SLC51B Antibody

What is SLC51B and what is its functional significance in cellular transport mechanisms?

SLC51B (Organic solute transporter subunit beta) is a 128-amino acid, single-transmembrane domain protein with a calculated molecular weight of approximately 14 kDa. It functions as an essential component of the OSTα-OSTβ heterodimeric complex . This complex serves as the intestinal basolateral transporter responsible for bile acid export from enterocytes into portal blood. Functionally, SLC51B modulates SLC51A (OSTα) glycosylation, membrane trafficking, and stability .

The OSTα-OSTβ complex efficiently transports major bile acid species, particularly taurocholate, and shows preferential transport of taurine conjugates compared to glycine-conjugated bile acids. Beyond bile acids, the complex transports steroids (e.g., estrone 3-sulfate, dehydroepiandrosterone 3-sulfate) and eicosanoids such as prostaglandin E2, thus playing a significant role in the enterohepatic circulation of sterols .

What is the expression pattern of SLC51B across different tissues and cellular compartments?

SLC51B exhibits a tissue-specific expression pattern with notable variations in expression levels:

At the subcellular level, SLC51B is primarily localized to the lateral and basal membranes of ileal enterocytes . This specific localization is critical for its function in the enterohepatic circulation of bile acids and sterols. Immunohistochemistry studies using anti-SLC51B antibodies consistently demonstrate this characteristic basolateral membrane localization pattern in epithelial cells of the small intestine, kidney, and liver .

What criteria should be considered when selecting an SLC51B antibody for specific experimental applications?

When selecting an SLC51B antibody, researchers should evaluate several critical parameters:

• Target Epitope: Consider antibodies targeting different regions of SLC51B:

  • N-terminal region (e.g., AA 1-35)

  • Central region (e.g., AA 57-128)

  • C-terminal region

  The search results indicate that antibodies targeting AA 57-128 are frequently used for multiple applications .

• Antibody Format:

  • Unconjugated antibodies for maximum flexibility

  • Fluorophore-conjugated antibodies (FITC, AbBy Fluor® 488, Cy3, etc.) for direct visualization

  • Enzyme-conjugated antibodies (HRP, etc.) for enhanced sensitivity in certain applications

• Validated Applications: Ensure the antibody has been validated for your specific application:

• Species Reactivity: Verify cross-reactivity with your species of interest. Many SLC51B antibodies react with human samples, while some also cross-react with mouse and rat orthologs .

What validation strategies can confirm the specificity and reliability of SLC51B antibodies?

Methodical validation is essential to ensure experimental reproducibility and data reliability:

• Multiple Antibody Approach: Compare results using antibodies targeting different SLC51B epitopes to confirm consistent detection patterns.

• Positive Control Tissues: Human small intestine tissue serves as an optimal positive control due to high SLC51B expression in ileal enterocytes .

• Knockout/Knockdown Validation: Compare antibody signals in:

  • Wildtype samples

  • Samples with genetic SLC51B knockdown

  • Samples from SLC51B knockout models (if available)

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide before application to samples. Signal reduction confirms specific binding.

• Western Blot Validation: Confirm detection of a single band at the expected molecular weight (~14-15 kDa) .

• Validation Reporting: Document all validation outcomes according to standards such as those proposed by the International Working Group for Antibody Validation (IWGAV).

How can SLC51B antibodies be optimized for multi-color immunofluorescence studies with OSTα?

To investigate the co-localization and interaction of SLC51B with SLC51A (OSTα), implement the following methodological approach:

• Antibody Selection: Choose antibodies raised in different host species:

  • Anti-SLC51B (rabbit polyclonal)

  • Anti-SLC51A (mouse monoclonal)

• Fluorophore Selection: Select fluorophores with minimal spectral overlap:

  • Anti-SLC51B: AbBy Fluor® 488 (excitation/emission: 499/515nm)

  • Anti-SLC51A: AbBy Fluor® 594 or AbBy Fluor® 647

• Sample Preparation:

  • Fix tissues with 4% paraformaldehyde

  • For membrane proteins like SLC51B, avoid harsh permeabilization

  • Perform antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0)

• Sequential Staining Protocol:

  • Block with 5% normal serum from the species of secondary antibodies

  • Incubate with anti-SLC51B antibody overnight at 4°C

  • Apply fluorophore-conjugated anti-rabbit secondary antibody

  • Block again with serum

  • Incubate with anti-SLC51A antibody

  • Apply differently-labeled secondary antibody

  • Counterstain nuclei with DAPI

• Controls:

  • Single-antibody controls to assess bleed-through

  • Secondary-only controls to assess non-specific binding

  • Positive control tissues with known co-expression

• Quantitative Analysis: Apply Pearson's or Mander's correlation coefficient to quantify co-localization.

What experimental approaches can elucidate the regulatory mechanisms controlling SLC51B expression in hypoxic conditions?

Based on research demonstrating hypoxia-induced changes in OSTα-OSTβ expression , the following methodology can be implemented:

• In Vitro Hypoxia Model Setup:

  • Culture cells in hypoxic chambers (1-3% O₂)

  • Use chemical hypoxia mimetics (e.g., CoCl₂, DFO)

  • Include VEGFa expression as a hypoxia marker control

• Transcriptional Analysis:

  • Perform RT-qPCR using validated SLC51B primers

  • Use TaqMan Gene Expression Assays (e.g., Hs00418306_m1 for human OSTβ)

  • Normalize to stable reference genes (e.g., β-actin)

• Protein Expression Analysis:

  • Western blot using anti-SLC51B antibodies

  • Immunofluorescence to assess subcellular localization changes

• HIF-1α Binding Assessment:

  • Electrophoretic Mobility Shift Assay (EMSA) with HIF-1α antibodies (e.g., sc13515)

  • Chromatin Immunoprecipitation (ChIP) to identify HIF-1α binding sites in the SLC51B promoter

• Functional Reporter Assays:

  • Construct luciferase reporters containing the SLC51B promoter

  • Evaluate promoter activity under normoxic vs. hypoxic conditions

  • Include mutated HIF binding sites as controls

How can SLC51B antibodies be employed to investigate the functional relationship between HNF1A and SLC51B in renal proximal tubule cells?

Research has established that SLC51B is a target of HNF1A involved in estrone sulfate (E1S) uptake in proximal tubule cells . To investigate this relationship:

• HNF1A-SLC51B Expression Correlation:

  • Co-immunostaining with anti-HNF1A and anti-SLC51B antibodies

  • Western blot analysis of HNF1A and SLC51B in wildtype vs. HNF1A-depleted cells

• ChIP-Sequencing Approach:

  • Perform HNF1A ChIP-Seq on human pluripotent stem cell-derived kidney organoids

  • Identify HNF1A binding sites in the SLC51B promoter region

  • Validate binding with targeted ChIP-qPCR

• Functional E1S Transport Assays:

  • Use radiolabeled or fluorescent-labeled E1S substrates

  • Compare uptake in control vs. HNF1A-depleted cells

  • Block transport with specific inhibitors to confirm specificity

• MODY3 Patient Model:

  • Analyze urinary E1S levels in patients with MODY3 (HNF1A mutations)

  • Correlate with SLC51B expression in patient-derived samples

  • Develop iPSC-derived kidney organoids from MODY3 patients

How should researchers address inconsistent or unexpected SLC51B antibody staining patterns?

When encountering unexpected staining patterns with SLC51B antibodies, implement this systematic troubleshooting approach:

• Antibody Validation Reassessment:

  • Verify antibody specificity using positive and negative controls

  • Test multiple antibodies targeting different epitopes

  • Confirm antibody performance with the supplier's validation data

• Sample-Specific Considerations:

  • Fixation artifacts: SLC51B is a membrane protein sensitive to overfixation

  • Antigen retrieval: Compare TE buffer pH 9.0 with citrate buffer pH 6.0

  • Sample storage conditions: Membrane proteins can degrade during improper storage

• Technical Optimization:

  • Titrate antibody concentration (typically 1:50-1:500 for IHC applications)

  • Modify blocking conditions to reduce background

  • Adjust incubation time and temperature

• Biological Interpretation Challenges:

  • SLC51B expression varies considerably between tissues

  • Pathological conditions may alter expression patterns

  • Consider post-translational modifications affecting epitope accessibility

• Western Blot Correlation:

  • Confirm protein expression by Western blot

  • Verify the molecular weight (~14-15 kDa)

  • Check for potential degradation products

What methodological approaches can resolve difficulties in detecting low-abundance SLC51B in non-intestinal tissues?

Detecting SLC51B in tissues with lower expression levels requires enhanced sensitivity approaches:

• Signal Amplification Techniques:

  • Tyramide Signal Amplification (TSA) for IHC/IF applications

  • Enhanced chemiluminescence systems for Western blot

  • Biotin-streptavidin amplification systems

• Sample Enrichment Strategies:

  • Membrane protein fractionation to concentrate SLC51B

  • Immunoprecipitation before Western blotting

  • Use detergents optimized for membrane protein extraction (e.g., CHAPS, DDM)

• Advanced Detection Systems:

  • Multiplexed fluorescence with spectral unmixing

  • Proximity ligation assay (PLA) to detect SLC51B-SLC51A complexes

  • Super-resolution microscopy techniques

• Alternative Assay Systems:

  • Ultrasensitive ELISA with optimized antibody pairs

  • Droplet Digital PCR for transcript quantification

  • Mass spectrometry-based proteomics approaches

How can SLC51B antibodies facilitate investigation of the pathophysiological consequences of SLC51B deficiency?

Studies have identified SLC51B deficiency in patients with congenital diarrhea and altered bile acid metabolism . To investigate these conditions:

• Clinical Sample Analysis:

  • Immunohistochemical staining of intestinal biopsies from patients

  • Western blot confirmation of SLC51B protein absence/reduction

  • Correlation with clinical parameters (bile acid malabsorption, steatorrhea)

• Functional Transport Studies:

  • Ex vivo transport assays using patient-derived intestinal organoids

  • Comparison with healthy control samples

  • Rescue experiments through SLC51B reconstitution

• Genotype-Phenotype Correlation:

  • Identify specific mutations (e.g., p.F27fs frameshift mutation)

  • Generate corresponding constructs for functional studies

  • Compare effects of different mutations on protein expression and function

• Translational Research Applications:

  • Develop diagnostic antibody panels for SLC51B deficiency

  • Establish prognostic markers for disease progression

  • Identify potential therapeutic targets within the bile acid transport pathway

What methodological considerations are important when using SLC51B antibodies to study the OSTα-OSTβ complex in non-traditional tissues?

While the OSTα-OSTβ complex is well-characterized in intestine and liver, investigating its presence and function in non-traditional tissues requires specialized approaches:

• Tissue-Specific Optimization:

  • Brain: Use specialized fixation to preserve membrane proteins across blood-brain barrier

  • Adrenal gland: Optimize antigen retrieval for steroidogenic cells

  • Kidney: Segment-specific analysis (proximal tubule vs. other segments)

• Co-Expression Analysis:

  • Multiplex immunofluorescence with tissue-specific markers

  • Single-cell analysis techniques to identify specific cell populations expressing SLC51B

  • Spatial transcriptomics coupled with protein detection

• Functional Assessment in Non-Traditional Tissues:

  • Transport assays adapted for neurosteroids in brain tissue

  • E1S uptake studies in kidney proximal tubule cells

  • Correlation with tissue-specific substrates

• Species Considerations:

  • Verify antibody cross-reactivity with the species of interest

  • Account for potential differences in expression patterns between species

  • Use species-specific positive control tissues