ABCC4 Antibody

What is ABCC4 and why is it a significant research target?

ABCC4, also known as MRP4 (Multidrug Resistance Protein 4), is an ATP-dependent transporter belonging to the ATP-binding cassette (ABC) family. This membrane protein actively extrudes physiological compounds and xenobiotics from cells . Its significance lies in its role in exporting nucleoside monophosphate derivatives and cyclic nucleotides, making it crucial in drug metabolism and cellular signaling pathways . ABCC4 is widely expressed across tissues, with particularly high levels in prostate, while being barely detectable in liver . Impaired ABCC4 function has been associated with increased hematopoietic toxicity during thiopurine therapy, highlighting its clinical relevance .

What structural characteristics define ABCC4 protein?

ABCC4 exhibits the typical domain organization of eukaryotic ABC transporters. In humans, the canonical protein consists of 1325 amino acid residues with a molecular mass of approximately 149.5 kDa . Its structure includes two membrane-spanning domains (MSD1 and MSD2), each containing six transmembrane helices, connected by cytoplasmic loops (CLs) . Additionally, ABCC4 contains two cytosolic nucleotide binding domains (NBDs) that bind and hydrolyze ATP to power substrate transport . The protein undergoes glycosylation as demonstrated by PNGase F treatment, which confirms its post-translational modification status . Up to four different isoforms have been reported for this protein, adding complexity to its structural analysis .

How should researchers select the appropriate ABCC4 antibody for their experiments?

When selecting an ABCC4 antibody, researchers should consider multiple factors including:

• Experimental application: Different applications require antibodies with specific characteristics. For Western blotting, high affinity and specificity are crucial, while immunohistochemistry may require antibodies that recognize native epitopes resistant to fixation procedures .

• Species reactivity: Confirm that the antibody recognizes ABCC4 in your experimental species. While orthologs exist in mouse, rat, frog, zebrafish, chimpanzee, and chicken, not all antibodies cross-react across species . For example, some human or mouse ABCC4 antibodies do not cross-react with zebrafish Abcc4 .

• Epitope location: Consider whether the antibody targets an extracellular domain (useful for flow cytometry on intact cells) or an intracellular domain (better for Western blotting and fixed-cell applications) .

• Validation data: Prioritize antibodies with extensive validation data including positive controls from tissues known to express ABCC4 (e.g., prostate) and negative controls from low-expressing tissues (e.g., liver) .

What are the optimal protocols for using ABCC4 antibodies in Western blotting?

For optimal Western blotting with ABCC4 antibodies, researchers should follow these methodological considerations:

• Sample preparation: Due to ABCC4's membrane localization, use strong detergent-based lysis buffers (e.g., RIPA) supplemented with protease inhibitors to efficiently extract the protein. For tissue samples, homogenization should be thorough to ensure complete membrane protein extraction .

• Gel selection: Given ABCC4's large size (149.5 kDa), use low percentage (6-8%) SDS-PAGE gels or gradient gels for better resolution of high molecular weight proteins .

• Transfer conditions: Employ wet transfer methods with extended transfer times (overnight at low voltage) to ensure complete transfer of large proteins like ABCC4 .

• Blocking and antibody incubation: 5% non-fat milk or BSA in TBST is typically effective. Primary antibody dilutions range from 1:500 to 1:2000 depending on the specific antibody, with overnight incubation at 4°C yielding best results .

• Detection considerations: ABCC4 often appears as a diffuse band due to glycosylation. To confirm specificity, PNGase F treatment can be used to demonstrate a mobility shift consistent with deglycosylation .

• Controls: Include positive control samples from high-expressing tissues (prostate) and negative controls from low-expressing tissues (liver) .

How can ABCC4 antibodies be effectively used in immunohistochemistry?

Effective immunohistochemistry (IHC) with ABCC4 antibodies requires specific methodological approaches:

What considerations are important when using ABCC4 antibodies in flow cytometry?

Flow cytometry with ABCC4 antibodies presents unique challenges due to the membrane localization of this protein:

• Cell preparation: Gentle cell dissociation methods (e.g., enzyme-free dissociation buffers) help preserve membrane integrity and surface epitopes .

• Fixation considerations: If fixation is necessary, use mild fixatives (0.5-2% paraformaldehyde) to maintain epitope accessibility while preserving cell morphology .

• Permeabilization strategies: For intracellular domains, selective permeabilization with saponin (0.1%) allows antibody access while maintaining membrane protein localization .

• Antibody selection: Choose antibodies specifically validated for flow cytometry applications, as not all ABCC4 antibodies perform consistently across different techniques .

• Gating strategy: Implement proper compensation and gating strategies to distinguish true ABCC4 signal from autofluorescence, particularly important in tissues with high endogenous fluorescence .

• Controls: Include fluorescence-minus-one (FMO) controls, isotype controls, and cells with known ABCC4 expression levels to establish detection thresholds and confirm specificity .

How can researchers use ABCC4 antibodies to study membrane localization and trafficking?

Investigating ABCC4 membrane localization and trafficking requires sophisticated approaches:

• Live cell imaging techniques: Combining ABCC4 antibodies with plasma membrane markers (e.g., wheat germ agglutinin-lectin) and subcellular compartment markers (e.g., calreticulin for ER) allows visualization of protein trafficking pathways .

• Co-localization analysis: Quantitative co-localization analysis using confocal microscopy can reveal relationships between ABCC4 and other cellular components. This approach has been valuable for evaluating ABCC4 mutants such as T796M, which demonstrates altered localization patterns compared to wild-type protein .

• Surface biotinylation assays: These assays distinguish plasma membrane-localized ABCC4 from intracellular pools, providing quantitative measures of trafficking efficiency .

• Temperature-sensitive trafficking studies: Conducting experiments at different temperatures (e.g., 28°C vs. 37°C) can reveal temperature-dependent folding and trafficking defects, as demonstrated with ABCC4 CL3 mutants .

• Pulse-chase experiments: These experiments track newly synthesized ABCC4 through the secretory pathway, allowing identification of trafficking bottlenecks or quality control checkpoints .

What methods are available to study ABCC4 post-translational modifications?

ABCC4 undergoes several post-translational modifications that can be studied using specialized approaches:

• Glycosylation analysis: Treatment with glycosidases like PNGase F followed by Western blotting reveals the extent of N-linked glycosylation. This approach has been used to demonstrate that both human and zebrafish ABCC4 are glycoproteins .

• Site-directed mutagenesis: Mutation of potential modification sites (e.g., glycosylation, phosphorylation) followed by functional analysis helps determine the significance of specific modifications .

• Mass spectrometry approaches: Immunoprecipitation of ABCC4 followed by mass spectrometry analysis can identify specific modification sites and their stoichiometry .

• Phospho-specific antibodies: For known phosphorylation sites, phospho-specific antibodies can track regulatory modifications in response to cellular signaling .

• In vitro translation systems: Comparing in vitro translated ABCC4 (lacking most post-translational modifications) with cellular expressed protein can highlight the contribution of modifications to protein size, stability, and localization .

How can researchers investigate protein-protein interactions involving ABCC4?

Studying ABCC4's interactions with other proteins requires specialized methodologies:

• Co-immunoprecipitation: Using ABCC4 antibodies to precipitate the protein complex followed by Western blotting for potential interacting partners. This technique has revealed interactions between cytoplasmic loops and nucleotide binding domains .

• Proximity ligation assays: These assays can detect protein interactions in situ with high sensitivity, allowing visualization of interaction events in their native cellular context .

• Molecular modeling and mutagenesis: Computational models can predict interaction sites (e.g., between CL3 and NBD1), which can then be verified through site-directed mutagenesis and functional assays .

• FRET/BRET approaches: These techniques detect protein interactions through energy transfer between fluorophores, allowing dynamic analysis of interactions in living cells .

• Domain mapping studies: Systematic analysis of the contribution of different ABCC4 domains to protein interactions, as demonstrated in studies of the cytoplasmic loop 3 (CL3) and its role in proper protein folding and expression .

What are common issues when using ABCC4 antibodies and how can they be resolved?

How should researchers interpret conflicting ABCC4 antibody data?

When faced with conflicting data from ABCC4 antibody experiments, researchers should:

• Evaluate antibody characteristics: Different antibodies may recognize distinct epitopes or isoforms. For example, antibodies targeting different domains may yield varying results if certain domains are masked in protein complexes or affected by post-translational modifications .

• Consider experimental conditions: Variations in sample preparation, fixation methods, and detection systems can significantly impact results. For instance, membrane proteins like ABCC4 are particularly sensitive to extraction and solubilization conditions .

• Validate with complementary approaches: Confirm antibody-based findings with alternative methods such as mRNA expression analysis, tagged protein expression, or functional assays .

• Account for biological variability: ABCC4 expression can vary across tissues, developmental stages, and disease states. The protein shows particularly high expression in prostate but is barely detectable in liver .

• Employ multiple antibodies: Using antibodies that recognize different epitopes can provide a more complete picture of ABCC4 expression and localization .

What essential controls should be included in ABCC4 antibody experiments?

Robust ABCC4 antibody experiments require comprehensive controls:

• Positive tissue controls: Include samples from tissues known to express high levels of ABCC4, such as prostate, to confirm detection capability .

• Negative tissue controls: Use tissues with minimal ABCC4 expression, such as liver, to establish background levels and specificity .

• Genetic controls: When available, utilize ABCC4 knockout or knockdown models to verify antibody specificity .

• Epitope blocking: Pre-incubation of antibodies with immunizing peptides should abolish specific staining in peptide competition assays .

• Expression constructs: Wild-type and mutant ABCC4 expression constructs can serve as defined positive controls, as demonstrated in studies of ABCC4 CL3 mutations .

• Processing controls: For glycoproteins like ABCC4, include deglycosylation controls (PNGase F treatment) to confirm protein identity based on molecular weight shifts .

How are ABCC4 antibodies used in cancer research?

ABCC4 antibodies have become instrumental in cancer research through several applications:

• Expression profiling: Immunohistochemical analysis of ABCC4 across different tumor types and stages helps establish correlations with clinical outcomes. The high expression of ABCC4 in prostate tissue makes it particularly relevant for prostate cancer studies .

• Drug resistance mechanisms: ABCC4 can export various anticancer drugs, and antibody-based detection helps track its expression changes during treatment and relapse. This approach has revealed ABCC4's role in resistance to nucleoside-based chemotherapeutics .

• Biomarker development: Quantitative analysis of ABCC4 expression using calibrated antibody-based assays can help identify patient subgroups likely to respond to specific therapies .

• Subcellular distribution analysis: Changes in ABCC4 localization (membrane versus cytoplasmic) may indicate altered function in cancer cells, detectable through careful immunostaining protocols .

• Therapeutic target validation: Antibody-based detection of ABCC4 helps validate the effectiveness of interventions designed to modulate its expression or function in cancer models .

What role does ABCC4 play in drug metabolism and resistance studies?

ABCC4's function as an efflux transporter makes it central to drug metabolism research:

• Thiopurine metabolism: ABCC4 antibodies help track protein expression in relation to thiopurine drug toxicity, where impaired ABCC4 function is associated with increased hematopoietic toxicity during therapy .

• Transport activity correlation: Combining antibody-based quantification of ABCC4 with functional transport assays helps establish structure-function relationships, particularly for variants with altered trafficking like the T796M mutation .

• Induction studies: Antibody-based detection methods reveal how ABCC4 expression changes in response to drug exposure, environmental factors, or disease conditions .

• Species differences: Comparing ABCC4 expression across species using species-specific antibodies helps translate findings between model organisms and humans. Important to note that some human or mouse ABCC4 antibodies do not cross-react with zebrafish Abcc4 .

• Polymorphism impact assessment: ABCC4 antibodies can help determine how genetic variants affect protein expression and membrane localization, which may explain interindividual differences in drug response .

How can researchers investigate ABCC4 structural domains and their functional significance?

Investigating ABCC4 structural domains requires sophisticated approaches:

• Domain-specific antibodies: Antibodies targeting specific domains (MSDs, NBDs, CLs) help determine their accessibility and structural integrity in different cellular contexts .

• Mutation analysis: Site-directed mutagenesis of conserved residues followed by antibody-based detection helps determine their importance for expression and localization. This approach revealed the crucial role of threonine in the conserved TLHN motif in cytoplasmic loop 3 .

• Chimeric protein analysis: Creating chimeric proteins between ABCC4 and related transporters helps map domains crucial for specific functions, with antibodies confirming expression and localization .

• Temperature-sensitive folding: Some domain mutations (e.g., in CL3) create temperature-sensitive folding defects, detectable as differences in antibody-detected expression at different temperatures (28°C vs. 37°C) .

• Molecular dynamics simulations: Computational approaches complemented by antibody-based validation help predict how specific amino acid substitutions affect domain stability and interactions. This approach successfully predicted the stability effects of various substitutions at position 804 in zebrafish Abcc4 .

What emerging technologies might enhance ABCC4 antibody-based research?

Several cutting-edge technologies promise to advance ABCC4 research:

• Super-resolution microscopy: Techniques like STORM and PALM can reveal nanoscale organization of ABCC4 in membrane microdomains, providing insights into functional clustering not visible with conventional microscopy .

• Single-cell proteomics: Antibody-based detection of ABCC4 at the single-cell level can reveal heterogeneity within tissues and tumors that may be masked in bulk analyses .

• Organoid models: Three-dimensional tissue cultures provide more physiologically relevant contexts for studying ABCC4 localization and function, with antibodies enabling detailed expression mapping .

• CRISPR-engineered reporter systems: Endogenously tagged ABCC4 allows correlation between antibody-based detection and functional studies without overexpression artifacts .

• Antibody engineering: Development of recombinant antibody fragments with enhanced tissue penetration and reduced background could improve detection sensitivity in complex samples .

How might understanding ABCC4 structure-function relationships lead to therapeutic applications?

Research into ABCC4 structure-function relationships has significant therapeutic implications:

• Small molecule modulator development: Detailed understanding of critical domains, such as the cytoplasmic loop 3 and its interaction with NBD1, provides targets for drugs aimed at modulating ABCC4 function .

• Personalized medicine approaches: Identifying how variants affect ABCC4 function helps predict individual responses to drugs transported by this protein, as demonstrated by the impact of CL3 mutations on protein expression and localization .

• Rescue of trafficking-deficient variants: Based on findings with temperature-sensitive mutants, chemical chaperones might rescue expression of certain ABCC4 variants, similar to approaches used with CFTR mutations .

• Combination therapy rationales: Understanding how ABCC4 contributes to drug resistance provides a scientific basis for combination therapies that include ABCC4 inhibitors .

• Biomarker development: ABCC4 expression or localization patterns detected by specific antibodies could serve as biomarkers for drug sensitivity or disease progression .