SLC51A Antibody

What is SLC51A and why is it important in research?

SLC51A (OSTα) is a 340-amino acid, 7-transmembrane domain protein encoded by the SLC51A gene. It combines with SLC51B (OSTβ) to form a functional heterodimeric transporter, designated as OSTα-OSTβ. This complex is expressed at the basolateral membrane of epithelium in intestine, kidney, liver, testis, and adrenal gland, where it mediates bile acids efflux .

The transporter is essential for intestinal bile acid absorption and dietary lipid absorption. Additionally, OSTα-OSTβ plays a central role in transporting conjugated steroids and related molecules across basolateral membranes of many epithelial cells . Its mechanism involves facilitated diffusion, allowing it to mediate either efflux or uptake depending on the substrates' electrochemical gradient. The importance of this protein in research stems from its implications in liver diseases, including nonalcoholic steatohepatitis, primary biliary cholangitis, and cholestatic drug-induced liver injury .

What applications are SLC51A antibodies suitable for?

SLC51A antibodies are versatile tools suitable for multiple research applications:

• Western blotting (WB): For detecting and quantifying SLC51A protein expression in tissue and cell lysates .

• Immunohistochemistry (IHC): For visualizing the spatial distribution of SLC51A in tissue sections, particularly useful in studying expression patterns in different organs .

• Immunofluorescence (IF): For subcellular localization studies and co-localization with other proteins .

• ELISA: For quantitative detection of SLC51A in various samples .

When selecting an application method, researchers should consider the specific antibody's validated applications. For example, the SLC51A Polyclonal Antibody (PACO47978) is validated for ELISA (recommended dilution 1:2000-1:10000), IHC (1:20-1:200), and IF (1:50-1:200) . Different applications may require different antibody concentrations and experimental conditions for optimal results.

How should SLC51A antibodies be stored and handled to maintain reactivity?

Proper storage and handling of SLC51A antibodies are critical for maintaining their reactivity and specificity:

• Storage temperature: Store antibodies at -20°C as received, unless otherwise specified by the manufacturer .

• Buffer conditions: Most SLC51A antibodies are supplied in PBS buffer with additives such as sodium azide and glycerol that help maintain stability. For example, some are preserved in 0.03% Proclin 300 with 50% Glycerol and 0.01M PBS at pH 7.4 .

• Aliquoting: To avoid repeated freeze-thaw cycles, divide the antibody solution into small aliquots upon first thaw.

• Thawing: Thaw antibodies completely on ice before use and mix gently but thoroughly.

• Working dilutions: Prepare working dilutions fresh each time and use within the same day.

• Stability: Most SLC51A antibodies remain stable for approximately 12 months from the date of receipt when stored properly .

Avoid contamination by using clean pipette tips and sterile containers. Always refer to the manufacturer's specific recommendations, as storage conditions may vary slightly between different antibody preparations.

How do post-translational modifications affect SLC51A detection with antibodies?

Post-translational modifications (PTMs) can significantly impact antibody recognition of SLC51A, potentially leading to variable results across different experimental conditions:

While the search results don't explicitly mention PTMs for SLC51A, membrane proteins commonly undergo modifications including phosphorylation, glycosylation, and ubiquitination that can affect epitope accessibility. When investigating SLC51A in different physiological or pathological contexts, researchers should consider:

• Epitope mapping: Understand which region of SLC51A your antibody targets. For instance, the OriGene antibody targets a synthetic peptide from the middle region of human OSTalpha (sequence: LLMLGPFQYAFLKITLTLVGLFLVPDGIYDPADISEGSTALWINTFLGVS) .

• Sample preparation: Different lysis buffers and conditions may preserve or destroy certain PTMs. For phosphorylation studies, phosphatase inhibitors are essential in extraction buffers.

• Confirmation strategies: Use multiple antibodies targeting different epitopes to confirm results, especially when studying novel conditions that might alter protein modifications.

• Deglycosylation assays: Consider enzymatic treatment of samples to remove glycosylation when investigating potential glycosylation effects on antibody binding.

In hypoxic conditions, for example, HIF-1α has been shown to regulate SLC51A expression , which might coincide with changes in post-translational modifications that could affect antibody detection.

How does the heterodimeric nature of OSTα-OSTβ impact experimental design when using SLC51A antibodies?

The functional unit of SLC51A (OSTα) requires heterodimerization with SLC51B (OSTβ) to form the OSTα-OSTβ complex. This presents unique considerations for experimental design:

• Co-expression analysis: When studying SLC51A function, researchers should simultaneously assess SLC51B expression, as the functional activity depends on both proteins forming a complex .

• Protein interaction studies: Co-immunoprecipitation experiments may require special considerations due to the membrane-embedded nature of the complex.

• Functional studies: Knockdown or overexpression of SLC51A alone may not provide complete insights into transporter function; both subunits should be considered.

• Interpreting localization data: The correct basolateral membrane localization of SLC51A depends on SLC51B expression. Without its partner, SLC51A may show atypical cellular distribution patterns .

• Tissue-specific considerations: The ratio of OSTα to OSTβ expression may vary across different tissues, potentially affecting antibody-based detection methods.

When designing siRNA knockdown experiments (similar to those mentioned in ), researchers should consider the impact on both subunits, even when targeting only one. For functional studies, monitoring both subunits provides more reliable insights into the biological significance of observed changes.

What controls are essential when validating SLC51A antibody specificity for research applications?

Rigorous validation requires multiple controls to ensure antibody specificity:

• Positive tissue controls: Human small intestine and liver tissues show high SLC51A expression and serve as excellent positive controls . Immunohistochemistry of human small intestine tissue has been validated with antibodies like PACO47978 .

• Negative controls:

  • Primary antibody omission

  • Isotype control (rabbit IgG for polyclonal rabbit antibodies)

  • Tissues known to lack SLC51A expression

  • Pre-absorption with immunizing peptide

• Genetic knockdown/knockout controls:

  • siRNA knockdown of SLC51A in cell lines such as Huh7 or HepG2

  • CRISPR-Cas9 knockout cells

  • Analysis of knockdown efficiency using RT-qPCR in parallel with protein detection

• Molecular weight verification:

  • The predicted protein size for SLC51A is 38 kDa , which should be confirmed in Western blot applications

  • Presence of multiple bands may indicate alternative splice variants, degradation products, or post-translational modifications

• Cross-reactivity assessment:

  • Test antibody against related transporters to ensure specificity

  • Consider species cross-reactivity when working with animal models

For RNA-level validation, techniques like those described in using TaqMan Gene Expression Assays (Hs00380895_m1 for human OSTα) with appropriate housekeeping genes (β-actin) can complement protein-level analysis.

What are the optimal protocols for Western blot detection of SLC51A?

Optimizing Western blot protocols for SLC51A detection requires attention to several key parameters:

• Sample preparation:

  • For membrane proteins like SLC51A, detergent selection is critical

  • Tissue homogenization in buffer containing protease inhibitors

  • For comparison with animal models, consider protocols used for rat liver samples in chronic renal failure studies

• Protein loading and separation:

  • Load 20-50 μg of total protein per lane

  • Use 10-12% SDS-PAGE gels for optimal resolution around the 38 kDa range

• Transfer conditions:

  • Wet transfer at 100V for 1 hour or 30V overnight at 4°C is recommended for membrane proteins

  • PVDF membranes may provide better results than nitrocellulose for hydrophobic proteins

• Blocking and antibody incubation:

  • Block membranes in 5% non-fat milk or BSA in PBS-T

  • Incubate with primary SLC51A antibody (dilution as recommended by manufacturer)

  • For horseradish peroxidase-conjugated secondary antibodies, a concentration of 10 ng/ml with 1-hour incubation at room temperature has been effective

• Detection:

  • Enhanced chemiluminescence systems like SuperSignal West Femto Maximum Sensitivity Substrate

  • Use systems like Fusion FX luminescence detector for visualization

• Controls:

  • Include β-actin (Ab8227) as loading control

  • Consider positive control samples from tissues known to express SLC51A (small intestine, liver)

For data analysis, normalize SLC51A signal to your loading control and perform statistical analysis using appropriate tests like one-sample t-test or unpaired t-test as described in previous studies .

How can researchers effectively use SLC51A antibodies to study disease models?

SLC51A antibodies provide valuable tools for investigating various disease models, particularly those related to liver pathology and bile acid metabolism:

• Nonalcoholic steatohepatitis (NASH):

  • IHC analysis of liver biopsies to assess SLC51A expression changes

  • Correlation with disease severity markers

  • Comparison with healthy controls using quantitative Western blot

• Primary biliary cholangitis:

  • Analysis of SLC51A expression in different stages of disease progression

  • Correlation with bile acid levels and cholestatic markers

  • Combination of IHC and IF to study cellular distribution changes

• Cholestatic drug-induced liver injury:

  • In vitro models using HepG2 cells treated with hepatotoxic compounds

  • Monitoring SLC51A expression changes using IF

  • Correlation with bile acid transport activity

• Chronic renal failure model:

  • Following protocols used in Sprague-Dawley rats with 5/6 nephrectomy

  • Immunoblot analysis of liver tissue for SLC51A expression changes

  • Comparison with sham surgery controls

• Hypoxia models:

  • Culture cells (e.g., Huh7) under normoxic or hypoxic conditions

  • Treatment with bile acids like CDCA (50 μM)

  • Analysis of SLC51A expression changes using real-time PCR and Western blot

  • Validation with VEGFa as a hypoxia marker

When studying these models, combine protein expression analysis with functional assays and mRNA quantification for comprehensive characterization. Normalize data appropriately and use statistical methods like t-tests to evaluate significance (p<0.05) .

What approaches can be used to study SLC51A-SLC51B interactions using antibodies?

The functional interaction between SLC51A and SLC51B is central to understanding the heterodimeric transporter. Several approaches can be employed:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate with anti-SLC51A antibody

  • Detect SLC51B in the precipitate using anti-SLC51B antibody

  • Consider membrane protein-specific IP protocols with appropriate detergents

  • Include controls for non-specific binding

• Proximity ligation assay (PLA):

  • Use primary antibodies against SLC51A and SLC51B from different species

  • Apply species-specific PLA probes

  • Visualize protein-protein interactions as fluorescent dots

  • Quantify interaction signals in different cellular compartments

• Dual immunofluorescence:

  • Use antibodies against both subunits labeled with different fluorophores

  • Analyze co-localization patterns using confocal microscopy

  • Calculate Pearson's correlation coefficient for quantitative assessment

  • Particularly useful in polarized cells to confirm basolateral membrane localization

• FRET/BRET analysis:

  • Tag SLC51A and SLC51B with appropriate fluorophores/luminophores

  • Measure energy transfer as an indicator of protein proximity

  • Control for expression levels of both proteins

• Cross-linking studies:

  • Apply membrane-permeable cross-linkers to stabilize protein-protein interactions

  • Detect complexes using SLC51A antibodies in Western blot

  • Identify higher molecular weight bands representing the heterodimer

When interpreting these studies, consider that the interaction may be affected by experimental conditions, disease states, or regulatory factors like HIF-1α, which has been shown to regulate SLC51A expression .

How can inconsistent SLC51A antibody results be resolved across different experimental conditions?

Inconsistencies in SLC51A antibody performance can stem from multiple factors. Here's a systematic approach to troubleshooting:

• Antibody-specific factors:

  • Verify antibody specificity with positive and negative controls

  • Ensure proper storage and handling as described in section 1.3

  • Consider lot-to-lot variations; test new lots against previous ones

  • For polyclonal antibodies like those described in , batch variations may occur

• Sample preparation issues:

  • Standardize extraction methods for consistent results

  • For membrane proteins like SLC51A, detergent selection is critical

  • Complete solubilization may require specialized buffers

  • Protect samples from proteolytic degradation with inhibitor cocktails

• Technical variables:

  • Standardize protein quantification methods

  • Maintain consistent blocking conditions and incubation times

  • Optimize antibody concentrations for each application

  • For IF, fixation methods significantly impact epitope accessibility

  • For IHC, antigen retrieval methods may need optimization

• Biological variables:

  • SLC51A expression varies by tissue; intestine and liver show high expression

  • Expression is regulated by factors including hypoxia via HIF-1α

  • Bile acids like CDCA (50 μM) can affect expression levels

  • Consider cell confluence and culture conditions for in vitro studies

• Analytical approach:

  • Apply multiple antibodies targeting different epitopes

  • Complement protein detection with mRNA analysis

  • Use quantitative methods with appropriate normalization

  • Statistical analysis should account for biological variability

If inconsistencies persist, consider contacting the antibody manufacturer for technical support, as they may have additional optimization protocols not included in standard documentation.

What factors should be considered when interpreting SLC51A expression changes in disease states?

Interpreting SLC51A expression changes in disease contexts requires careful consideration of multiple factors:

• Baseline expression patterns:

  • SLC51A shows tissue-specific expression, predominantly in intestine, kidney, liver, testis, and adrenal gland

  • Expression levels vary between different cell types within the same organ

  • Basolateral membrane localization is characteristic and functionally relevant

• Regulatory mechanisms:

  • Transcriptional regulation by factors such as HIF-1α under hypoxic conditions

  • Bile acid-mediated regulation, with compounds like CDCA affecting expression

  • Consider enterohepatic circulation effects on expression patterns

• Relationship with SLC51B:

  • Functional activity requires both subunits of the heterodimer

  • Changes in SLC51A may not correlate with functional changes if SLC51B is unchanged

  • Ratio between subunits may vary in disease states

• Disease-specific considerations:

  • In liver diseases like nonalcoholic steatohepatitis and primary biliary cholangitis, increased expression has been observed

  • Consider compensatory mechanisms in response to altered bile acid homeostasis

  • Changes may reflect adaptation rather than primary pathology

• Methodological validation:

  • Confirm protein changes with mRNA quantification using validated assays (e.g., TaqMan Gene Expression Assay Hs00380895_m1)

  • Use appropriate controls (e.g., VEGFa as hypoxia marker)

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

• Statistical analysis:

  • Apply appropriate statistical tests (t-test for two-group comparisons)

  • Consider p<0.05 as statistically significant

  • Report both statistical significance and biological relevance

For comprehensive interpretation, correlate SLC51A expression changes with functional assays, clinical parameters, and other related transporters involved in bile acid homeostasis.

How do different detection systems affect the sensitivity and specificity of SLC51A antibody-based assays?

Detection systems significantly impact the performance of SLC51A antibody-based assays:

• Western blot detection systems:

  • Enhanced chemiluminescence systems like SuperSignal West Femto Maximum Sensitivity Substrate provide high sensitivity

  • Luminescence detector systems (e.g., Fusion FX) offer quantitative capabilities

  • Fluorescence-based Western blot systems may provide better linear range

  • Digital imaging systems enable more accurate quantification than film-based methods

• Immunohistochemistry detection:

  • Enzyme-based systems (HRP/DAB) provide permanent staining but lower sensitivity

  • Tyramide signal amplification can enhance sensitivity for low-abundance proteins

  • Chromogenic multiplex IHC allows simultaneous detection of SLC51A and related proteins

  • Digital pathology platforms enable quantitative analysis of expression patterns

• Immunofluorescence considerations:

  • Direct vs. indirect detection methods affect signal strength

  • Fluorophore selection impacts sensitivity, with newer generations providing better signal-to-noise ratio

  • Confocal microscopy offers superior resolution for membrane localization studies

  • Super-resolution microscopy may reveal substructures not visible with conventional microscopy

• ELISA detection options:

  • Colorimetric vs. fluorometric vs. chemiluminescent substrates differ in sensitivity

  • Signal amplification systems can lower detection limits

  • Sandwich ELISA formats typically provide better specificity than direct coating methods

• Technical considerations across methods:

  • Signal-to-noise ratio optimization is critical for membrane proteins

  • Background reduction strategies (e.g., blocking optimizations)

  • Appropriate negative controls for each detection system

  • Calibration standards for quantitative applications

When selecting a detection system, consider not only sensitivity requirements but also the dynamic range needed, as SLC51A expression can vary significantly between different experimental conditions, such as hypoxia versus normoxia or CDCA treatment versus vehicle control .

What emerging technologies might enhance SLC51A antibody-based research?

Several emerging technologies hold promise for advancing SLC51A antibody-based research:

• Single-cell proteomics:

  • Analysis of SLC51A expression at the single-cell level to identify cell-specific expression patterns

  • Correlation with functional bile acid transport in individual cells

  • Integration with single-cell transcriptomics data

• Advanced imaging techniques:

  • Super-resolution microscopy for detailed subcellular localization

  • Live-cell imaging to track dynamic changes in SLC51A-SLC51B interactions

  • Expansion microscopy for enhanced visualization of membrane protein organization

• Spatial proteomics:

  • Techniques like Imaging Mass Cytometry or CODEX for multiplexed protein detection

  • Spatial transcriptomics integration to correlate protein and mRNA distributions

  • Analysis of SLC51A expression in the tissue microenvironment context

• Antibody engineering approaches:

  • Development of recombinant antibodies with improved specificity

  • Single-domain antibodies for accessing restricted epitopes

  • Bispecific antibodies targeting both SLC51A and SLC51B simultaneously

• Functional antibody applications:

  • Conformation-specific antibodies to detect active versus inactive transporter states

  • Antibody-based biosensors for real-time monitoring of bile acid transport

  • Antibody-drug conjugates for targeting cells with aberrant SLC51A expression

• Computational approaches:

  • AI-assisted image analysis for quantitative assessment of expression patterns

  • Predictive modeling of antibody-epitope interactions

  • Systems biology integration of SLC51A function in enterohepatic circulation

These technologies could particularly enhance understanding of SLC51A's role in disease models and its regulation under conditions like hypoxia, where HIF-1α has been shown to influence its expression .

How can SLC51A antibodies be used to investigate regulatory mechanisms of transporter expression?

SLC51A antibodies provide valuable tools for exploring the complex regulatory mechanisms governing this important transporter:

• Transcription factor studies:

  • Investigate HIF-1α regulation of SLC51A using approaches similar to the EMSA (Electrophoretic Mobility Shift Assay) experiments described in

  • Combine with ChIP assays to confirm direct binding to the SLC51A promoter

  • Correlate protein expression changes with transcription factor activity

• Signal transduction pathway analysis:

  • Use pharmacological inhibitors or genetic approaches to target specific pathways

  • Monitor SLC51A protein expression changes using Western blot

  • Perform IF to assess changes in subcellular localization

• Post-transcriptional regulation:

  • Investigate miRNA-mediated regulation by correlating miRNA and SLC51A expression

  • RNA-protein interaction studies to identify regulatory RNA-binding proteins

  • Compare mRNA and protein levels to identify post-transcriptional control points

• Post-translational modification analysis:

  • Phosphorylation-specific antibodies to detect regulatory modifications

  • Mass spectrometry-based approaches to identify novel modifications

  • Correlation of modifications with functional activity

• Bile acid-mediated regulation:

  • Treatment with bile acids like CDCA (50 μM) as described in

  • Time-course and dose-response studies using Western blot

  • Combine with functional transport assays to correlate expression and activity

• Oxygen-dependent regulation:

  • Compare normoxic and hypoxic conditions as described in

  • Monitor SLC51A expression along with validated hypoxia markers like VEGFa

  • Investigate the mechanism through HIF-1α binding studies

For these studies, combining antibody-based protein detection with mRNA quantification using validated assays (e.g., TaqMan Gene Expression Assay Hs00380895_m1 for human OSTα) provides comprehensive insights into regulatory mechanisms at multiple levels.