SLC51A Antibody, HRP conjugated

What is SLC51A and why is it a significant research target?

SLC51A encodes the alpha subunit of the heteromeric organic solute transporter alpha-beta (OSTα-OSTβ), which functions as a critical intestinal basolateral transporter responsible for bile acid export from enterocytes into portal blood . This transporter plays an essential role in the enterohepatic circulation of bile acids, significantly impacting lipid metabolism and cholesterol homeostasis . The dysregulation of SLC51A has been linked to several liver diseases, including cholestasis and non-alcoholic fatty liver disease, making it an important target for therapeutic interventions . Research into SLC51A function provides valuable insights into bile acid transport mechanisms and potential treatments for associated disorders.

What advantages does HRP conjugation offer in SLC51A antibody applications?

HRP (Horseradish Peroxidase) conjugation offers significant advantages in experimental applications by enabling direct enzymatic detection without requiring a secondary antibody step . This conjugation reduces experimental time, minimizes background noise, and decreases potential cross-reactivity issues that might arise with separate primary and secondary antibody incubations . The HRP enzyme catalyzes colorimetric, chemiluminescent, or fluorescent reactions depending on the substrate used, providing flexible detection options across multiple experimental platforms . The recommended dilution for HRP-conjugated SLC51A antibodies ranges from 1:100-500 for Western blotting to 1:1000 for ELISA applications, allowing for optimization based on specific experimental requirements .

What are the standard applications for SLC51A antibody, HRP conjugated?

SLC51A antibody with HRP conjugation can be utilized across multiple experimental platforms including:

• Western Blotting: Typically used at 1:100-500 dilution for direct detection of SLC51A protein expression in tissue or cell lysates .

• ELISA: Employed at approximately 1:1000 dilution for quantitative measurement of SLC51A in solution samples .

• Immunohistochemistry (IHC): Applied at 1:20-200 dilution to visualize SLC51A expression patterns in tissue sections, particularly in intestinal and liver tissues where expression is highest .

• Immunofluorescence (IF): Used at 1:50-200 dilution, often in hepatocyte cell lines like HepG2 to determine subcellular localization .

The direct HRP conjugation eliminates the need for secondary antibody incubation steps, streamlining these experimental procedures significantly .

What sample types can be effectively analyzed using SLC51A antibody, HRP conjugated?

SLC51A antibody with HRP conjugation has demonstrated effective detection across multiple sample types including:

• Human Tissue Samples: Particularly effective in small intestine sections, where SLC51A is predominantly expressed at the basolateral membrane of enterocytes .

• Hepatic Cell Lines: Successfully applied in HepG2 cells for immunofluorescence applications, revealing subcellular localization patterns .

• Serum Samples: Can be utilized in ELISA-based detection methods for quantifying circulating levels in experimental bile acid transport studies .

• Recombinant Protein Preparations: Valuable for validation studies using purified recombinant SLC51A proteins .

The antibody shows specific reactivity with human samples according to manufacturer specifications, making it particularly suitable for translational research involving human tissues and cell lines .

How can SLC51A antibody, HRP conjugated be effectively used to investigate bile acid transport mechanisms?

To effectively investigate bile acid transport mechanisms using SLC51A antibody with HRP conjugation:

• Comparative Expression Analysis: Use Western blotting (1:100-500 dilution) to quantify SLC51A expression across different experimental conditions, such as comparing normal versus cholestatic liver samples . This approach allows researchers to establish correlations between transporter expression and bile acid metabolism dysregulation.

• Co-localization Studies: Combine SLC51A antibody-HRP with other markers of bile acid transport (such as SLC51B antibodies) in immunofluorescence experiments to visualize the heterodimeric complex formation at the basolateral membrane . This method reveals insights into the trafficking and membrane localization of the complete transporter complex.

• Functional Correlation: After measuring bile acid profiles using mass spectrometry (as seen in the taurine-conjugated/unconjugated bile acid ratio data), correlate these profiles with SLC51A expression levels detected via Western blotting or immunohistochemistry . This integrated approach helps establish direct relationships between transporter expression and functional outcomes.

• Knockout Validation: Compare antibody signal in wild-type versus SLC51A knockout models to confirm specificity and to investigate compensatory mechanisms in bile acid transport .

The methodological workflow should include appropriate controls, particularly negative controls omitting the primary antibody and positive controls using tissues known to express high levels of SLC51A (small intestine and liver samples) .

What are the optimal conditions for Western blot analysis using SLC51A antibody, HRP conjugated?

For optimal Western blot analysis using SLC51A antibody with HRP conjugation:

• Sample Preparation:

  • Use RIPA buffer supplemented with protease inhibitors for efficient protein extraction

  • Load 20-50 μg of total protein per lane

  • Include reducing agents (β-mercaptoethanol) in the sample buffer to properly denature the transmembrane SLC51A protein

• Gel Electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal separation of SLC51A (expected molecular weight range)

  • Include positive control samples from tissues known to express SLC51A (small intestine or liver extracts)

• Transfer and Blocking:

  • Transfer to PVDF membrane at 100V for 60-90 minutes

  • Block with 5% non-fat dry milk in TBST for at least 1 hour at room temperature

• Antibody Incubation:

  • Dilute SLC51A-HRP antibody at 1:100-500 in blocking buffer

  • Incubate overnight at 4°C for maximum sensitivity and specificity

  • Wash extensively with TBST (at least 3 × 10 minutes)

• Detection:

  • Use enhanced chemiluminescence (ECL) substrate optimized for HRP

  • Expose to X-ray film or use digital imaging systems

  • For quantitative analysis, include housekeeping protein controls

This optimized protocol minimizes background while maximizing specific signal, allowing for reliable detection of SLC51A protein in complex biological samples.

How can researchers troubleshoot non-specific binding or weak signals when using SLC51A antibody, HRP conjugated?

When troubleshooting non-specific binding or weak signals with SLC51A antibody-HRP:

• For Non-specific Binding:

  • Increase Blocking Time/Concentration: Extend blocking to 2 hours or increase BSA/milk concentration to 5-7%

  • Optimize Antibody Dilution: Test serial dilutions beyond the recommended 1:100-500 range to find the optimal concentration

  • Add Detergent: Increase Tween-20 concentration in wash buffer to 0.1-0.2%

  • Pre-absorb Antibody: Incubate with non-target tissue lysate to remove cross-reactive antibodies

  • Validate Specificity: Include SLC51A-knockout or siRNA-knockdown samples as negative controls

• For Weak Signals:

  • Increase Protein Loading: Load up to 50-70 μg of total protein

  • Optimize Extraction: Use membrane protein enrichment methods (e.g., Triton X-114 phase separation)

  • Reduce Antibody Dilution: Test more concentrated antibody solutions (1:50-1:100)

  • Extended Exposure: Increase ECL substrate incubation time and imaging exposure

  • Enhance Detection: Switch to more sensitive substrates like SuperSignal West Femto

  • Verify Target Expression: Confirm SLC51A expression in your sample type with qPCR before protein analysis

• Technical Optimizations:

  • Sample Preparation: Prevent protein degradation by using fresh samples and maintaining cold temperatures

  • Transfer Efficiency: Verify complete protein transfer using reversible total protein stains

  • Membrane Selection: PVDF membranes may provide better protein retention than nitrocellulose

  • HRP Activity: Ensure the antibody has been stored properly to maintain HRP enzymatic activity

Systematic implementation of these troubleshooting steps can significantly improve both specificity and sensitivity when working with SLC51A antibody-HRP conjugates.

What are the critical differences in detection sensitivity between SLC51A antibody-HRP and unconjugated primary antibodies with secondary detection?

The critical differences in detection sensitivity between directly HRP-conjugated SLC51A antibodies and unconjugated primary antibodies with secondary detection include:

• Signal Amplification:

  • Secondary Detection System: Provides signal amplification as multiple secondary antibodies can bind to each primary antibody, potentially offering 2-10 fold signal enhancement

  • Direct HRP Conjugation: Limited to 1:1 ratio of enzyme to antibody, resulting in lower theoretical signal ceiling

• Background Noise:

  • Secondary Detection: May introduce additional background from non-specific binding of secondary antibodies

  • Direct HRP Conjugation: Typically produces cleaner backgrounds with reduced non-specific signals

• Experimental Variables:

  • Secondary Detection: Introduces additional optimization steps (secondary antibody dilution, incubation time)

  • Direct HRP Conjugation: Reduces experimental variables, leading to more consistent results across replicates

• Experimental Timeline:

  • Secondary Detection: Requires additional incubation (1-2 hours) and wash steps

  • Direct HRP Conjugation: Streamlines protocols by eliminating secondary antibody steps, reducing total experimental time by 2-3 hours

• Cross-Reactivity Issues:

  • Secondary Detection: Potential cross-reactivity with endogenous immunoglobulins in samples

  • Direct HRP Conjugation: Eliminates cross-reactivity concerns related to secondary antibodies

For SLC51A detection specifically, the HRP-conjugated antibody provides sufficient sensitivity for standard Western blot applications (1:100-500 dilution range) while offering advantages in experimental simplicity and background reduction .

How can SLC51A antibody, HRP conjugated be used to investigate the relationship between SLC51A and SLC51B in the functional transporter complex?

To investigate the relationship between SLC51A and SLC51B in the functional transporter complex using SLC51A antibody-HRP:

• Co-expression Analysis:

  • Perform dual immunostaining using SLC51A antibody-HRP and an SLC51B antibody with a different reporter system

  • Quantify the co-localization coefficient in different cell types or under various experimental conditions

  • Compare expression ratios in normal versus disease models to understand stoichiometric requirements

• Protein-Protein Interaction Studies:

  • Use SLC51A antibody-HRP in co-immunoprecipitation experiments followed by detection of SLC51B

  • Apply proximity ligation assays (PLA) to visualize and quantify SLC51A-SLC51B interaction events in situ

  • Employ FRET-based approaches using SLC51A antibody-HRP as donor and fluorescently-labeled SLC51B antibody as acceptor

• Functional Correlation Analysis:

  • Correlate the expression levels of both subunits (detected via respective antibodies) with transporter activity measurements

  • Investigate how alterations in SLC51A glycosylation impact complex formation and function

  • Examine how SLC51B modulates SLC51A trafficking and stability using pulse-chase experiments with immunodetection

• Knockdown/Knockout Experiments:

  • Use SLC51A antibody-HRP to quantify how SLC51B knockdown affects SLC51A expression, glycosylation, and subcellular localization

  • Similarly, investigate how SLC51A manipulation affects SLC51B using specific antibodies

• Disease Model Applications:

  • Apply co-detection methods in cholestasis models to understand how pathological conditions affect the stoichiometry and localization of the complex

  • Investigate the correlation between SLC51A/SLC51B expression ratio and bile acid profiles in hepatic diseases

This multifaceted approach leverages the specificity of the SLC51A antibody-HRP to generate insights into the molecular determinants of OSTα-OSTβ complex formation, stability, and function in both physiological and pathological contexts.

How can SLC51A antibody, HRP conjugated be utilized in investigating liver disease mechanisms?

SLC51A antibody with HRP conjugation can be strategically employed to investigate liver disease mechanisms through multiple experimental approaches:

• Comparative Expression Analysis in Disease Models:

  • Quantify SLC51A expression via Western blotting (1:100-500 dilution) in tissue samples from various liver disease models

  • Compare expression patterns between healthy controls and pathological states such as cholestasis, non-alcoholic fatty liver disease (NAFLD), and biliary atresia

  • Correlate expression changes with disease progression markers and bile acid profiles

• Cellular Localization in Pathological States:

  • Perform immunohistochemistry (1:20-200 dilution) on liver biopsies to examine changes in subcellular localization during disease

  • Investigate potential internalization or mislocalization of SLC51A in response to cholestatic injury

  • Combine with markers of cellular stress to understand the relationship between ER stress, inflammation, and transporter trafficking

• Functional Correlations:

  • Correlate SLC51A expression detected by the antibody with altered bile acid ratios, particularly focusing on taurine-conjugated to unconjugated bile acid ratios which show significant elevation in disease states

  • Analyze the relationship between SLC51A expression and specific bile acid species that show pathological accumulation (as shown in the table from source where ratios like TCA/CA increased from 0.15±0.14 to 1.63±1.33 in disease models)

• Therapeutic Intervention Studies:

  • Use the antibody to monitor SLC51A expression changes in response to therapeutic interventions (such as the ABZ treatment mentioned in source )

  • Track normalization of expression patterns following successful treatment

• Mechanistic Investigations:

  • Apply the antibody in conjunction with modulators of bile acid synthesis, transport, or signaling to elucidate regulatory mechanisms

  • Investigate relationships between FXR activation, SLC51A expression, and disease phenotypes

This multifaceted approach using SLC51A antibody-HRP can reveal critical insights into the role of this transporter in liver disease pathogenesis, potentially identifying new therapeutic targets or biomarkers for clinical applications.

What experimental protocols are recommended for SLC51A detection in patient-derived samples?

For detection of SLC51A in patient-derived samples using HRP-conjugated antibodies, the following optimized protocols are recommended:

• Liver Biopsy Immunohistochemistry:

  • Fix tissue in 10% neutral buffered formalin for 24 hours

  • Process, embed in paraffin, and section at 4-5 μm thickness

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes

  • Block endogenous peroxidase using 3% H₂O₂ for 10 minutes

  • Apply protein block (5% normal goat serum) for 1 hour

  • Incubate with SLC51A antibody-HRP at 1:20-1:200 dilution overnight at 4°C

  • Develop with DAB substrate and counterstain with hematoxylin

  • Score expression patterns using established pathological criteria

• Serum ELISA for Circulating SLC51A:

  • Coat high-binding microplates with capture antibody against SLC51A

  • Block with 5% BSA in PBS for 2 hours at room temperature

  • Apply patient serum samples (diluted 1:5 to 1:20)

  • Incubate with SLC51A antibody-HRP (1:1000 dilution)

  • Develop with TMB substrate and measure absorbance at 450 nm

  • Quantify against standard curve of recombinant SLC51A protein

• Western Blot Analysis of Tissue Extracts:

  • Homogenize tissue samples in RIPA buffer with protease inhibitor cocktail

  • Centrifuge at 14,000g for 15 minutes at 4°C

  • Quantify protein concentration using BCA assay

  • Load 40-50 μg protein per lane on 10-12% SDS-PAGE gels

  • Transfer to PVDF membrane at 100V for 90 minutes

  • Block with 5% non-fat milk in TBST for 1 hour

  • Apply SLC51A antibody-HRP at 1:100-500 dilution overnight at 4°C

  • Wash extensively with TBST (3 × 10 minutes)

  • Develop with enhanced chemiluminescence substrate

• Isolation and Analysis of Patient-Derived Enterocytes:

  • Obtain intestinal biopsy samples during endoscopic procedures

  • Isolate enterocytes using EDTA-based isolation methods

  • Prepare membrane fractions via ultracentrifugation

  • Analyze SLC51A expression via Western blotting as described above

  • Compare expression profiles between patient groups with different bile acid-related disorders

These protocols have been optimized to maximize sensitivity while minimizing background in patient-derived samples, which often contain complex protein mixtures and potential interfering substances.

How does SLC51A expression correlate with bile acid metabolism in experimental models, and how can the antibody be used to monitor these correlations?

SLC51A expression shows significant correlations with bile acid metabolism in experimental models, which can be monitored using HRP-conjugated antibodies through several methodological approaches:

• Correlation Analysis Between Protein Expression and Bile Acid Profiles:

  • Quantify SLC51A protein levels via Western blotting with the HRP-conjugated antibody (1:100-500 dilution)

  • Simultaneously measure bile acid profiles using liquid chromatography-mass spectrometry

  • Analyze the statistical correlation between SLC51A expression and specific bile acid ratios, particularly:

    • Total taurine-conjugated/unconjugated bile acids (reference values: normal 0.20±0.09 vs. disease 1.38±0.86)

    • TCA/CA ratios (reference values: normal 0.15±0.14 vs. disease 1.63±1.33)

    • TαMCA/αMCA and TβMCA/βMCA ratios, which can increase approximately 10-fold in disease states

• Time-Course Experiments to Track Expression Changes:

  • Use the antibody in time-resolved studies following bile acid pool manipulation

  • Monitor SLC51A expression changes during progression of cholestasis or recovery

  • Correlate expression dynamics with temporal changes in bile acid composition

• Tissue-Specific Expression Analysis:

  • Apply immunohistochemistry (1:20-200 dilution) to examine regional differences in SLC51A expression across the intestinal-hepatic axis

  • Compare expression gradients with tissue-specific bile acid concentrations

  • Identify regulatory feedback mechanisms between bile acid levels and transporter expression

• Intervention Studies:

  • Monitor SLC51A expression changes in response to therapeutic agents that modulate bile acid metabolism

  • Track normalization of both expression and bile acid profiles after successful intervention

  • Establish predictive relationships between early changes in SLC51A expression and subsequent normalization of bile acid homeostasis

• Mechanistic Investigations Using Genetic Models:

  • Compare SLC51A expression using the antibody in wild-type versus genetically modified models with altered bile acid metabolism

  • Correlate expression changes with functional outcomes measured through bile acid transport assays

  • Establish causative relationships between SLC51A expression levels and specific aspects of bile acid homeostasis

This integrated approach leveraging SLC51A antibody-HRP provides critical insights into the regulatory interplay between bile acid transporter expression and metabolic outcomes, potentially identifying new therapeutic targets for bile acid-related disorders.

What are the most effective protocols for dual immunostaining to visualize SLC51A and SLC51B co-localization?

For effective dual immunostaining to visualize SLC51A and SLC51B co-localization, the following optimized protocol is recommended:

• Sample Preparation:

  • Fix tissue sections or cultured cells with 4% paraformaldehyde for 15 minutes

  • For tissues: perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 20 minutes

  • For cells: permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

  • Block with 5% normal serum (matching the species of secondary antibody) with 1% BSA in PBS for 1 hour at room temperature

• Primary Antibody Application (Sequential Method):

  • First Immunostaining Round:

    • Apply SLC51A antibody-HRP (1:50-200 dilution) in blocking buffer overnight at 4°C

    • Wash extensively with PBS (3 × 10 minutes)

    • Develop with tyramide signal amplification (TSA) fluorescent substrate (FITC or Cy3)

    • Wash thoroughly and perform antibody elution using glycine buffer (pH 2.2) for 10 minutes

  • Second Immunostaining Round:

    • Apply unconjugated SLC51B rabbit polyclonal antibody (1:100-200 dilution) overnight at 4°C

    • Wash with PBS (3 × 10 minutes)

    • Incubate with fluorophore-conjugated secondary antibody (with spectrally distinct fluorophore from first round) for 1 hour at room temperature

    • Wash extensively and counterstain nuclei with DAPI

• Controls and Validation:

  • Include single-stained controls for each antibody to verify signal specificity

  • Apply isotype controls to assess non-specific binding

  • Use samples with known expression patterns (small intestine sections) as positive controls

  • Include absorption controls by pre-incubating antibodies with respective recombinant proteins

• Image Acquisition and Analysis:

  • Capture images using confocal microscopy with appropriate filter sets

  • Analyze co-localization using specialized software (e.g., JACoP plugin for ImageJ)

  • Quantify Pearson's correlation coefficient or Manders' overlap coefficient

  • Generate intensity correlation plots to visualize co-occurrence patterns

• Alternative Approaches for Challenging Samples:

  • For samples with high autofluorescence, consider using chromogenic detection for one antibody and fluorescent for the other

  • For highly cross-reactive antibodies, implement the Zenon labeling technology to directly label primary antibodies

  • For ultra-resolution requirements, apply proximity ligation assay (PLA) to visualize actual protein-protein interactions

This protocol provides robust visualization of SLC51A and SLC51B co-localization while minimizing cross-reactivity and background issues commonly encountered in dual immunostaining procedures.

How do different recombinant fragments of SLC51A affect antibody recognition and experimental outcomes?

Different recombinant fragments of SLC51A can significantly impact antibody recognition and experimental outcomes in several important ways:

• Epitope Accessibility and Antibody Specificity:

  • C-terminal Epitope Antibodies: The anti-SLC51A rabbit polyclonal antibody targeting the C-terminal epitope typically demonstrates superior specificity in Western blot applications (1:100-500 dilution) . This region appears less conserved across species and more immunogenic, resulting in more distinctive binding patterns.

  • Full-length vs. Fragment Recognition: Antibodies raised against full-length SLC51A may recognize multiple epitopes, potentially increasing sensitivity but sometimes at the cost of specificity. In contrast, antibodies targeting specific fragments (such as the recombinant fragment from amino acids 1-48) offer enhanced specificity but may miss conformational epitopes .

• Recombinant Fragment Production Considerations:

  • Research indicates that selecting regions with high immunogenicity and hydrophilicity for recombinant protein production yields more effective antibodies. For example, the methodology described in source utilized antibody epitope prediction and hydrophilicity analysis to select optimal regions for recombinant production .

  • The PCR amplification of target regions followed by subcloning into expression vectors (like pET-23a(+)) with 6× histidine tags facilitates efficient purification and subsequent antibody production .

• Application-Specific Performance:

  • Western Blotting: Antibodies against linear epitopes in recombinant fragments perform well in Western blot applications where proteins are denatured .

  • Immunohistochemistry: For applications requiring recognition of native protein conformations, antibodies generated against carefully selected fragments that maintain secondary structure elements show superior performance .

  • ELISA Applications: The recombinant fragment used for immunization significantly impacts ELISA sensitivity, with antibodies against more exposed regions demonstrating better performance in solution-phase detection (1:2000-1:10000 dilution range) .

• Cross-Reactivity Considerations:

  • Researchers must carefully select recombinant fragments with minimal sequence homology to other proteins, particularly other SLC family members, to avoid cross-reactivity .

  • The recombinant Human Organic solute transporter subunit α protein (1-48AA) fragment used in one antibody preparation demonstrated excellent specificity with minimal cross-reactivity to related transporters .

This knowledge allows researchers to strategically select antibodies raised against specific SLC51A recombinant fragments based on their intended experimental applications, optimizing both sensitivity and specificity.

What are the key considerations when comparing SLC51A antibody performance across different species models?

When comparing SLC51A antibody performance across different species models, researchers should consider several critical factors:

• Sequence Homology and Epitope Conservation:

  • Human SLC51A antibodies may show variable cross-reactivity with rodent or other mammalian models depending on sequence conservation at the epitope region

  • The C-terminal region targeted by some commercial antibodies shows less conservation across species compared to transmembrane domains

  • Researchers should perform sequence alignment analysis between human and target species before antibody selection

• Application-Specific Cross-Reactivity:

  • Western blot applications typically require higher sequence identity at the epitope region compared to immunohistochemistry

  • While the primary antibody examined in the search results is specifically reactive with human samples, researchers working with mouse models should validate cross-reactivity experimentally

  • Consider using species-specific positive controls (e.g., human small intestine versus mouse small intestine) to verify signal specificity

• Optimization Requirements for Non-Human Models:

  • Protocol modifications are often necessary when adapting human-reactive antibodies to other species:

    • Increased antibody concentration (often 2-5 fold higher)

    • Extended incubation times (overnight at 4°C instead of 2 hours at room temperature)

    • Modified antigen retrieval methods for tissue sections

    • Alternative blocking reagents to minimize species-specific background

• Expression Pattern Differences:

  • SLC51A expression patterns vary across species, with some showing broader tissue distribution

  • Expected molecular weight may differ due to species-specific post-translational modifications

  • Different bile acid profiles across species (as shown in source ) may affect SLC51A regulation and expression levels

• Validation Strategies for Cross-Species Applications:

  • Perform side-by-side comparison with species-specific antibodies when available

  • Include genetic controls (knockout or knockdown) to confirm specificity

  • Use recombinant protein controls from the target species

  • Consider peptide blocking experiments to verify epitope specificity

These considerations are essential for researchers seeking to translate findings between human and animal models, ensuring valid cross-species comparisons and appropriate interpretation of experimental results.

How can SLC51A antibody, HRP conjugated be combined with bile acid profiling techniques for comprehensive metabolic studies?

Integrating SLC51A antibody-HRP detection with bile acid profiling techniques creates a powerful approach for comprehensive metabolic studies through several methodological strategies:

• Correlation Analysis Between Protein Expression and Bile Acid Signatures:

  • Quantify SLC51A protein levels via Western blotting (1:100-500 dilution) in hepatic and intestinal samples

  • Perform liquid chromatography-mass spectrometry (LC-MS/MS) analysis of bile acid profiles in matched samples

  • Establish statistical correlations between SLC51A expression and specific bile acid parameters, such as:

    • Total bile acid pool size

    • Conjugated/unconjugated bile acid ratios (reference values from source : normal 0.20±0.09 vs. disease 1.38±0.86)

    • Individual bile acid species proportions (e.g., TCA/CA, TαMCA/αMCA)

• Intervention Study Design:

  • Monitor both SLC51A expression and bile acid profiles before and after pharmacological interventions

  • Analyze how changes in SLC51A expression (detected with antibody-HRP) temporally correlate with alterations in bile acid composition

  • Develop predictive models linking early changes in transporter expression with subsequent metabolic adaptations

• Cell Type-Specific Analysis Using Immunohistochemistry:

  • Perform immunohistochemistry (1:20-200 dilution) to identify cell types expressing SLC51A

  • Microdissect specific cell populations (e.g., enterocytes vs. hepatocytes) for targeted bile acid profiling

  • Correlate cell type-specific expression patterns with local bile acid composition

• Integrated Multi-Omics Approach:

  • Combine antibody-based protein quantification with transcriptomics and bile acid metabolomics

  • Implement computational modeling to identify regulatory networks linking SLC51A expression to bile acid homeostasis

  • Generate testable hypotheses regarding the molecular mechanisms of SLC51A regulation

• Disease Model Applications:

  • Apply this integrated approach in models of cholestasis, non-alcoholic fatty liver disease, or inflammatory bowel disease

  • Map the relationship between transporter dysfunction (detected via antibody) and pathological bile acid profiles

  • Identify potential therapeutic targets based on correlation patterns

This integrated approach leveraging SLC51A antibody-HRP alongside advanced bile acid profiling techniques provides unprecedented insights into the molecular mechanisms regulating bile acid transport and metabolism in both physiological and pathological states.

What are the key differences in experimental approach when studying SLC51A in enterohepatic circulation versus inflammatory conditions?

The experimental approaches for studying SLC51A differ significantly when investigating enterohepatic circulation versus inflammatory conditions:

• Sample Selection and Processing:

  • Enterohepatic Circulation Studies:

    • Focus on intact enterohepatic axis (ileum, portal blood, liver)

    • Require careful preservation of tissue architecture for transport studies

    • Typically employ bile duct cannulation techniques to collect bile samples

  • Inflammatory Condition Studies:

    • Include inflammatory cell populations (macrophages, neutrophils)

    • Require preservation of cytokine signaling molecules

    • Often utilize induced inflammation models (DSS colitis, TNBS, LPS challenge)

• Antibody Application Protocols:

  • Enterohepatic Circulation:

    • Membrane fraction enrichment prior to Western blotting to concentrate transporter proteins

    • Immunohistochemistry (1:20-200 dilution) focusing on basolateral membrane localization

    • Co-localization studies with SLC51B to assess functional complex formation

  • Inflammatory Conditions:

    • Total protein extraction to capture potential cytoplasmic redistribution

    • Dual immunostaining with inflammatory markers (NF-κB, TNF-α receptors)

    • Higher antibody concentrations may be required due to potential protein degradation

• Functional Correlation Analysis:

  • Enterohepatic Circulation:

    • Correlate SLC51A expression with bile acid transport kinetics

    • Analyze relationship with conjugated/unconjugated bile acid ratios (normal: 0.20±0.09)

    • Focus on taurine and glycine conjugation patterns

  • Inflammatory Conditions:

    • Correlate expression with inflammatory cytokine levels

    • Analyze relationship with oxidative stress markers

    • Examine membrane integrity and transporter internalization

• Time-Course Considerations:

  • Enterohepatic Circulation:

    • Longer experimental timelines (days to weeks) to capture adaptive responses

    • Circadian rhythm considerations due to diurnal variations in bile acid production

    • Postprandial vs. fasting state comparisons

  • Inflammatory Conditions:

    • Shorter experimental windows (hours to days) focusing on acute responses

    • Critical timing of sample collection relative to inflammatory stimulus

    • Resolution phase sampling to capture recovery dynamics

• Mechanistic Investigation Approaches:

  • Enterohepatic Circulation:

    • Focus on FXR-mediated transcriptional regulation

    • Investigate post-translational modifications affecting membrane trafficking

    • Analyze heterodimer formation with SLC51B

  • Inflammatory Conditions:

    • Examine NF-κB and AP-1 transcriptional regulation pathways

    • Investigate role of inflammatory mediators in transporter endocytosis

    • Analyze protective mechanisms against inflammatory damage

This tailored approach ensures that the experimental design appropriately addresses the distinct biological questions and technical challenges associated with studying SLC51A in different physiological and pathological contexts.

What emerging technologies can enhance the utility of SLC51A antibody, HRP conjugated in translational research?

Several emerging technologies can significantly enhance the utility of SLC51A antibody-HRP conjugated in translational research:

• Multiplexed Imaging Technologies:

  • CODEX (CO-Detection by indEXing): Allows simultaneous detection of SLC51A alongside dozens of other proteins using antibody-based DNA barcoding in the same tissue section

  • Imaging Mass Cytometry (IMC): Combines the SLC51A antibody with metal isotope tags for high-dimensional spatial analysis of protein expression in relation to up to 40 other markers

  • Multiplex Immunofluorescence with Spectral Unmixing: Enables co-detection of SLC51A with multiple bile acid transporters and regulatory proteins by overcoming fluorescence spectral overlap limitations

• Single-Cell Analysis Integration:

  • Combining HRP-Antibody Detection with Single-Cell RNA-Seq: Correlating protein-level expression with transcriptomic profiles at single-cell resolution

  • Mass Cytometry (CyTOF) with Metal-Conjugated Antibodies: Adapting SLC51A detection to metal-conjugated formats for high-dimensional single-cell protein expression analysis

  • Spatial Transcriptomics with Protein Detection: Integrating SLC51A antibody-based detection with spatial transcriptomics to correlate protein localization with gene expression patterns

• Advanced Tissue Clearing and 3D Imaging:

  • CLARITY and iDISCO Compatibility: Adapting SLC51A antibody-HRP protocols for whole-organ clearing techniques to visualize transporter distribution throughout intact tissues

  • Light-Sheet Microscopy Integration: Enabling rapid 3D imaging of SLC51A distribution across the entire enterohepatic axis

  • Expansion Microscopy: Physically expanding tissue samples to achieve super-resolution imaging of SLC51A subcellular localization using standard confocal microscopy

• Microfluidic and Organ-on-Chip Platforms:

  • Gut-Liver-on-Chip Models: Incorporating SLC51A antibody-based detection in microfluidic platforms that recapitulate the enterohepatic circulation

  • Real-Time Monitoring: Developing non-destructive imaging approaches for tracking SLC51A expression dynamics in living organoid systems

  • High-Throughput Screening Integration: Adapting antibody-based detection for automated analysis of SLC51A modulation in response to therapeutic candidates

• Computational Biology Integration:

  • Machine Learning for Image Analysis: Developing algorithms to automatically quantify SLC51A expression patterns and subcellular localization from immunostaining data

  • Systems Biology Modeling: Incorporating antibody-derived quantitative data into computational models of bile acid homeostasis

  • Digital Pathology Integration: Creating automated scoring systems for SLC51A expression in patient samples to support clinical decision-making

These emerging technologies can transform how researchers utilize SLC51A antibodies, moving beyond traditional applications toward integrated, high-dimensional analyses that provide unprecedented insights into bile acid transport mechanisms in health and disease.

What are the most important considerations for researchers selecting SLC51A antibody, HRP conjugated for their studies?

When selecting SLC51A antibody with HRP conjugation for research applications, investigators should prioritize several critical considerations to ensure optimal experimental outcomes:

• Experimental Application Compatibility:

  • Ensure the antibody has been validated for your specific application, as performance varies significantly between techniques

  • Verify the recommended dilution ranges for each application: Western blot (1:100-500), ELISA (1:1000-10000), immunohistochemistry (1:20-200), and immunofluorescence (1:50-200)

  • Consider whether direct HRP conjugation is advantageous for your specific experimental design, weighing the benefits of protocol simplification against potential signal amplification limitations

• Epitope Specificity and Recognition:

  • Confirm the exact epitope region recognized by the antibody (e.g., C-terminal, as in source )

  • Evaluate whether the epitope accessibility might be compromised in your experimental system, particularly in native protein conformations or membrane-embedded contexts

  • Consider whether post-translational modifications might affect epitope recognition in your biological system

• Validation Status and Quality Controls:

  • Review the validation data provided by manufacturers, including Western blot images and immunostaining patterns

  • Check for cross-reactivity testing against related transporters, particularly other SLC family members

  • Verify species reactivity claims with experimental evidence, especially for cross-species applications

• Technical Specifications and Format:

  • Confirm storage buffer compatibility with your experimental system (typically 50% glycerol, 0.01M PBS, pH 7.4)

  • Verify the HRP enzyme activity retention during shipping and storage

  • Check antibody concentration and total protein amount to ensure sufficient material for planned experiments

• Experimental Design Considerations:

  • Include appropriate positive controls (small intestine or liver samples)

  • Implement negative controls (peptide blocking, isotype controls) to verify specificity

  • Plan for optimization steps, particularly when adapting the antibody to new biological systems or modified protocols

Careful consideration of these factors during antibody selection will significantly enhance experimental success rates, data quality, and interpretability when investigating SLC51A biology across diverse research applications.

How is the field of SLC51A research evolving, and what future directions might require optimized antibody-based detection methods?

The field of SLC51A research is rapidly evolving along several key trajectories that will require increasingly sophisticated antibody-based detection methods:

• Expanding Understanding of Physiological Roles:

  • Beyond Bile Acid Transport: Emerging research indicates SLC51A may transport additional substrates including steroids and eicosanoids like prostaglandin E2 . Future studies will require highly specific antibodies to correlate transporter expression with these diverse functions.

  • Tissue-Specific Functions: While initially characterized in intestinal and liver tissues, SLC51A's role in additional tissues requires investigation with optimized immunohistochemistry protocols using HRP-conjugated antibodies (1:20-200 dilution) .

  • Regulatory Networks: Antibody-based approaches will be crucial for mapping the complex regulatory networks controlling SLC51A expression across different physiological states .

• Disease Relevance and Therapeutic Potential:

  • Expanded Disease Associations: Beyond established connections to cholestasis and NAFLD, SLC51A is being investigated in inflammatory bowel disease, metabolic syndrome, and cancer. These applications will require antibodies validated across diverse pathological samples .

  • Biomarker Development: Quantitative immunoassays using HRP-conjugated antibodies may enable development of circulating SLC51A as a biomarker for liver and intestinal disorders .

  • Therapeutic Modulation: As SLC51A emerges as a potential therapeutic target, antibody-based detection will be essential for screening compound effects on expression and localization .

• Technological Integration:

  • Single-Cell Resolution Studies: Future research will require antibodies compatible with single-cell proteomics approaches to understand cellular heterogeneity in SLC51A expression.

  • Spatial Biology Applications: Integration with spatial transcriptomics will necessitate antibodies optimized for multiplexed detection alongside other markers .

  • Live-Cell Imaging: Development of non-toxic detection methods using antibody fragments for tracking SLC51A dynamics in living systems .

• Translational Research Directions:

  • Patient Stratification: Immunohistochemistry protocols using SLC51A antibodies may help stratify patients for targeted therapies in liver and intestinal disorders .

  • Pharmacodynamic Biomarkers: Antibody-based detection of SLC51A modulation might serve as a pharmacodynamic biomarker in clinical trials targeting bile acid metabolism .

  • Personalized Medicine Applications: Correlating individual variations in SLC51A expression with treatment responses will require highly reproducible antibody-based quantification methods .

• Methodological Advances:

  • Super-Resolution Microscopy Compatibility: Next-generation studies of SLC51A membrane organization will require antibodies optimized for super-resolution techniques like STORM and PALM.

  • Multiplexed Detection Systems: Antibodies designed for simultaneous detection of multiple components of the bile acid transport machinery .

  • Rapid Point-of-Care Diagnostics: Development of clinical applications may require adaptation of research-grade antibodies to simplified detection formats .