SLCO1B1 Antibody, FITC conjugated

What is SLCO1B1 and why is it significant for antibody-based detection methods?

SLCO1B1 (Solute carrier organic anion transporter family member 1B1) is a transmembrane hepatic uptake transporter that plays a crucial role in the Na(+)-independent transport of various organic anions. It is highly expressed in the liver, specifically at the basolateral membranes of centrilobular hepatocytes . The protein consists of 691 amino acid residues with a molecular mass of approximately 76.4 kDa .

This transporter is particularly significant in pharmacogenomic research because:

• It mediates the uptake of numerous clinically important drugs, including statins, methotrexate, and various endogenous compounds such as bile acids

• Genetic variants in SLCO1B1 are associated with altered drug pharmacokinetics and increased risk of adverse effects, particularly statin-associated muscle symptoms (SAMS)

• The detection and characterization of SLCO1B1 expression patterns using antibody-based methods provides critical insights into drug disposition and personalized medicine approaches

For antibody-based detection, researchers should consider that SLCO1B1 undergoes post-translational modifications, including glycosylation, which may affect epitope recognition .

What are the technical considerations when working with FITC-conjugated antibodies for SLCO1B1 detection?

When working with SLCO1B1 antibody, FITC conjugated, researchers should address several technical considerations:

Light sensitivity and storage:

• FITC-conjugated antibodies are highly sensitive to light exposure, which can cause gradual loss of fluorescence

• Store the antibody in the dark at recommended temperatures (typically -20°C)

Optimal dilution factors:

• For immunofluorescence on mammalian cells, the recommended dilution is typically 1:500 in PBS containing 10% fetal bovine serum

• Empirical determination of optimal dilution may be necessary depending on your specific application, sample type, or cell line

Blocking procedures:

• Use PBS containing 10% fetal bovine serum as a blocking solution to reduce non-specific binding

• Block for approximately 20 minutes at room temperature before antibody application

Fluorescence detection parameters:

• Use appropriate filter sets optimized for FITC detection (excitation ~495 nm, emission ~520 nm)

• Consider photobleaching effects during imaging, particularly for quantitative applications

Background reduction:

• Include appropriate washing steps (typically 2 × 5 minutes with PBS) after antibody incubation

• Consider autofluorescence controls, especially when working with tissues with high intrinsic fluorescence

How can SLCO1B1 antibody, FITC conjugated be validated for experimental use?

Proper validation of SLCO1B1 antibody, FITC conjugated should follow these methodological approaches:

Expression system controls:

• Test the antibody on cells known to express SLCO1B1 (e.g., hepatocytes) versus cells with minimal expression

• Consider using SLCO1B1-overexpressing cell lines (such as transfected HEK293 cells) as positive controls

Validation in multiple applications:

• Cross-validate across different detection methods, such as immunofluorescence, Western blotting, and flow cytometry

• The SLCO1B1 antibody has been validated for applications including ELISA, Western blot, and immunohistochemistry

Specificity testing:

• Use peptide competition assays with the immunogen peptide (amino acids 426-537 of SLCO1B1 for certain commercial antibodies)

• Include genetic knockdown/knockout controls where possible to confirm specificity

Cross-reactivity assessment:

• Test for cross-reactivity with related transporters (e.g., OATP1B3, OATP2B1)

• Validate across different species if working with non-human models (available antibodies have reported reactivity with human, mouse, and rat SLCO1B1)

A comprehensive validation approach enhances confidence in experimental results and strengthens the reliability of downstream analyses.

What protocol should be followed for immunofluorescence studies using FITC-conjugated SLCO1B1 antibody?

For optimal immunofluorescence results with SLCO1B1 antibody, FITC conjugated, follow this detailed protocol:

Sample preparation:

• Culture cells on sterile glass coverslips or appropriate chamber slides

• Fix cells using 4% paraformaldehyde for 10-15 minutes at room temperature

• Wash cells 3 times with PBS

Permeabilization (for intracellular epitopes):

• Treat with 0.1-0.2% Triton X-100 in PBS for 5-10 minutes

• Wash 3 times with PBS

Blocking and antibody incubation:

• Apply blocking solution (PBS containing 10% fetal bovine serum) for 20 minutes at room temperature

• Remove blocking solution and add 1 mL of PBS/10% FBS containing SLCO1B1 antibody, FITC conjugated (1:500 dilution)

• Incubate for 1 hour at room temperature in the dark

• Wash cells 2 × 5 minutes with PBS

Mounting and visualization:

• Mount coverslips using anti-fade mounting medium (preferably containing DAPI for nuclear counterstaining)

• Seal edges with nail polish to prevent drying

• Observe using a fluorescence microscope equipped with appropriate FITC filters

• Store slides at 4°C in the dark when not being examined

Controls to include:

• Secondary antibody-only control (to assess background)

• Positive control (tissue/cells known to express SLCO1B1)

• Negative control (tissue/cells known not to express SLCO1B1)

This protocol has been successfully used for detection of epitope-tagged fusion proteins in cultured CHO cells and can be adapted for SLCO1B1 detection .

How does SLCO1B1 genetic variation impact protein expression and antibody detection?

SLCO1B1 genetic variations can significantly impact protein expression and consequently affect antibody detection in several ways:

Altered protein abundance:

• Certain variants, particularly those affecting protein folding, can result in protein degradation and decreased transporter expression

• Deep Mutational Scanning (DMS) has identified variants that display less than 25% of wild-type protein expression, which would result in reduced antibody signal intensity

Subcellular localization changes:

• Some variants may affect trafficking to the plasma membrane, resulting in altered localization patterns when visualized by immunofluorescence

• Researchers should use appropriate microscopy techniques to distinguish between membrane-localized and intracellular protein pools

Epitope accessibility:

• Certain mutations might impact the accessibility of epitopes recognized by the antibody

• For example, if using an antibody targeting amino acids 426-537 , variants within this region could theoretically affect binding efficiency

Experimental applications:

• The GFP/mCherry ratio in DMS systems has been used as an indicator of SLCO1B1 protein expression

• The SLCO1B1*2 variant showed 61.5% of wild-type GFP/mCherry ratio, correlating with decreased protein quantity observed in Western blot analyses

Variant classification examples:

• c.629G>T (p.G210V) abolishes transport of multiple OATP1B1 substrates and has drastically reduced expression

• c.317T>C, c.633A>G, c.639T>A, c.820A>G, and c.2005A>C are considered normal-function variants despite some altered kinetic parameters

When interpreting antibody detection results, researchers should consider these genetic influences on expression levels and integrate genotyping data when available.

What advanced applications exist for SLCO1B1 antibody, FITC conjugated in pharmacogenomic research?

SLCO1B1 antibody, FITC conjugated offers several advanced applications in pharmacogenomic research:

Flow cytometry-based variant screening:

• Fluorescence-activated cell sorting (FACS) can be used to isolate cells expressing different SLCO1B1 variants based on fluorescence intensity

• This approach enables high-throughput functional characterization of genetic variants

• Cells can be sorted into different "bins" based on GFP/mCherry ratios as indicators of protein expression

Live-cell imaging of transport dynamics:

• FITC-conjugated antibodies against extracellular epitopes of SLCO1B1 can be used to track dynamic changes in transporter localization

• Combined with substrate fluorescence assays, this allows real-time monitoring of transport activity in relation to transporter expression

Multiplex imaging with drug substrates:

• Co-localization studies combining FITC-conjugated SLCO1B1 antibodies with fluorescently labeled drug substrates

• This approach can reveal spatial relationships between transporter expression and substrate accumulation

Quantitative analysis of SLCO1B1 expression:

• Precise quantification of SLCO1B1 expression levels in patient-derived samples

• Correlation with genotype data to establish expression-genotype-phenotype relationships

• Potential biomarker development for predicting drug response or adverse effects

Integration with Deep Mutational Scanning:

• The "landing pad cell-based system" described for DMS of SLCO1B1 variants can be combined with antibody detection

• This integration allows validation of GFP fusion protein findings with native protein detection

These advanced applications extend beyond basic detection and enable mechanistic insights into how genetic variation affects transporter function in clinically relevant contexts.

How can researchers troubleshoot common issues with FITC-conjugated antibody detection of SLCO1B1?

When troubleshooting FITC-conjugated antibody detection of SLCO1B1, researchers should systematically address these common issues:

Low signal intensity:

High background:

Inconsistent results:

Validation recommendations:

• Confirm antibody specificity using SLCO1B1 knockout/knockdown controls

• Compare results with alternative detection methods (e.g., Western blot, qPCR)

• Include known genetic variants of SLCO1B1 as reference standards

By systematically addressing these issues, researchers can optimize detection protocols and ensure reliable results with FITC-conjugated SLCO1B1 antibodies.

What methods can be used to quantify SLCO1B1 expression levels using FITC-conjugated antibodies?

Accurate quantification of SLCO1B1 expression using FITC-conjugated antibodies requires rigorous methodological approaches:

Flow cytometry quantification:

• Measure mean fluorescence intensity (MFI) as an indicator of SLCO1B1 expression level

• Use quantitative beads with known numbers of fluorophores to establish calibration curves

• Express results as molecules of equivalent soluble fluorochrome (MESF) or antibody binding capacity (ABC)

• Include isotype controls and unstained samples for background correction

Quantitative microscopy approaches:

• Integrated density measurements from defined regions of interest

• Use internal reference standards with known FITC molecule numbers

• Apply background subtraction and flatfield correction to compensate for optical artifacts

• Consider photobleaching correction for time-course or z-stack imaging

Normalization strategies:

• Ratio SLCO1B1 signal to a housekeeping protein (e.g., Na+/K+-ATPase for membrane proteins)

• Use dual labeling approaches, similar to the GFP/mCherry ratio system employed in DMS

• Account for cell size/surface area variations in comparative analyses

Advanced quantitative techniques:

• Fluorescence correlation spectroscopy (FCS) for absolute concentration determination

• Förster resonance energy transfer (FRET) for proximity-based quantification

• Fluorescence lifetime imaging microscopy (FLIM) to distinguish specific from non-specific signals

Validation with absolute quantification methods:

• LC-MS/MS-based quantitative targeted absolute proteomics (QTAP) analysis can validate fluorescence-based quantification

• Western blotting with recombinant protein standards can provide complementary validation

When reporting quantitative data, researchers should clearly describe all normalization procedures, control measurements, and calibration approaches to ensure reproducibility and facilitate cross-study comparisons.

How does SLCO1B1 antibody, FITC conjugated compare with other detection methods for studying transporter function?

SLCO1B1 antibody, FITC conjugated offers distinct advantages and limitations compared to other detection methods:

Comparison with other antibody conjugates:

Comparison with non-antibody detection methods:

Complementary approaches for comprehensive analysis:

• Combine FITC-antibody detection with functional assays to correlate expression with activity

• Integrate genetic information (variants) with protein expression data for mechanistic insights

• Use multiple detection methods to overcome the limitations of any single approach

The choice between these methods should be guided by the specific research question, available equipment, required sensitivity, and whether spatial information about protein localization is needed.

What role does SLCO1B1 detection play in understanding statin-induced myopathy mechanisms?

SLCO1B1 detection using antibody-based approaches provides critical insights into statin-induced myopathy mechanisms:

Genetic-protein expression correlation:

• The SLCO1B1*5 allele (containing the c.521T>C variant) is strongly associated with statin-induced myopathy and statin discontinuation

• Detection of SLCO1B1 protein levels in patients with different genotypes helps establish the molecular basis for this association

• Multivariate analysis has shown that SLCO1B1 genotype contributes significantly (8.8%) to atorvastatin discontinuation

Tissue-specific expression patterns:

• SLCO1B1 is highly expressed in the liver (basolateral membranes of centrilobular hepatocytes)

• The relative expression in muscle tissue versus liver can help explain why certain variants predispose to myopathy

• Fluorescence-based detection can reveal subtle differences in transporter localization and abundance

Variant-specific functional changes:

• Different SLCO1B1 variants have distinct effects on protein expression and function:

  • c.629G>T (p.G210V) abolishes transport activity comparable to the c.521T>C variant

  • Other variants (c.317T>C, c.633A>G, c.639T>A, c.820A>G, c.2005A>C) are considered normal-function despite some altered kinetic parameters

• Immunodetection combined with functional assays helps classify variants accurately

Mechanistic models:

• Reduced hepatic uptake of statins due to SLCO1B1 dysfunction leads to increased systemic exposure

• Higher plasma concentrations increase muscle exposure and toxicity risk

• Immunofluorescence studies can track statin distribution in relation to transporter expression

Clinical applications:

• The Clinical Pharmacogenetics Implementation Consortium (CPIC) recommends limiting statin doses in patients with decreased/poor function SLCO1B1 phenotypes

• Simvastatin should be avoided altogether in poor function phenotypes

• Antibody-based detection helps validate functional classifications of novel variants

Understanding the relationship between SLCO1B1 genotype, protein expression, and statin distribution provides a molecular foundation for personalized approaches to lipid-lowering therapy and myopathy risk prediction.

What advanced experimental designs can leverage SLCO1B1 antibody, FITC conjugated for mechanistic studies?

Sophisticated experimental designs using SLCO1B1 antibody, FITC conjugated can advance mechanistic understanding in multiple areas:

Time-resolved trafficking studies:

• Pulse-chase experimental designs to track SLCO1B1 internalization and recycling

• Live-cell imaging of FITC-labeled external epitopes combined with pH-sensitive dyes

• Correlate transporter localization changes with substrate uptake kinetics

Co-localization with regulatory proteins:

• Dual immunofluorescence combining SLCO1B1 antibody, FITC conjugated with antibodies against regulatory proteins

• Investigate physical interactions using proximity ligation assays or FRET approaches

• Examine post-translational modification effects (e.g., phosphorylation, glycosylation) on transporter localization

3D tissue architecture analysis:

• Confocal imaging of SLCO1B1 in complex tissue environments like liver slices

• Correlate zonal expression patterns with metabolic gradients and drug distribution

• Volume rendering to understand spatial relationships between transporters and cellular structures

Single-cell heterogeneity assessment:

• Flow cytometry analysis of SLCO1B1 expression across cell populations

• Imaging flow cytometry to correlate morphological features with transporter expression

• Single-cell sequencing integration to link genetic variation with protein expression

Patient-derived models:

• Primary hepatocyte cultures from patients with different SLCO1B1 genotypes

• Induced pluripotent stem cell (iPSC)-derived hepatocytes expressing variant transporters

• Humanized mouse models with variant SLCO1B1 alleles

Functional correlation experimental design example:

• Approach: Simultaneous detection of SLCO1B1 expression and substrate transport

• Method:

  • Plate hepatocytes on gridded coverslips

  • Perform live-cell imaging with fluorescent SLCO1B1 substrates (e.g., fluorescently-labeled statins)

  • Fix cells and perform immunofluorescence with SLCO1B1 antibody, FITC conjugated

  • Relocate the same cells using grid coordinates

  • Correlate substrate uptake with transporter expression at single-cell level

• Controls:

  • Include SLCO1B1 inhibitors (e.g., cyclosporine) as negative controls

  • Use cells with known SLCO1B1 variants as reference standards

• Analysis:

  • Quantify correlation between transporter expression and substrate accumulation

  • Apply machine learning algorithms to identify patterns beyond simple correlations

These advanced experimental designs can provide mechanistic insights that go beyond basic detection of SLCO1B1, contributing to fundamental understanding of transporter biology and pharmacogenomics.

How can Duplex Fluorescence Melting Curve Analysis (DFMCA) complement antibody-based SLCO1B1 detection methods?

Duplex Fluorescence Melting Curve Analysis (DFMCA) and antibody-based detection methods provide complementary approaches to SLCO1B1 investigation:

DFMCA methodology overview:
DFMCA is a rapid genotyping method for detecting SLCO1B1 polymorphisms that utilizes:

• PCR amplification of allelic regions

• Fluorescent probes designed for specific SLCO1B1 variants (e.g., rs2306283 and rs4149056)

• Melting temperature shifts to differentiate genotypes

• Concurrent detection of multiple polymorphisms within 2 hours

Complementary integration with antibody detection:

Integrated experimental design example:

• Initial screening: Use DFMCA to rapidly identify individuals with various SLCO1B1 genotypes (rs2306283, rs4149056)

• Sample selection: Group samples based on genotype (e.g., wild-type, heterozygous, homozygous variant)

• Protein expression analysis: Apply SLCO1B1 antibody, FITC conjugated to detect transporter expression in patient-derived samples

• Correlation analysis: Analyze relationships between:

  • Genotype (from DFMCA)

  • Protein expression (from antibody detection)

  • Clinical phenotypes (e.g., drug response, adverse effects)

Technical advantages of integration:

• DFMCA requires minimal DNA (~3.125 ng) , allowing analysis of samples too small for protein studies

• FITC-conjugated antibody detection provides spatial information not available from genetic analysis

• Combined approach enables validation of novel variants identified by either method

Future directions for integrated approaches:

• Development of multiplexed systems combining genetic and protein detection

• High-throughput platforms for simultaneous analysis of multiple transporters

• Artificial intelligence algorithms to predict protein expression patterns from genetic data