SLC5A6 Antibody

What is SLC5A6 and what functional significance does it have in transport systems?

SLC5A6 encodes the sodium-dependent multivitamin transporter (SMVT), a crucial membrane protein belonging to the sodium:solute symporter family. This transporter is responsible for the Na⁺-dependent uptake of several essential micronutrients, including biotin (vitamin B7), pantothenic acid (vitamin B5), the vitamin-like substance α-lipoic acid, and iodide . The transporter utilizes sodium gradients to facilitate the movement of these substrates across cell membranes. SMVT plays a critical role in normal growth and development through ensuring proper absorption and cellular uptake of these essential micronutrients .

Research has demonstrated that genetic variants in SLC5A6 can lead to systemic deficiencies of these micronutrients, resulting in multisystemic disorders with variable phenotypes including developmental delay, neurological disorders, growth delay, skin and hair changes, metabolic abnormalities, and immunological dysfunctions .

What are the optimal applications for SLC5A6 antibodies in experimental research?

SLC5A6 antibodies have been successfully employed in several experimental techniques:

• Immunohistochemistry (IHC): SLC5A6 antibodies work effectively at dilutions of 1:50-1:200, allowing visualization of the transporter in tissue specimens . This technique is particularly valuable for studying tissue distribution and expression patterns.

• Western blotting: For protein quantification and molecular weight confirmation, SLC5A6 antibodies can be used in combination with β-actin antibodies as loading controls to provide reliable relative expression data .

• Immunofluorescence: SLC5A6 antibodies perform well at concentrations of 0.25-2 μg/mL for subcellular localization studies .

These applications support diverse research objectives including protein expression profiling, cellular distribution assessment, and functional characterization of the SMVT transporter across different experimental systems.

How should researchers validate SLC5A6 antibody specificity in their experimental systems?

Validating antibody specificity is essential for reliable research outcomes. For SLC5A6 antibodies, a multi-step validation approach is recommended:

• Genetic validation: Compare antibody reactivity between wild-type tissues and those with confirmed genetic deletion of SLC5A6. Conditional knockout models, such as the intestinal-specific SMVT knockout mice, provide excellent controls for antibody validation .

• Western blot verification: A properly validated SLC5A6 antibody should detect a protein of the expected molecular weight. SLC5A6 detection should be performed alongside β-actin as a loading control, with quantification using infrared imaging systems for accurate relative expression analysis .

• Peptide competition assay: Pre-incubation of the antibody with the immunogen peptide (e.g., "LNPATIYPVLPKLLSLLPLSCQKRLHCRSYGQDHLDTGLFPEKPRNGVLGDSRDKEAMALDGTAYQGSSSTCILQET") should abolish specific signals .

• Cross-validation: Compare results between different antibody clones or detection methods to confirm consistency of findings.

• Positive and negative tissue controls: Based on known expression patterns, select appropriate positive control tissues with high SLC5A6 expression and negative control tissues with minimal expression.

What methodological approaches can be used to study SLC5A6-mediated transport kinetics?

Several sophisticated methodologies can be employed to characterize SLC5A6-mediated transport:

• Radiotracer uptake assays: Use radiolabeled substrates such as [³H]-biotin or [¹¹C]biotin to measure transport activity in vitro and in vivo . For in vitro studies, cells expressing SLC5A6 (either endogenously or through heterologous expression) are incubated with radiotracer in an appropriate assay buffer, followed by washing and scintillation counting to measure accumulated radioactivity .

• Electrophysiological recordings: Two-electrode voltage clamp techniques in Xenopus oocytes expressing SLC5A6 can directly measure substrate-induced currents. This approach allows real-time assessment of transport activity and characterization of kinetic parameters .

• Fluorescent substrate analogs: Biotin-conjugated fluorescent probes can be utilized to monitor transport activity through fluorescence microscopy or flow cytometry, though care must be taken as structural modifications may alter substrate recognition .

• PET imaging: For in vivo studies, positron emission tomography using [¹¹C]biotin provides a non-invasive method to study biotin trafficking and, by extension, SLC5A6 activity in living subjects .

Each of these methodologies offers distinct advantages and can be selected based on the specific research questions being addressed. For quantitative kinetic analysis, nonlinear regression fitting of concentration-dependent uptake data allows determination of parameters such as Km and Vmax .

How can researchers design effective conditional knockout experiments for SLC5A6?

Designing conditional knockout experiments for SLC5A6 requires careful planning:

• Targeting strategy: Design a targeting vector that places loxP sites flanking critical exons of the SLC5A6 gene. Based on previous successful approaches, placing loxP sites around exons 4-6 has proven effective . The targeting vector should include:

  • Short homology arm (SA): approximately 1.9 kb

  • Long homology arm (LA): approximately 8.4 kb

  • FRT-flanked Neomycin resistance cassette for selection

• Primer design for genotyping: Design specific PCR primers to distinguish between wild-type, floxed, and deleted alleles. For example:

  • For Neo cassette: Forward 5′-AGGGAGAACGTGGACTCTGAAGAG-3′ and reverse 5′-CCAGAGGCCACTTGTGTAGC-3′

  • For loxP sites: Forward 5′-TGCTGGTGTTCCGAATGTGACTTG-3′ and reverse 5′-GCAGCAGGGTTGAGGCAGATAGC-3′

• Cre-driver selection: Choose appropriate Cre-recombinase expressing mouse lines based on research objectives. For intestinal-specific deletion, villin promoter-driven Cre expression has been successfully employed .

• Validation of knockout efficiency:

  • Genomic PCR to confirm presence of Cre-recombinase and loxP sites

  • RT-PCR using tissue-specific mRNA to verify deletion

  • Western blot analysis to confirm protein depletion using validated antibodies

• Experimental controls: Include appropriate controls in all experiments, such as Cre-negative littermates with loxP sites (SLC5A6+/+loxP) and wild-type mice.

Following this approach ensures generation of viable conditional knockout models for studying tissue-specific functions of SLC5A6.

What are the critical factors for optimizing SLC5A6 antibody performance in Western blot applications?

Optimizing SLC5A6 antibody performance in Western blots requires attention to several technical details:

• Sample preparation:

  • Use RIPA buffer containing complete protease inhibitor cocktail for cell/tissue homogenization

  • Isolate the soluble fraction by centrifugation at 8,000 g for 5 minutes

  • Ensure equal protein loading (typically 20-50 μg per lane)

• Gel selection and transfer conditions:

  • 10% polyacrylamide gels provide appropriate resolution for SLC5A6 detection

  • For optimal transfer of membrane proteins, PVDF membranes are preferred over nitrocellulose

• Antibody incubation:

  • Primary antibody: Use affinity-purified SLC5A6 antibodies at manufacturer-recommended dilutions (typically 1:1000 to 1:5000)

  • Consider simultaneous probing with mouse monoclonal β-actin antibody as loading control

  • Secondary antibodies: For dual detection, infrared dye-conjugated secondary antibodies (e.g., anti-rabbit IR 800 dye and anti-mouse IR 680 dye) at 1:25,000 dilution provide quantifiable results

• Detection and quantification:

  • Infrared imaging systems allow precise quantification of relative expression

  • Use appropriate software (e.g., Odyssey application software) to normalize SLC5A6 signals to corresponding β-actin levels

• Troubleshooting common issues:

  • Weak or absent signals: Increase antibody concentration, extend incubation time, or enhance signal with amplification systems

  • High background: Increase blocking time/concentration, use more stringent washing, or decrease antibody concentration

  • Multiple bands: Verify specificity with knockout controls, optimize sample preparation to prevent degradation, or try alternative antibody clones

How can SLC5A6 antibodies contribute to understanding disease mechanisms in patients with SLC5A6 variants?

SLC5A6 antibodies provide valuable tools for investigating pathogenesis of diseases associated with SLC5A6 variants:

• Protein expression analysis: Western blotting using SLC5A6 antibodies can determine if variants affect protein expression levels or stability in patient-derived samples such as fibroblasts or lymphoblasts .

• Subcellular localization studies: Immunofluorescence microscopy with SLC5A6 antibodies can reveal whether pathogenic variants affect the plasma membrane localization of the transporter. For instance, previous studies demonstrated that the p.(Tyr162Cys) and p.(Ser429Gly) missense variants did not affect plasma membrane localization but likely reduced catalytic activity .

• Tissue distribution assessment: Immunohistochemistry on patient biopsy samples can highlight tissue-specific alterations in SLC5A6 expression patterns compared to healthy controls .

• Structure-function correlations: By combining antibody-based protein detection with 3D homology modeling data of SMVT structure (which contains 13 transmembrane helices), researchers can better understand how specific variants disrupt transporter function .

• Therapeutic monitoring: SLC5A6 antibodies can be used to track changes in transporter expression in response to multivitamin supplementation therapy, which has been shown to improve clinical outcomes in patients with SLC5A6 variants .

These approaches contribute to early diagnosis and development of targeted therapeutic interventions, which have demonstrated clinical improvement in affected individuals .

What methodological approaches can researchers employ to study the relationship between SLC5A6 dysfunction and clinical phenotypes?

Investigating connections between SLC5A6 dysfunction and clinical manifestations requires integrated methodological approaches:

• Genetic-functional correlation studies:

  • Compare protein expression and function between different variant types (missense vs. truncating)

  • Correlate variant location within the protein structure with clinical severity

  • Use SLC5A6 antibodies to assess expression and localization in patient-derived cells

• Transport function analysis:

  • Measure uptake of radiolabeled substrates (biotin, pantothenic acid, lipoic acid) in patient cells

  • Quantify substrate-induced currents using electrophysiological techniques

  • Compare transport kinetics between wild-type and variant transporters

• Biomarker assessment:

  • Measure metabolic indicators of vitamin deficiency in patient samples

  • Utilize SLC5A6 antibodies in combination with biomarkers of cellular stress or mitochondrial dysfunction

  • Track changes in biomarker levels in response to supplementation therapy

• Animal model validation:

  • Generate knock-in models with specific patient variants

  • Use conditional knockout models to study tissue-specific effects

  • Apply SLC5A6 antibodies to compare protein expression between models and patient samples

• Treatment response monitoring:

  • Use SLC5A6 antibodies to track potential compensatory changes in transporter expression during supplementation

  • Correlate clinical improvement with molecular/cellular changes

  • Establish personalized supplementation protocols based on variant-specific dysfunction patterns

This integrated approach has proven valuable in establishing that early supplementation with biotin and pantothenic acid can stabilize the clinical picture and potentially prevent life-threatening events in patients with pathogenic SLC5A6 variants .

What controls should be included when performing experiments with SLC5A6 antibodies?

Robust experimental design with SLC5A6 antibodies requires comprehensive controls:

• Negative controls:

  • Tissue/cells with confirmed absence of SLC5A6 expression

  • Conditional knockout samples (e.g., intestinal-specific SMVT KO mouse tissues)

  • Primary antibody omission control to assess secondary antibody specificity

  • Isotype control antibody (rabbit IgG at equivalent concentration)

• Positive controls:

  • Tissues with known high SLC5A6 expression

  • Recombinant SLC5A6 protein

  • Overexpression systems with verified SLC5A6 expression

• Technical controls:

  • Loading controls for Western blots (β-actin recommended)

  • Housekeeping gene controls for RT-PCR

  • Peptide competition assay using the immunogen sequence (LNPATIYPVLPKLLSLLPLSCQKRLHCRSYGQDHLDTGLFPEKPRNGVLGDSRDKEAMALDGTAYQGSSSTCILQET)

• Validation controls:

  • Multiple antibody clones targeting different epitopes

  • Alternative detection methods (e.g., comparing immunohistochemistry with RNA in situ hybridization)

  • Correlation with mRNA expression data

• Experimental treatment controls:

  • Vehicle-only conditions

  • Dose-response validations

  • Time-course studies

Implementing these controls ensures reliable and reproducible results when working with SLC5A6 antibodies across different experimental platforms.

How can researchers investigate the interaction between SLC5A6 and other membrane transporters?

Investigating interactions between SLC5A6 and other membrane transporters requires specialized approaches:

• Co-immunoprecipitation (Co-IP):

  • Use SLC5A6 antibodies to pull down protein complexes

  • Identify interacting partners through mass spectrometry

  • Confirm specific interactions with reciprocal Co-IP experiments

• Proximity ligation assay (PLA):

  • Utilize SLC5A6 antibodies in combination with antibodies against suspected interacting transporters

  • PLA signals indicate protein proximity (<40 nm) in situ

  • Quantify interaction signals across different experimental conditions

• FRET/BRET analysis:

  • Generate fluorescent or bioluminescent fusion proteins

  • Measure energy transfer between SLC5A6 and potential partners

  • Calculate interaction efficiency under various conditions

• Functional interaction studies:

  • Measure substrate transport in systems with manipulated expression of SLC5A6 and other transporters

  • Compare uptake kinetics in single vs. double transfectants

  • Assess how inhibiting one transporter affects the function of others

• Membrane raft isolation:

  • Isolate lipid raft fractions using detergent resistance methods

  • Use SLC5A6 antibodies to detect co-localization with other transporters in specific membrane domains

  • Determine if disrupting membrane organization affects transporter interactions

These methodologies can reveal whether SLC5A6 functions independently or as part of larger transport complexes, providing insights into the regulation of vitamin and micronutrient homeostasis at the cellular level.

What emerging technologies could enhance SLC5A6 research beyond current antibody-based approaches?

Several cutting-edge technologies offer promising avenues for advancing SLC5A6 research:

• CRISPR-Cas9 genome editing:

  • Generate knock-in reporter lines with fluorescent or affinity tags

  • Create precise disease-associated mutations for functional studies

  • Develop high-throughput screening platforms to identify regulators of SLC5A6 expression and function

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize SLC5A6 distribution at nanoscale resolution

  • Live-cell imaging with genetically encoded biosensors to monitor substrate transport in real-time

  • Correlative light and electron microscopy to link SLC5A6 localization with ultrastructural features

• Single-cell analysis:

  • Single-cell RNA-seq to identify cell populations with differential SLC5A6 expression

  • Single-cell proteomics to correlate SLC5A6 protein levels with cellular phenotypes

  • Spatial transcriptomics to map expression patterns within complex tissues

• Novel tracers for in vivo studies:

  • Development of additional PET radiotracers beyond [¹¹C]biotin to study other SLC5A6 substrates

  • Multimodal imaging probes combining PET with optical or MRI contrast

  • Substrate analogs with improved specificity for SLC5A6 over other transporters

• Systems biology approaches:

  • Multi-omics integration to understand SLC5A6 in broader metabolic networks

  • Mathematical modeling of vitamin transport and utilization

  • Network analysis to identify disease-relevant pathways affected by SLC5A6 dysfunction

These technologies complement antibody-based approaches and will provide deeper insights into SLC5A6 biology and pathophysiology.

How can researchers design studies to address the Na⁺ coupling mechanism of SLC5A6?

Investigating the Na⁺ coupling mechanism of SLC5A6 requires specialized experimental designs:

• Site-directed mutagenesis studies:

  • Target conserved residues in transmembrane domains, particularly those in TM4 and TM11 where important residues like Tyr162 and Ser429 are located

  • Create alanine scanning libraries of potential Na⁺ binding sites

  • Generate chimeric transporters with other Na⁺-dependent transporters to identify regions critical for coupling

• Electrophysiological approaches:

  • Two-electrode voltage clamp recordings in Xenopus oocytes expressing wild-type or mutant SLC5A6

  • Measure substrate-induced currents at varying membrane potentials

  • Determine Na⁺:substrate stoichiometry by analyzing current-voltage relationships

  • Investigate potential Na⁺ leak currents in the absence of substrate

• Ion replacement studies:

  • Systematically replace Na⁺ with other cations (Li⁺, K⁺, etc.) in transport assays

  • Measure uptake of radiolabeled substrates under different ionic conditions

  • Determine the Na⁺ concentration dependence of transport activity

• Structural biology approaches:

  • Utilize the 13-transmembrane helix model of SMVT for in silico docking studies

  • Apply molecular dynamics simulations to model Na⁺ and substrate binding events

  • Compare with crystal structures of related transporters in the sodium:solute symporter family

• pH dependence studies:

  • Measure transport activity across a range of extracellular and intracellular pH values

  • Identify potential proton-coupled transport components

  • Explore the interplay between Na⁺ gradients and pH gradients in driving substrate uptake

These approaches can provide valuable insights into the molecular mechanisms underlying SLC5A6-mediated transport and may identify novel targets for therapeutic intervention in related disorders.