SLC19A2 Antibody, Biotin conjugated

What is SLC19A2 and why are antibodies against it important for research?

SLC19A2 (Solute Carrier Family 19 Member 2) functions as a thiamine transporter, specifically known as thiamine transporter 1 (THTR-1). This integral membrane protein plays a crucial role in cellular thiamine uptake mechanisms across various tissues. Antibodies targeting SLC19A2 are essential research tools for investigating thiamine transport pathways, examining protein expression patterns in different tissues, and studying disorders related to thiamine metabolism. Unlike SLC19A3 (thiamine transporter 2), which demonstrates adaptive regulation in response to extracellular thiamine levels, SLC19A2 exhibits different regulatory patterns, making it an important comparative model in transport studies . Research with these antibodies facilitates understanding of cellular nutrient acquisition mechanisms and pathological conditions associated with thiamine transport deficiencies.

What are the key specifications of commercially available SLC19A2 antibody with biotin conjugation?

The polyclonal SLC19A2 antibody (AA 209-285) conjugated to biotin is derived from rabbit hosts and specifically targets amino acids 209-285 of the human SLC19A2 protein . The antibody undergoes Protein G purification with >95% purity and is generated using recombinant human thiamine transporter 1 protein fragment (amino acids 209-285) as the immunogen . This IgG isotype antibody has confirmed reactivity against human samples and is validated for enzyme-linked immunosorbent assay (ELISA) applications . The biotin conjugation enables versatile detection methods through streptavidin-based systems, providing flexibility for various experimental protocols including western blotting, immunohistochemistry, and protein-protein interaction studies.

How does the amino acid specificity (AA 209-285) of SLC19A2 antibody affect its application range?

The amino acid specificity of the SLC19A2 antibody targeting residues 209-285 has significant implications for experimental applications. This region represents a specific domain within the SLC19A2 protein structure that may be differentially accessible depending on the protein's conformational state or its interactions with other cellular components. When selecting this antibody, researchers should consider that:

• The targeted region (209-285AA) likely represents an extracellular or intracellular domain that maintains accessibility in native protein conformations.

• The epitope may be conserved across human samples but potentially differs in other species, explaining its specific reactivity to human targets.

• Post-translational modifications within this region might affect antibody binding efficiency.

• Protein structural alterations in different experimental conditions could impact epitope accessibility.

For comprehensive studies, researchers might need complementary antibodies targeting different epitopes, such as those specific to SLC19A2 AA 1-497 or AA 21-120, to validate findings and ensure reliable protein detection across various experimental conditions .

What are the optimal protocols for using biotin-conjugated SLC19A2 antibody in Western blot analyses?

For optimal Western blot analysis using biotin-conjugated SLC19A2 antibody, researchers should implement the following methodological approach:

• Sample preparation: Prepare cell or tissue lysates using a buffer containing protease inhibitors to prevent protein degradation. For SLC19A2 studies, particularly when examining thiamine-dependent regulation, condition cells with different thiamine concentrations (0-1 mM) for appropriate periods (7-9 days) before lysis.

• Gel electrophoresis: Separate proteins using NuPAGE 4-12% Bis-Tris gradient minigels or equivalent gradient gels that provide optimal resolution for membrane proteins like SLC19A2.

• Transfer: Transfer proteins to polyvinylidene difluoride (PVDF) membranes using appropriate transfer buffer systems optimized for transmembrane proteins.

• Blocking: Block membranes with 5% non-fat dry milk or 3% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 hour at room temperature.

• Antibody incubation: Dilute biotin-conjugated SLC19A2 antibody (1:500 to 1:1000) in blocking buffer and incubate membranes overnight at 4°C.

• Detection system: Utilize streptavidin-conjugated fluorophores (like IRDye-800) or streptavidin-horseradish peroxidase (HRP) for detection. For comparative studies, include β-actin (1:3,000 dilution) as a loading control.

• Visualization: For fluorescent detection, use systems like Odyssey infrared imaging; for chemiluminescence, use appropriate substrates followed by autoradiography or digital imaging.

This protocol has been demonstrated effective in detecting thiamine transporter expression changes in cell culture models exposed to varying thiamine concentrations .

How can biotin-conjugated SLC19A2 antibody be utilized in Electrophoretic Mobility Shift Assays (EMSA) for studying transcription factor binding?

Biotin-conjugated SLC19A2 antibody can be employed in EMSA studies to investigate transcription factor interactions with the SLC19A2 promoter, particularly when examining regulatory mechanisms of thiamine transport. The methodology involves:

• Nuclear extract preparation: Isolate nuclear proteins from cells cultured under varying conditions (such as thiamine-deficient versus thiamine-oversupplemented) using established nuclear extraction protocols.

• DNA probe design: Design and biotin-label DNA probes corresponding to potential regulatory regions of the SLC19A2 promoter. For comparison, include probes from the SLC19A3 promoter region, which contains known thiamine-responsive elements (between nucleotides -77 and -29) .

• Binding reaction setup: Perform binding reactions with 3 μg nuclear extract, 20 fmol biotin-labeled DNA probe, and 50 ng/μl poly(dI·dC) for 30 minutes at room temperature.

• Competition analysis: Include 200-fold molar excess of unlabeled probe to verify binding specificity.

• Supershift assay: Pretreat nuclear extracts with specific antibodies (e.g., anti-SP1 monoclonal antibody) to identify bound transcription factors.

• Electrophoresis and transfer: Separate DNA-protein complexes on 6% DNA retardation gels and transfer to nylon membranes.

• Detection: Visualize using chemiluminescence methods suitable for biotin-labeled nucleic acids.

This approach allows researchers to investigate differential regulation of SLC19A2 versus SLC19A3 promoters under varying thiamine conditions, contributing to understanding transcriptional control mechanisms of thiamine transporters .

What strategies can be employed to optimize immunohistochemistry protocols using biotin-conjugated SLC19A2 antibody?

Optimizing immunohistochemistry (IHC) protocols with biotin-conjugated SLC19A2 antibody requires addressing several methodological considerations:

• Antigen retrieval optimization: Test multiple retrieval methods (heat-induced epitope retrieval using citrate buffer pH 6.0, EDTA buffer pH 9.0, or enzymatic retrieval) to determine which best exposes the epitope (AA 209-285) while preserving tissue morphology.

• Endogenous biotin blocking: Critical for biotin-conjugated antibodies, implement an endogenous biotin blocking step using avidin-biotin blocking kits to prevent false-positive signals, particularly in biotin-rich tissues (liver, kidney).

• Signal amplification systems: Employ tyramide signal amplification (TSA) for detecting low abundance SLC19A2 protein while maintaining specificity.

• Multi-labeling protocols: For co-localization studies with other thiamine pathway components, use sequential labeling approaches with appropriate fluorophore combinations that minimize spectral overlap.

• Validation controls:

  • Positive control: Include tissues with known SLC19A2 expression

  • Negative control: Omit primary antibody

  • Absorption control: Pre-incubate antibody with immunizing peptide (AA 209-285)

  • Comparative analysis: When possible, compare results with unconjugated SLC19A2 antibodies

• Alternative detection strategies: If high background persists, consider using streptavidin-conjugated quantum dots or nanogold particles for superior signal-to-noise ratios.

• Quantification approaches: Implement digital image analysis using appropriate software to quantify membrane versus cytoplasmic staining patterns, particularly useful when comparing SLC19A2 expression in normal versus pathological tissue samples.

How should experiments be designed to study SLC19A2 expression modulation under varying thiamine conditions?

Designing experiments to study SLC19A2 expression modulation under varying thiamine conditions requires careful consideration of multiple factors:

• Cell model selection: Choose appropriate cell lines that endogenously express SLC19A2, such as intestinal epithelial cells (Caco-2) that have been demonstrated to model thiamine transport mechanisms effectively .

• Thiamine concentration range: Establish experimental conditions with precisely defined thiamine concentrations:

  • Deficient condition: Culture media without thiamine supplementation

  • Normal condition: Physiological thiamine levels (approximately 10-20 nM)

  • Oversupplemented condition: High thiamine concentration (1 mM)

• Exposure duration: Maintain cells under defined thiamine conditions for sufficient time (7-9 days) to allow for adaptive responses in transporter expression patterns .

• Multi-level analysis approach:

  • Protein expression: Western blot analysis using biotin-conjugated SLC19A2 antibody

  • mRNA quantification: RT-qPCR for SLC19A2 transcript levels

  • Promoter activity: Luciferase reporter assays with SLC19A2 promoter constructs

  • Functional transport: [3H]thiamine uptake measurements

  • Cellular localization: Immunofluorescence using biotin-conjugated SLC19A2 antibody

• Comparative analysis: Include parallel examination of SLC19A3 (THTR-2) expression, which has demonstrated adaptive regulation in response to extracellular thiamine levels, unlike the relatively stable expression pattern of SLC19A2 under varying thiamine conditions .

• Validation steps: Confirm antibody specificity using recombinant SLC19A2 expression systems and knockdown/knockout models to ensure accurate interpretation of expression data.

This comprehensive experimental approach facilitates understanding of the differential regulation mechanisms between SLC19A2 and SLC19A3 transporters in response to substrate availability.

What are common pitfalls when using biotin-conjugated antibodies and how can they be addressed in SLC19A2 research?

When working with biotin-conjugated SLC19A2 antibodies, researchers should be aware of and address these common methodological challenges:

• Endogenous biotin interference: Tissues and cells contain natural biotin that can interact with detection systems.

  • Solution: Implement stringent blocking protocols using avidin/biotin blocking kits before antibody application.

  • Alternative: Consider using tissues from biotin-deficient experimental models for validation studies.

• Excessive signal amplification: Biotin-streptavidin systems provide significant amplification that can obscure subtle expression differences.

  • Solution: Titrate detection reagent concentrations and optimize exposure times.

  • Alternative: Use direct fluorophore conjugates for scenarios requiring quantitative comparisons.

• Non-specific binding: Polyclonal biotin-conjugated antibodies may exhibit cross-reactivity with related proteins.

  • Solution: Validate antibody specificity using recombinant SLC19A2 expression systems and knockdown controls.

  • Alternative: Compare results with monoclonal alternatives when available.

• Biotin conjugation variability: Lot-to-lot variations in biotin:antibody ratios can affect binding kinetics and signal intensity.

  • Solution: Standardize protocols using consistent lot numbers for longitudinal studies.

  • Alternative: Include internal standards to normalize between experiments.

• Biotin-conjugated antibody stability issues: Extended storage can lead to decreased performance.

  • Solution: Aliquot antibodies upon receipt and follow manufacturer's storage recommendations.

  • Alternative: Validate each new lot against historical controls.

• False localization patterns: Artifactual staining patterns may emerge in fixed tissues.

  • Solution: Compare multiple fixation protocols (paraformaldehyde, methanol) to ensure consistent localization patterns.

  • Alternative: Verify results with different detection methods like subcellular fractionation.

• Co-labeling complications: Streptavidin systems limit options for multiple biotin-conjugated antibodies.

  • Solution: Implement sequential labeling protocols with intermediate blocking steps.

  • Alternative: Use different conjugates (HRP, FITC) for co-labeling studies.

These methodological considerations are particularly important when investigating membrane proteins like SLC19A2, where accurate subcellular localization is critical for understanding transporter function.

How can researchers design experiments to compare and contrast SLC19A2 and SLC19A3 expression and regulation?

To effectively compare and contrast SLC19A2 and SLC19A3 expression and regulation, researchers should implement a comprehensive experimental framework:

• Differential expression analysis:

  • Simultaneous quantification of both transporters across diverse tissue panels using biotin-conjugated antibodies specific to each transporter

  • Cross-validation with mRNA expression analysis

  • Development of tissue expression maps highlighting predominant transporter patterns

• Thiamine responsiveness assays:

  • Culture cellular models in defined thiamine concentrations (deficient, normal, oversupplemented)

  • Compare protein and mRNA expression patterns of both transporters under each condition

  • Focus on differential responsiveness, as SLC19A3 demonstrates adaptive regulation to thiamine levels while SLC19A2 remains relatively stable

• Promoter structure-function analysis:

  • Generate luciferase reporter constructs containing promoter regions of both genes

  • Create 5'-deletion constructs to identify regulatory elements

  • Test promoter activities under varying thiamine conditions

  • For SLC19A3, focus on the thiamine-responsive region between -77 and -29, particularly the SP1/guanosine cytidine box that mediates thiamine responsiveness

  • Perform parallel analyses on SLC19A2 promoter to identify regulatory differences

• Transcription factor binding analysis:

  • Design biotin-labeled DNA probes corresponding to key promoter regions of both genes

  • Perform electrophoretic mobility shift assays (EMSA) to identify differential transcription factor binding patterns

  • Conduct supershift assays with antibodies against candidate transcription factors (e.g., SP1)

  • Correlate binding patterns with differential thiamine responsiveness

• Functional transport studies:

  • Measure [3H]thiamine uptake in cells with selective knockdown of each transporter

  • Analyze kinetic parameters (Km, Vmax) under different thiamine pretreatment conditions

  • Assess compensatory mechanisms when one transporter is deficient

This integrated approach enables researchers to elucidate the complementary and distinct roles of these related thiamine transporters in maintaining cellular thiamine homeostasis.

How should researchers interpret conflicting data between protein localization and functional studies of SLC19A2?

When confronted with discrepancies between SLC19A2 protein localization (determined using biotin-conjugated antibodies) and functional transport studies, researchers should implement a systematic analytical approach:

• Methodological validation:

  • Verify antibody specificity through multiple controls including absorption tests with immunizing peptide (AA 209-285)

  • Confirm functional assay specificity using selective inhibitors or competitive substrates

  • Test multiple fixation and permeabilization protocols that may affect epitope accessibility

• Biological explanations:

  • Consider post-translational modifications that might affect antibody recognition without altering function

  • Evaluate potential protein trafficking mechanisms where intracellular reserves may not represent the functionally active population

  • Assess whether detected protein represents mature versus immature forms of the transporter

• Resolution strategies:

  • Implement subcellular fractionation followed by Western blotting to quantify transporter distribution across membrane compartments

  • Utilize surface biotinylation assays to specifically label and quantify plasma membrane-localized transporters

  • Develop transport-deficient SLC19A2 mutants to dissociate protein presence from functional capacity

  • Employ proximity ligation assays to investigate protein-protein interactions that might regulate localization or function

• Integrated data interpretation:

  • Construct comprehensive models that incorporate both localization and functional data

  • Consider temporal dynamics where protein localization may precede functional activity

  • Evaluate whether experimental conditions (such as thiamine availability) differentially affect localization versus function

  • Acknowledge technical limitations of both approaches in final interpretations

This systematic approach helps researchers develop more accurate models of SLC19A2 biology that reconcile apparently conflicting experimental observations.

What statistical approaches are recommended for analyzing SLC19A2 expression data across multiple experimental conditions?

For robust statistical analysis of SLC19A2 expression data across multiple experimental conditions, researchers should implement the following analytical framework:

This comprehensive statistical approach enhances reproducibility and facilitates meaningful interpretation of SLC19A2 expression patterns across experimental conditions.

How can researchers distinguish between direct and indirect effects on SLC19A2 expression in complex experimental systems?

Distinguishing between direct and indirect effects on SLC19A2 expression in complex experimental systems requires implementing multiple complementary approaches:

• Temporal analysis:

  • Perform time-course experiments measuring SLC19A2 expression at multiple intervals following experimental manipulation

  • Identify sequential changes in regulatory factors preceding SLC19A2 expression changes

  • Plot temporal relationships to establish cause-effect sequences

• Pharmacological interventions:

  • Utilize selective inhibitors of candidate regulatory pathways

  • Implement dose-response studies to correlate pathway inhibition with SLC19A2 expression

  • Consider antagonist/agonist pairs to confirm bidirectional regulation

• Genetic manipulation strategies:

  • Employ targeted knockdown/knockout of candidate regulatory factors

  • Implement rescue experiments to confirm specificity

  • Utilize inducible expression systems for temporal control

• Promoter analysis:

  • Unlike SLC19A3, which contains a thiamine-responsive promoter region (between -77 and -29), SLC19A2 promoter activity shows minimal response to extracellular thiamine levels

  • Construct reporter assays with wild-type and mutated SLC19A2 promoter regions

  • Identify transcription factor binding sites through in silico analysis followed by site-directed mutagenesis

  • Perform chromatin immunoprecipitation (ChIP) to detect direct transcription factor binding

• Protein-protein interaction studies:

  • Investigate direct interactions through co-immunoprecipitation using biotin-conjugated SLC19A2 antibodies

  • Confirm specificity through reverse immunoprecipitation

  • Employ proximity ligation assays to visualize interactions in situ

• Mathematical modeling:

  • Develop pathway models incorporating known regulatory components

  • Test model predictions through targeted experimental validation

  • Refine models iteratively based on experimental outcomes

• Multi-omics integration:

  • Correlate transcriptomic, proteomic, and metabolomic datasets

  • Identify convergent patterns suggesting direct versus secondary regulatory mechanisms

  • Apply network analysis to visualize regulatory hierarchies

This systematic approach enables researchers to differentiate between primary regulatory mechanisms directly affecting SLC19A2 expression and secondary effects mediated through intermediate factors or compensatory responses.

What are the key considerations for developing robust experimental protocols with SLC19A2 antibody, biotin conjugated?

Developing robust experimental protocols with biotin-conjugated SLC19A2 antibody requires careful attention to multiple technical and biological considerations throughout the experimental workflow:

• Experimental design fundamentals:

  • Include appropriate positive and negative controls for each application

  • Implement biological replicates (minimum n=3) to account for natural variation

  • Design experiments that facilitate direct comparison between SLC19A2 and SLC19A3 where relevant

• Antibody validation requirements:

  • Confirm specificity through multiple approaches (Western blot, immunoprecipitation, immunofluorescence)

  • Validate recognition of the target epitope (AA 209-285) in both denatured and native states

  • Test cross-reactivity with related transporters, particularly SLC19A3

• Biotin conjugation considerations:

  • Address endogenous biotin through appropriate blocking strategies

  • Evaluate batch-to-batch variation in conjugation efficiency

  • Consider signal-to-noise ratios across different detection systems

• Application-specific optimizations:

  • For Western blotting: Optimize membrane transfer conditions for this integral membrane protein

  • For immunohistochemistry: Determine optimal antigen retrieval methods that preserve epitope integrity

  • For immunofluorescence: Implement appropriate permeabilization protocols that maintain membrane architecture

• Data interpretation frameworks:

  • Develop standardized quantification methodologies

  • Establish appropriate normalization strategies

  • Implement statistical analysis approaches that account for technical and biological variability

• Integration with functional studies:

  • Correlate protein expression with thiamine transport capacity

  • Investigate structure-function relationships through targeted mutagenesis

  • Consider regulatory mechanisms in response to varying thiamine conditions