SLCO1A2 Antibody

What is SLCO1A2 and why is it important in research?

SLCO1A2, also known as OATP1A2, OATP-A, SLC21A3, OATP-1, and organic anion transporting polypeptide A, is a sodium-independent transporter that mediates the cellular uptake of organic anions, including numerous clinically important drugs . The protein is approximately 74.1 kilodaltons in mass and consists of 670 amino acids with 12 transmembrane domains . SLCO1A2 is primarily expressed in epithelial cells from multiple tissues, including the apical surface of the intestinal epithelium, renal tubules, brain capillary endothelium, and biliary cholangiocytes . Due to its critical role in drug absorption, distribution, and elimination, SLCO1A2 is an important research target for pharmacokinetic and pharmacodynamic studies .

What are the main applications of SLCO1A2 antibodies in research?

SLCO1A2 antibodies serve multiple purposes in laboratory research:

• Protein detection and quantification via Western blotting

• Cellular localization studies through immunohistochemistry (IHC) and immunocytochemistry (ICC)

• Trafficking and expression analysis using immunofluorescence techniques

• Validation of transporter variants in experimental models

• Investigation of polymorphism effects on membrane targeting

When studying SLCO1A2 localization, researchers typically fix cells in 4% paraformaldehyde, permeabilize them with 0.1% Triton X-100, and incubate with anti-SLCO1A2 primary antibody (approximately 10 μg/mL), followed by detection with fluorophore-conjugated secondary antibodies such as Alexa Fluor® 594 .

What types of SLCO1A2 antibodies are available for research?

These antibodies target different epitopes within the SLCO1A2 protein, with many focusing on the middle region or specific domains like N286 . When selecting an antibody, researchers should consider the specific application requirements and the need for cross-reactivity with orthologs from other species .

How should I optimize Western blot protocols for SLCO1A2 detection?

Western blotting for SLCO1A2 requires careful optimization due to the protein's hydrophobic nature and multiple transmembrane domains. Follow these methodological steps:

• Sample preparation: Use specialized membrane protein extraction buffers containing mild detergents like 1% Triton X-100 or RIPA buffer supplemented with protease inhibitors.

• Gel electrophoresis: Avoid excessive heating of samples (heat only to 37°C rather than boiling) to prevent protein aggregation. Use 8-10% SDS-PAGE gels for optimal separation of the 74.1 kDa protein.

• Transfer conditions: Employ wet transfer methods with methanol-containing buffers to facilitate the transfer of hydrophobic membrane proteins.

• Blocking: Use 5% non-fat milk or BSA in TBS-T for 1-2 hours at room temperature.

• Antibody incubation: Most SLCO1A2 antibodies work optimally at 1:500-1:1000 dilution when incubated overnight at 4°C.

• Detection: For low abundance expression, enhance sensitivity using HRP-conjugated secondary antibodies with chemiluminescent substrates.

When analyzing expression levels in variant studies, compare band intensities to a housekeeping protein control such as β-actin or GAPDH for proper normalization .

What controls should be included when validating SLCO1A2 antibody specificity?

Proper experimental controls are crucial for validating SLCO1A2 antibody specificity:

• Positive control: Include lysates from tissues known to express SLCO1A2 (liver, brain, intestine) or from cells transfected with SLCO1A2 expression constructs.

• Negative control: Use tissues or cells with no or minimal SLCO1A2 expression, or SLCO1A2 knockout models.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to verify signal specificity.

• Alternative antibody validation: Compare results with a different antibody targeting a distinct SLCO1A2 epitope.

• siRNA knockdown: Demonstrate signal reduction in cells treated with SLCO1A2-specific siRNA.

Researchers studying transporter variants should include wild-type SLCO1A2 as a reference standard to assess relative expression levels and subcellular localization patterns .

How can I assess SLCO1A2 protein localization in tissue or cell samples?

To effectively visualize SLCO1A2 localization:

• Immunofluorescence in cultured cells:

  • Fix cells using 4% paraformaldehyde for 20 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

  • Block with 5% goat serum in PBS for 30 minutes

  • Incubate with anti-SLCO1A2 primary antibody (10 μg/mL) for 2 hours

  • Detect with appropriate fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor® 594 at 1:1000 dilution)

  • Mount using specialized mounting medium containing nuclear counterstain

• Immunohistochemistry in tissue sections:

  • For formalin-fixed, paraffin-embedded tissues, use appropriate antigen retrieval methods (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Block endogenous peroxidase activity with 3% hydrogen peroxide

  • Apply optimized antibody dilution as determined by titration experiments

  • Use positive control tissues (brain capillary endothelium, intestinal epithelia) to confirm staining patterns

Co-localization studies with membrane markers (Na⁺/K⁺-ATPase, E-cadherin) or intracellular compartment markers (calnexin for ER, GM130 for Golgi) can provide additional insights into trafficking of wild-type versus variant SLCO1A2 proteins .

How can I investigate the impact of SLCO1A2 polymorphisms on transporter function?

To comprehensively characterize SLCO1A2 variants:

• Identification of polymorphisms:

  • PCR-amplify coding exons from genomic DNA using KAPA HiFi PCR Kits or similar high-fidelity systems

  • Design primers to cover all coding regions (exons 3-16, as exons 1-2 are non-coding)

  • Perform bidirectional sequencing to identify novel or known variants

• Functional analysis in expression systems:

  • Create variant constructs using site-directed mutagenesis

  • Express wild-type and variant transporters in appropriate cell lines (HEK-293, MDCK)

  • Measure substrate transport using radiolabeled or fluorescent substrates

  • Analyze kinetic parameters (Km, Vmax) to quantify functional alterations

• Protein expression and localization:

  • Compare membrane versus intracellular expression using cell surface biotinylation assays

  • Visualize localization differences through immunofluorescence microscopy

  • Quantify protein expression levels via Western blotting

Research has identified several novel SLCO1A2 polymorphisms, including G763A (V255I), G862A (D288N), and A775C (T259P), which affect transporter stability and membrane targeting . Studies suggest that charged residues at positions 184, 185, and 288 may form intramolecular ionic interactions that stabilize transporter structure, while bulky substituents at position 259 may disrupt protein stability .

What techniques can be used to study SLCO1A2 interactions with drug substrates?

Several advanced techniques can elucidate SLCO1A2-drug interactions:

• Substrate transport assays:

  • Direct measurement using radioisotope-labeled compounds

  • Fluorescent substrate accumulation assays (e.g., with BODIPY-labeled bile acids)

  • LC-MS/MS quantification of substrate uptake

• Binding assays:

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Photoaffinity labeling to identify binding sites

• Structural studies:

  • Site-directed mutagenesis combined with functional assays to map substrate binding domains

  • Homology modeling based on related transporters

  • Molecular docking simulations to predict binding modes

• Competition assays:

  • IC50 determination for different substrates

  • Transport inhibition profiles to characterize substrate specificity

SLCO1A2 transports numerous drug substrates including imatinib, fexofenadine, methotrexate, HIV protease inhibitors, and HMG-CoA reductase inhibitors . For accurate assessment of transport kinetics, researchers should account for passive diffusion by performing parallel experiments at 4°C or in the presence of specific inhibitors.

How can SLCO1A2 antibodies be used in clinical research applications?

SLCO1A2 antibodies offer valuable tools for translational and clinical research:

• Pharmacogenomic correlations:

  • Analyze SLCO1A2 expression in patient tissue samples

  • Correlate expression levels with drug response phenotypes

  • Investigate the impact of gene polymorphisms on protein expression

• Biomarker development:

  • Evaluate SLCO1A2 as a potential biomarker for drug response

  • Assess expression changes in disease states

  • Develop immunohistochemical scoring systems for standardized evaluation

• Precision medicine applications:

  • Screen patient samples for transporter expression before drug therapy

  • Identify individuals at risk for altered drug pharmacokinetics

  • Select optimal drug regimens based on transporter expression profiles

Research has demonstrated that individuals carrying novel SNPs in the SLCO1A2 gene may be at risk of impaired efficacy or enhanced toxicity during treatment with SLCO1A2 substrate drugs . This suggests potential clinical utility for SLCO1A2 expression analysis in personalized medicine approaches.

What are common problems encountered when using SLCO1A2 antibodies and how can they be resolved?

When working with SLCO1A2 antibodies, remember that membrane proteins often require special handling. Consider using mild detergents for extraction, avoid excessive heating of samples, and optimize transfer conditions for hydrophobic proteins .

How should researchers interpret conflicting results with different SLCO1A2 antibodies?

When facing discrepancies between different SLCO1A2 antibodies:

• Evaluate antibody characteristics:

  • Compare the epitopes targeted by each antibody

  • Assess validation data provided by manufacturers

  • Consider antibody format (polyclonal vs. monoclonal)

• Verify specificity:

  • Perform peptide competition assays

  • Use SLCO1A2 knockout or knockdown controls

  • Compare with orthogonal detection methods (e.g., mass spectrometry)

• Analyze experimental conditions:

  • Different fixation protocols may affect epitope accessibility

  • Sample preparation methods might influence protein conformation

  • Buffer compositions can impact antibody-antigen interactions

• Validate functional correlation:

  • Correlate protein detection with functional transport assays

  • Combine protein expression data with mRNA quantification

  • Use different antibodies for complementary detection methods

Remember that antibodies recognizing different domains may yield divergent results, especially when studying protein variants with domain-specific alterations . In such cases, using multiple antibodies targeting distinct epitopes can provide more comprehensive understanding of protein expression and localization.

How are SLCO1A2 antibodies being used in blood-brain barrier research?

SLCO1A2 is expressed in brain capillary endothelium and may play a critical role in drug uptake into the brain . Recent research applications include:

• Blood-brain barrier (BBB) transport studies:

  • Immunolocalization of SLCO1A2 in human brain microvessels

  • Co-localization with other BBB markers

  • Quantification of expression in different brain regions

• CNS drug delivery investigations:

  • Correlation of transporter expression with drug penetration

  • Development of drug delivery strategies targeting SLCO1A2

  • Assessment of transporter regulation under pathological conditions

• Disease-related research:

  • Evaluation of SLCO1A2 expression changes in neurological disorders

  • Investigation of polymorphisms affecting CNS drug delivery

  • Correlation with treatment outcomes for brain-targeted therapies

Researchers can employ SLCO1A2 antibodies in immunofluorescence studies of brain capillary isolates or brain sections to assess expression patterns and potential regulatory mechanisms affecting drug transport across the BBB .

What role do SLCO1A2 antibodies play in studying drug-drug interactions and personalized medicine?

SLCO1A2 antibodies facilitate crucial research in pharmacogenomics and personalized medicine:

• Transporter-mediated drug-drug interactions:

  • Visualization of competitive binding at the protein level

  • Correlation of expression levels with interaction potential

  • Assessment of transporter regulation following drug exposure

• Personalized drug therapy approaches:

  • Stratification of patients based on transporter expression profiles

  • Correlation of polymorphisms with protein function and drug response

  • Development of predictive biomarkers for drug efficacy and toxicity

• Regulatory science applications:

  • Evaluation of transporter involvement in drug disposition

  • Assessment of polymorphic variants affecting drug safety

  • Investigation of population differences in transporter expression

Research has shown that SLCO1A2 transports numerous clinically important drugs, including imatinib, fexofenadine, methotrexate, HIV protease inhibitors, and HMG-CoA reductase inhibitors . Individuals carrying specific SNPs in the SLCO1A2 gene may experience impaired drug efficacy or enhanced toxicity, highlighting the importance of transporter research in personalized medicine approaches .