ABCC2 Antibody

What is ABCC2 and why is it significant in research?

ABCC2 is a member of the ATP-binding cassette transporter family that exports a broad spectrum of substrates, including anticancer drugs such as vincristine, cisplatin, doxorubicin, methotrexate, irinotecan, and paclitaxel. It plays a crucial role in detoxification by mediating the export of endogenous and exogenous organic anions conjugated with glutathione, sulfate, or glucuronide . Its significance lies in its association with multidrug resistance in cancer treatment and its physiological role in biliary transport. ABCC2 is specifically expressed in the apical membrane of hepatocytes, renal proximal tubule cells, and enterocytes of the duodenum and jejunum , making it an important research target for understanding drug metabolism and resistance mechanisms.

What are the common applications of ABCC2 antibodies in research?

ABCC2 antibodies are widely employed in various experimental techniques including Western blotting, immunocytochemistry (ICC), immunohistochemistry (IHC), and flow cytometry . Researchers typically use these antibodies to:

• Detect and quantify ABCC2 expression levels in different cell types

• Investigate subcellular localization using ICC/IF techniques

• Assess ABCC2 upregulation in drug-resistant cancer cell lines

• Study the correlation between ABCC2 expression and drug response

• Examine the effects of genetic knockdown or pharmacological inhibition of ABCC2

How do I select the appropriate ABCC2 antibody for my specific experimental needs?

When selecting an ABCC2 antibody, consider the following methodological approach:

• Reactive species compatibility: Confirm the antibody has been validated for your species of interest (e.g., human, mouse, rat) .

• Application suitability: Verify the antibody is validated for your specific application (WB, IHC, ICC, flow cytometry) .

• Epitope specificity: Choose antibodies that target unique epitopes of ABCC2 to avoid cross-reactivity with other ABC transporters.

• Clonality consideration:

  • Polyclonal antibodies offer higher sensitivity but potentially lower specificity

  • Monoclonal antibodies provide higher specificity but may have limited epitope recognition

• Validation data: Review immunoblots, immunostaining images, and flow cytometry histograms provided by manufacturers to assess performance .

Optimal working dilutions vary by application, for example: WB (1:500-1:1,000), ICC (1:50-1:200), IHC (1:50-1:200), and flow cytometry (1:50-1:100) .

What are the best practices for validating ABCC2 antibody specificity?

A comprehensive validation approach should include:

• Positive and negative controls:

  • Use cells known to express high levels of ABCC2 (e.g., HepG2) as positive controls

  • Use cells with low/no ABCC2 expression or ABCC2 knockdown cells as negative controls

• Multiple detection methods:

  • Compare protein detection across different techniques (WB, ICC, flow cytometry)

  • Verify consistent molecular weight detection (~174 kDa)

• Knockdown/knockout validation:

  • Perform siRNA or shRNA experiments targeting ABCC2 to confirm antibody specificity

  • In A549/DDP cells, ABCC2 knockdown should show decreased detection by the antibody

• Peptide competition assay:

  • Pre-incubate antibody with blocking peptide before application

  • Signal should be significantly reduced if antibody is specific

• Correlation with mRNA expression:

  • Compare protein detection with qRT-PCR results for ABCC2 expression

  • Consistent correlation strengthens validation evidence

How should I optimize immunocytochemistry protocols for ABCC2 detection?

For optimal ICC detection of ABCC2, follow this methodological approach:

• Cell fixation:

  • Use paraformaldehyde (4%) for 15-20 minutes at room temperature

  • This preserves membrane protein structure while maintaining cellular architecture

• Permeabilization optimization:

  • Use 0.1-0.3% Triton X-100 for intracellular domains

  • For membrane localization studies, use milder detergents (0.1% saponin)

• Blocking parameters:

  • Block with 5-10% normal serum (same species as secondary antibody)

  • Include 1% BSA to reduce non-specific binding

• Antibody incubation:

  • Primary: Use 1:50-1:200 dilution at 4°C overnight

  • Secondary: Use 1:200-1:500 dilution for 1-2 hours at room temperature

• Nuclear counterstaining:

  • DAPI staining helps visualize subcellular localization relative to the nucleus

• Confocal microscopy settings:

  • Use z-stack imaging to confirm membrane localization

  • Employ proper negative controls and single-stained samples for establishing threshold settings

Successful staining should reveal ABCC2 primarily at the cell membrane, with particular enrichment at apical domains in polarized cell types such as HepG2 .

What experimental strategies can effectively assess ABCC2 transport function?

To evaluate ABCC2 transport function rather than merely expression:

• Drug accumulation assays:

  • Measure intracellular concentration of fluorescent ABCC2 substrates (e.g., CDCF)

  • Compare accumulation in the presence and absence of ABCC2 inhibitors

• Drug sensitivity testing:

  • Conduct MTT assays with ABCC2 substrates like cisplatin in wild-type versus ABCC2-knockdown cells

  • IC50 values should decrease in ABCC2-knockdown cells if transport is inhibited

• Transport assays in recombinant cell lines:

  • Use transwell systems with ABCC2-expressing cells (e.g., LLC-PK1 with ABCC2 variants)

  • Measure directional transport of substrates across cell monolayers

• Vesicular transport assays:

  • Isolate membrane vesicles from ABCC2-expressing cells

  • Quantify ATP-dependent uptake of radiolabeled substrates

• In vivo xenograft models:

  • Compare drug efficacy in xenografts with normal versus knockdown ABCC2 expression

  • Measure tumor growth inhibition and drug concentration in tissues

How can I address inconsistent ABCC2 antibody staining patterns?

When encountering variable staining patterns:

• Epitope accessibility issues:

  • Problem: Membrane protein epitopes may be masked by fixation

  • Solution: Test different fixation methods or antigen retrieval techniques

• Expression level variability:

  • Problem: ABCC2 expression varies with cell density and growth conditions

  • Solution: Standardize culture conditions and document confluence at collection

• Cell type-specific localization:

  • Problem: ABCC2 shows different localization patterns between cell types

  • Solution: Compare with published localization patterns for your specific cell model

  • Expected pattern: Apical membrane localization in polarized cells like hepatocytes

• Polymorphism interference:

  • Problem: ABCC2 polymorphisms may affect antibody binding

  • Solution: Verify which epitope your antibody recognizes and check if it spans polymorphic regions

• Storage and handling effects:

  • Problem: Antibody efficacy decreases with repeated freeze-thaw cycles

  • Solution: Aliquot antibodies upon receipt and store at recommended temperatures (-20°C for long-term; 4°C for up to one month)

What are the common pitfalls in interpreting ABCC2 expression data in drug resistance studies?

To avoid misinterpretation in drug resistance studies:

How should I analyze ABCC2 polymorphism effects on protein function?

When studying ABCC2 polymorphic variants:

• Integrated analytical approach:

  • Collect genotype data (e.g., rs717620, G1249A)

  • Correlate with protein expression levels (Western blot)

  • Assess membrane localization (ICC/IF)

  • Measure substrate transport capability

• Statistical analysis framework:

  • Use multivariate analysis to adjust for confounding factors

  • Report adjusted odds ratios with confidence intervals

  • Example from research: ABCC2 rs717620 T variant showed adjusted OR: 3.83 (95% CI: 1.73-8.48, p = 0.001) for hyperbilirubinemia risk

• Validation strategies:

  • Test findings in independent datasets

  • In the example study, ABCC2 rs717620 T variant association was verified in a validation dataset (adjusted OR: 4.91, 95% CI: 1.36-17.73, p = 0.015)

• Functional verification:

  • Create recombinant cell lines expressing wild-type versus polymorphic ABCC2

  • Compare transport kinetics of relevant substrates

  • Assess differences in drug sensitivity using dose-response curves

• Clinical correlation:

  • Examine associations with clinical outcomes (e.g., drug toxicity, treatment efficacy)

  • Consider interaction with other genetic and environmental factors

How can ABCC2 antibodies be utilized in cancer chemoresistance research?

Advanced applications in chemoresistance research include:

• Dynamic expression monitoring:

  • Track ABCC2 expression changes during development of drug resistance

  • Correlate with acquisition of cross-resistance to multiple agents

• Predictive biomarker development:

  • Quantify ABCC2 levels in patient samples before chemotherapy

  • Correlate with treatment response to develop predictive algorithms

• Combination therapy optimization:

  • Use ABCC2 antibodies to monitor effects of transporter inhibitors

  • Develop rational drug combinations targeting ABCC2-mediated resistance

• Mechanistic pathway investigation:

  • Combine ABCC2 immunoprecipitation with mass spectrometry to identify interacting partners

  • Map signaling pathways regulating ABCC2 expression in resistant cells

• Targeted therapy approaches:

  • Develop antibody-drug conjugates targeting cells with high ABCC2 expression

  • Create ABCC2 antibody-based delivery systems to overcome efflux-mediated resistance

Research evidence demonstrates that ABCC2 knockdown reverses cisplatin resistance in NSCLC cells by:

• Promoting G1 phase cell cycle arrest

• Activating PARP and caspase-3

• Enhancing the cytotoxicity of cisplatin in vivo in subcutaneous tumor models

What novel approaches exist for studying ABCC2 structure-function relationships?

Advanced structural and functional research approaches include:

• Cryo-EM structural analysis:

  • Utilize antibody fragments to stabilize ABCC2 for structural determination

  • Map conformational changes during transport cycle

• CRISPR/Cas9 engineered models:

  • Create precise mutations in ABCC2 domains

  • Correlate structural modifications with transport function

  • Generate tagged ABCC2 variants for live-cell imaging

• Single-molecule tracking:

  • Use antibody fragments to track ABCC2 mobility in the membrane

  • Correlate dynamic behavior with transport activity

• Domain-specific antibodies:

  • Develop antibodies targeting specific ABCC2 functional domains

  • Use as tools to inhibit or probe distinct aspects of transporter function

• Conformational sensors:

  • Design antibodies recognizing specific conformational states of ABCC2

  • Monitor transport cycle dynamics in real-time

Current research indicates that ABCC2 possesses a broad substrate specificity that enables it to export numerous anticancer drugs and conjugated organic anions, making it a critical player in both physiological detoxification and pathological drug resistance .

How can researchers integrate ABCC2 antibody data with other omics approaches?

For multi-omics integration strategies:

• Transcriptomics correlation:

  • Compare ABCC2 protein levels (by Western blot) with RNA-seq data

  • Identify potential post-transcriptional regulatory mechanisms

  • Example finding: Upregulation of ABCC2 in cisplatin-resistant cells at both mRNA and protein levels

• Proteomics integration:

  • Combine ABCC2 immunoprecipitation with mass spectrometry

  • Map the ABCC2 interactome in different cellular states

  • Identify changes in protein-protein interactions during drug resistance development

• Metabolomics correlation:

  • Link ABCC2 expression levels with metabolite profiles

  • Track changes in ABCC2 substrates and their metabolites

  • Create predictive models of ABCC2 transport activity

• Network analysis approaches:

  • Integrate antibody-based ABCC2 quantification with regulatory network data

  • Identify master regulators of ABCC2 expression

  • Map complete resistance pathways involving ABCC2

• Clinical data integration:

  • Correlate ABCC2 expression in patient samples with:

    • Treatment outcomes

    • Genetic polymorphisms

    • Disease progression markers

  • Example clinical finding: ABCC2 rs717620 T variant increases risk of hyperbilirubinemia in drug-induced liver injury patients (OR: 3.83, p = 0.001)

What are the latest developments in targeting ABCC2 for cancer therapy?

Current research frontiers include:

• Selective ABCC2 inhibitors:

  • Development of specific small molecule inhibitors

  • Antibody-based blocking strategies

  • Non-competitive allosteric modulators

• Gene therapy approaches:

  • CRISPR/Cas9-mediated ABCC2 knockout or modification

  • siRNA/shRNA therapeutic delivery systems

  • Example from research: shRNA ABCC2 knockdown enhanced cisplatin cytotoxicity both in vitro and in vivo

• Nanoparticle-based strategies:

  • Design of drug delivery systems evading ABCC2-mediated efflux

  • Co-delivery of chemotherapeutics with ABCC2 inhibitors

  • Targeted nanoparticles for ABCC2-expressing cancer cells

• Biomarker development:

  • ABCC2 antibody-based diagnostic tools

  • Liquid biopsy approaches for monitoring ABCC2 expression

  • Predictive algorithms incorporating ABCC2 status

• Precision medicine applications:

  • ABCC2 genotype-guided therapy selection

  • Polymorphism-based dose adjustment

  • Combined inhibition of multiple ABC transporters based on expression profiles

Research evidence shows that targeting ABCC2 can significantly reverse chemoresistance, particularly for platinum-based therapies in non-small cell lung cancer .

How does ABCC2 function vary across different tissue and cellular contexts?

Understanding contextual variations requires:

• Tissue-specific expression mapping:

  • Compare ABCC2 levels across tissues using tissue microarrays

  • Document differential subcellular localization patterns

  • ABCC2 is primarily expressed in:

    • Apical membrane of hepatocytes

    • Renal proximal tubule cells

    • Enterocytes of duodenum and jejunum

• Co-expression network analysis:

  • Identify tissue-specific co-regulators of ABCC2

  • Map functional partners varying between tissues

  • Correlate with tissue-specific substrate profiles

• Single-cell resolution studies:

  • Use antibodies for flow cytometry and single-cell sorting

  • Perform single-cell RNA-seq with ABCC2 protein correlation

  • Identify cellular heterogeneity within tissues

• Physiological context variations:

  • Compare ABCC2 function under normal vs. pathological conditions

  • Assess regulation during inflammation, oxidative stress, and disease states

  • Document adaptive responses to xenobiotic exposure

• Developmental expression patterns:

  • Map ABCC2 expression changes during organ development

  • Correlate with acquisition of detoxification capacity

  • Compare with other ABC transporters' developmental regulation