

• Immunohistochemistry Quantification:

  • Use standardized staining protocols with consistent reagent lots

  • Include calibration slides in each batch

  • Employ digital image analysis with defined intensity thresholds

  • Measure both staining intensity and percent positive area

  • Report results as H-score or quantitative immunohistochemistry (qIHC) values

• ELISA Approaches:

  • Develop sandwich ELISA using biotin-conjugated SLC11A2 antibody as detection antibody

  • Include recombinant SLC11A2 protein standards

  • Validate with knockout/knockdown samples

  • Determine optimal sample dilution to ensure measurements within linear range

• Flow Cytometry:

  • Use streptavidin-fluorophore conjugates for detection

  • Include compensation controls when multiplexing

  • Report as median fluorescence intensity (MFI)

  • Validate with isotype controls and knockdown samples

The choice of quantification method should align with the specific research question, considering factors such as need for spatial information, sensitivity requirements, and availability of appropriate controls.