SLC10A2 Antibody

What is SLC10A2/ASBT and why is it important in research?

SLC10A2, also known as ASBT (Apical Sodium-dependent Bile acid Transporter), ISBT, or NTCP2, is a sodium/bile acid cotransporter primarily responsible for the uptake of bile acids by apical cells in the distal ileum . This protein plays a critical role in the sodium-dependent reabsorption of bile acids from the lumen of the small intestine and has key functions in cholesterol metabolism . Mutations in SLC10A2 cause primary bile acid malabsorption (PBAM) and may be associated with other diseases of the liver and intestines, such as familial hypertriglyceridemia (FHTG) . Due to its importance in bile acid transport and cholesterol homeostasis, SLC10A2/ASBT is a significant target for research in gastrointestinal physiology, liver function, and metabolic disorders.

What are the typical molecular characteristics of SLC10A2/ASBT that researchers should be aware of?

SLC10A2/ASBT is a membrane protein with the following characteristics:

SLC10A2/ASBT typically appears as a pair of unglycosylated (~38 kDa) and glycosylated (~41 kDa) immunoreactive bands on Western blots . Researchers should expect this pattern when analyzing experimental results to correctly identify the protein. The protein contains multiple transmembrane domains, with research supporting a 7-transmembrane domain topology with an extracellular N-terminus and cytoplasmic C-terminus .

What applications are SLC10A2 antibodies typically used for in research?

SLC10A2 antibodies are commonly used in several research applications:

Researchers should note that these applications have been validated with specific antibodies (e.g., 25245-1-AP, bs-23146R) and may require optimization for different experimental conditions . It is recommended that each reagent should be titrated in each testing system to obtain optimal results.

How can researchers validate the specificity of SLC10A2 antibodies in their experimental systems?

Validating antibody specificity is critical for obtaining reliable results. For SLC10A2 antibodies, researchers should consider the following approaches:

• Positive Control Selection: Use tissues known to express SLC10A2/ASBT, such as mouse small intestine or kidney tissue, as positive controls . The antibody should detect bands at the expected molecular weight (38-40 kDa).

• Knockout/Knockdown Validation: Compare antibody signal in wild-type samples versus those where SLC10A2 has been knocked out or knocked down. The specific signal should be absent or significantly reduced in the knockout/knockdown samples.

• Epitope Competition Assay: Pre-incubate the antibody with its immunizing peptide before application to samples. This should block specific binding and eliminate true positive signals.

• Cross-Reactivity Assessment: Test the antibody against samples from different species to confirm expected reactivity patterns. For example, antibody 25245-1-AP shows reactivity with human and mouse samples , while bs-23146R reacts with human and mouse with predicted reactivity for rat, dog, cow, horse, and rabbit .

• Multiple Antibody Comparison: Use multiple antibodies targeting different epitopes of SLC10A2 to confirm consistent detection patterns.

What factors influence the membrane topology determination of SLC10A2/ASBT, and how does this affect antibody selection?

The membrane topology of SLC10A2/ASBT has been a subject of controversy, with different models proposing between seven to nine transmembrane domains (TMDs) . This topological ambiguity has important implications for antibody selection and experimental design:

• Conflicting Topological Models: N-glycosylation analysis supports a 7 TMD model, while membrane insertion scanning supports a 9 TMD model. Recent research using dual label epitope insertion strongly supports the 7 TMD topology .

• Epitope Accessibility: In the 7 TMD model, loops 1 (99-130), 2 (180-191), and 3 (253-287) are extracellular, while loops 1 (50-73), 2 (150-160), and 3 (215-227) are intracellular . Antibodies targeting extracellular epitopes will be accessible in non-permeabilized cells, while those targeting intracellular epitopes require cell permeabilization.

• Experimental Validation: Studies using epitope insertion at positions 116, 120, 186, 270, and 284 showed accessibility to antibodies in non-permeabilized cells, confirming their extracellular localization. Conversely, epitopes at positions 56, 92, 156, and 221 were only detected after permeabilization with saponin, confirming their intracellular localization .

When selecting antibodies for experiments:

• For live-cell surface labeling, choose antibodies targeting extracellular epitopes

• For total protein detection, antibodies targeting any epitope can be used with proper sample preparation

• Consider the specific topology when interpreting localization or functional studies

How can epitope tagging approaches be used to study SLC10A2/ASBT structure and function?

Epitope tagging is a powerful approach for studying SLC10A2/ASBT structure and function, as demonstrated in topological studies:

• Strategic Tag Placement: Insert small epitope tags (e.g., HA, FLAG) at strategic positions within the protein sequence to probe specific regions without significantly disrupting function .

• Dual Label Approach: Use two distinct epitopes (e.g., HA and FLAG) to simultaneously probe different regions of the protein, allowing for comparative analysis of accessibility .

• Tag Detection Methods:

  • For membrane topology: Compare antibody accessibility in permeabilized versus non-permeabilized cells

  • For trafficking: Use surface biotinylation followed by detection with epitope-specific antibodies

  • For localization: Employ confocal microscopy with epitope-specific antibodies

• Functional Assessment: Validate that tagged constructs retain function using appropriate assays (e.g., sodium-dependent taurocholate uptake). Some insertion sites may disrupt function, providing insights into critical regions .

An experimental approach using this method might include:

• Generate multiple constructs with tags at different positions

• Express in appropriate cell lines (e.g., COS-1, MDCK)

• Validate expression by Western blot

• Assess membrane localization by surface biotinylation

• Confirm topology by immunofluorescence in permeabilized vs. non-permeabilized cells

• Validate function through transport assays

What are common challenges in detecting SLC10A2/ASBT using antibodies, and how can they be addressed?

Researchers often encounter several challenges when working with SLC10A2/ASBT antibodies:

• Post-translational Modifications: SLC10A2/ASBT exists in both glycosylated (~41 kDa) and unglycosylated (~38 kDa) forms . To distinguish these forms:

  • Use glycosidase treatments to confirm glycosylation status

  • Include positive controls from tissues known to express both forms

  • Use gradient gels to achieve better resolution of closely migrating bands

• Membrane Protein Extraction: As a multi-pass membrane protein, SLC10A2/ASBT can be difficult to extract effectively:

  • Use appropriate detergents (e.g., Triton X-100, SDS, or NP-40)

  • Avoid excessive heating that may cause aggregation

  • Include protease inhibitors to prevent degradation

  • Consider specialized membrane protein extraction kits

• Antibody Accessibility Issues: Depending on the epitope location, some regions of SLC10A2/ASBT may be inaccessible:

  • For intracellular epitopes (positions 56, 92, 156, 221), ensure complete cell permeabilization

  • For extracellular epitopes (positions 116, 120, 186, 270, 284), surface labeling of intact cells is feasible

  • Consider using denatured vs. native conditions depending on epitope accessibility

• Specificity Validation: When signals are ambiguous:

  • Include appropriate positive controls (e.g., mouse small intestine tissue for WB, mouse kidney tissue for IP)

  • Use multiple antibodies targeting different epitopes

  • Perform peptide competition assays

What are the optimal storage and handling conditions for SLC10A2 antibodies to maintain activity?

Proper storage and handling of SLC10A2 antibodies is crucial for maintaining their activity and specificity:

• Storage Temperature: Most SLC10A2 antibodies should be stored at -20°C and are typically stable for one year after shipment . Avoid repeated freeze-thaw cycles.

• Buffer Composition: SLC10A2 antibodies are commonly stored in:

  • PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

  • TBS (pH 7.4) with 1% BSA, 0.02% Proclin300, and 50% Glycerol

• Aliquoting Recommendations: For the 25245-1-AP antibody, aliquoting is noted as unnecessary for -20°C storage, but this may vary for other antibodies . Small-volume aliquots (20 μl) may contain 0.1% BSA as a stabilizer.

• Working Dilution Preparation: When preparing working dilutions:

  • Use fresh, cold buffer

  • Prepare immediately before use

  • Keep on ice during experiments

  • Do not store diluted antibody for extended periods

• Quality Control Practices:

  • Check for signs of precipitation or contamination before use

  • Validate activity periodically with positive controls

  • Record lot numbers and correlate with experimental outcomes

How are SLC10A2 antibodies being used to investigate bile acid transport mechanisms?

SLC10A2 antibodies have become instrumental in elucidating the mechanisms of bile acid transport:

• Topological Analysis: Researchers have used epitope-tagged constructs to determine that human ASBT has a 7-transmembrane domain structure rather than the previously proposed 9-transmembrane model . This clarification is crucial for understanding the structural basis of bile acid transport.

• Localization Studies: SLC10A2 antibodies enable precise localization of the transporter in tissues:

  • Confirmed apical membrane localization in ileal enterocytes

  • Identified expression in cholangiocytes

  • Detected differential expression patterns in various disease states

• Protein-Protein Interactions: Immunoprecipitation with SLC10A2 antibodies allows researchers to:

  • Identify binding partners involved in trafficking

  • Characterize regulatory protein interactions

  • Study complex formation with other transport proteins

• Expression Analysis in Disease Models: Western blotting with SLC10A2 antibodies has been used to:

  • Quantify expression changes in intestinal inflammation

  • Assess transporter levels in cholestatic conditions

  • Monitor changes in expression during drug treatments

What advanced imaging techniques can be combined with SLC10A2 antibodies for subcellular localization studies?

Several advanced imaging techniques can be employed with SLC10A2 antibodies to investigate subcellular localization:

• Confocal Microscopy: Used to confirm membrane localization of epitope-tagged SLC10A2/ASBT in both permeabilized and non-permeabilized cells . This technique provides optical sections that eliminate out-of-focus blur.

• Super-Resolution Microscopy: Techniques such as STORM, PALM, or STED can resolve the distribution of SLC10A2/ASBT at the nanoscale level, potentially revealing clustering patterns or specific membrane domain localization.

• Live-Cell Imaging: Using antibodies against extracellular epitopes or fluorescently tagged SLC10A2 constructs enables:

  • Real-time trafficking studies

  • Internalization and recycling analysis

  • Monitoring of dynamic changes in response to stimuli

• Correlative Light and Electron Microscopy (CLEM): Combines the specificity of fluorescent antibody labeling with ultrastructural context:

  • Precise localization at the ultrastructural level

  • Visualization of transporter in relation to membrane microdomains

  • Detection in specialized membrane structures

• Proximity Ligation Assay (PLA): Enables detection of protein-protein interactions in situ:

  • Identify interactions between SLC10A2 and regulatory proteins

  • Detect complexes with other transport proteins

  • Visualize changes in interaction patterns under different conditions

When designing imaging experiments, researchers should consider:

• The accessibility of epitopes based on the 7 TMD topology model

• The need for permeabilization depending on epitope location

• The potential effect of fixation methods on epitope recognition

• The use of appropriate controls to validate specificity

How can SLC10A2 antibodies contribute to understanding disease mechanisms?

SLC10A2 antibodies are increasingly being used to investigate the role of this transporter in various diseases:

• Primary Bile Acid Malabsorption (PBAM): Mutations in SLC10A2 cause PBAM . Antibodies can be used to:

  • Detect altered expression levels in patient samples

  • Characterize the cellular fate of mutant transporters

  • Evaluate the impact of therapeutic interventions on expression and localization

• Inflammatory Bowel Disease (IBD): SLC10A2 expression is often altered in IBD. Antibodies enable:

  • Quantification of changes in expression during inflammation

  • Assessment of the impact of anti-inflammatory therapies

  • Correlation of transporter levels with disease severity

• Cholestatic Liver Diseases: By examining changes in SLC10A2 expression:

  • Researchers can explore compensatory mechanisms during cholestasis

  • Monitor adaptations in bile acid transport

  • Evaluate potential therapeutic targets

• Metabolic Disorders: Given SLC10A2's role in cholesterol metabolism , antibodies help:

  • Investigate transporter levels in hypercholesterolemia

  • Assess responses to lipid-lowering therapies

  • Explore connections to familial hypertriglyceridemia (FHTG)

What are the considerations for using SLC10A2 antibodies in translational research and drug development?

SLC10A2 antibodies play a crucial role in translational research and drug development:

• Target Validation: Antibodies help validate SLC10A2 as a therapeutic target by:

  • Confirming expression in relevant tissues

  • Examining changes in disease states

  • Correlating expression levels with clinical parameters

• Drug Screening: In high-throughput screening for SLC10A2 inhibitors, antibodies enable:

  • Evaluation of compound effects on protein expression

  • Assessment of subcellular localization changes

  • Detection of potential compensatory mechanisms

• Biomarker Development: SLC10A2 antibodies may contribute to biomarker development:

  • Detection of transporter levels in accessible samples

  • Correlation with disease progression or treatment response

  • Development of diagnostic assays

• Personalized Medicine Approaches: By characterizing individual variations in SLC10A2:

  • Predict response to bile acid sequestrants

  • Identify patients who might benefit from targeted therapies

  • Monitor treatment efficacy at the molecular level

• Safety Assessment: During drug development, antibodies help:

  • Evaluate off-target effects on bile acid transport

  • Assess potential drug-induced cholestasis

  • Monitor compensatory changes in transporter expression