ABCC4 Antibody

What are the common applications of ABCC4 antibodies in research?

ABCC4 antibodies are widely used in multiple applications including Western blot (WB), immunohistochemistry (IHC-P), immunofluorescence/immunocytochemistry (IF/ICC), and enzyme-linked immunosorbent assay (ELISA) . Western blotting is particularly common for detecting ABCC4 protein expression levels in various cell and tissue types. These antibodies enable researchers to investigate ABCC4's role in drug resistance, cellular localization, and physiological functions across different experimental models.

What is the subcellular localization of ABCC4 and how can antibodies help determine this?

ABCC4 primarily localizes to the plasma membrane where it functions as an efflux transporter . Antibodies are crucial tools for determining ABCC4's subcellular localization through techniques such as immunofluorescence microscopy and cellular fractionation followed by Western blotting. For instance, researchers have used surface membrane biotinylation with NHS-SS-biotin followed by streptavidin bead isolation and immunoblotting with ABCC4 antibodies to confirm the membrane localization of ABCC4 in platelets . Confocal microscopy with ABCC4 antibodies has also been used to visualize its distribution in three dimensions, showing its presence at the plasma membrane .

Which species reactivity should I consider when selecting an ABCC4 antibody?

When selecting an ABCC4 antibody, consider the species compatibility based on your experimental model. Many commercially available ABCC4 antibodies show reactivity with human, mouse, and rat samples . It's worth noting that some antibodies might not cross-react between species - for example, some human/mouse ABCC4 antibodies don't recognize zebrafish Abcc4 . Always verify the species reactivity in the antibody datasheet and, if possible, validate it with positive and negative controls from your species of interest.

How do I validate the specificity of an ABCC4 antibody?

Validating ABCC4 antibody specificity involves multiple approaches:

• Genetic controls: Use ABCC4 knockout/knockdown models alongside wild-type samples. Research shows that CRISPR-Cas9 knockout of ABCC4 in K562 cells eliminated antibody detection, confirming specificity .

• Recombinant overexpression: Compare cells transfected with ABCC4 expression vectors to control cells. Increased signal in ABCC4-overexpressing cells supports antibody specificity .

• Epitope analysis: Check if the antibody recognizes a unique epitope within ABCC4. For example, antibodies raised against amino acids 1067-1325 of human ABCC4 target a specific region of the protein .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide before staining to block specific binding.

• Multiple detection methods: Confirm results across different techniques (e.g., WB, IHC, IF) to ensure consistent findings.

What are the differences between monoclonal and polyclonal ABCC4 antibodies, and when should I use each?

Choose monoclonal antibodies when:

• High specificity is crucial (e.g., distinguishing ABCC4 from other ABC transporters)

• Reproducibility across experiments is essential

• Quantitative analysis is planned

Choose polyclonal antibodies when:

• Higher sensitivity is needed to detect low abundance ABCC4

• Different conformational states of ABCC4 need to be recognized

• Working with species where ABCC4 epitope conservation is uncertain

How can I ensure my ABCC4 antibody recognizes the correct isoform?

ABCC4 can exist in multiple isoforms , and ensuring antibody recognition of the correct isoform requires several considerations:

• Epitope location: Check whether the antibody epitope is present in all isoforms or is isoform-specific. The exact antibody epitope should be provided in product documentation.

• Isoform-specific controls: When studying specific isoforms, use positive controls expressing only that isoform (e.g., recombinant expression systems).

• Western blot analysis: Look for distinct molecular weight bands corresponding to different isoforms. The canonical human ABCC4 protein has 1325 amino acid residues with a mass of 149.5 kDa .

• RT-PCR verification: Complement antibody studies with RT-PCR using isoform-specific primers to confirm the presence of specific isoform mRNAs in your experimental system.

• Immunoprecipitation followed by mass spectrometry: For definitive isoform identification, immunoprecipitate ABCC4 with your antibody and analyze the protein by mass spectrometry.

What is the optimal protocol for detecting ABCC4 by Western blot?

Based on published research protocols , an optimized Western blot procedure for ABCC4 detection includes:

• Sample preparation:

  • For cell lines: Lyse cells in buffer containing 1% SDS, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and protease inhibitors

  • For tissues: Homogenize in the same buffer (1:10 w/v)

  • For membrane enrichment: Consider isolating membrane fractions

• Protein separation:

  • Use 7.5% or 8% SDS-PAGE gels (ABCC4 is a large protein ~149.5 kDa)

  • Load 20-50 μg of total protein per lane

  • Include positive and negative controls

• Transfer conditions:

  • Transfer to PVDF or nitrocellulose membranes (PVDF often preferred for large proteins)

  • Use wet transfer at 100V for 2 hours or overnight at 30V, 4°C

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with primary ABCC4 antibody (typically 1:500-1:1000 dilution) overnight at 4°C

  • Wash 3-5 times with TBST

  • Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) detection system

  • Exposure time varies depending on protein abundance

How can I optimize ABCC4 detection in immunohistochemistry?

For optimal ABCC4 detection in formalin-fixed paraffin-embedded (FFPE) tissues:

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Pressure cook for 10-20 minutes or microwave for 15-20 minutes

• Blocking:

  • Block endogenous peroxidase activity with 3% H₂O₂ for 10-15 minutes

  • Block non-specific binding with 5-10% normal serum from secondary antibody species for 1 hour

• Primary antibody:

  • Use ABCC4 antibody at 1:50-1:200 dilution

  • Incubate overnight at 4°C in a humidified chamber

• Detection system:

  • Use appropriate secondary antibody system (e.g., polymer detection system)

  • Develop with DAB chromogen

  • Counterstain with hematoxylin

• Controls:

  • Include positive control tissues (prostate is known to express high levels of ABCC4)

  • Include negative controls (primary antibody omitted)

  • Consider using ABCC4 knockout/knockdown tissues as specificity controls

What is the best way to detect ABCC4 subcellular localization?

To accurately determine ABCC4 subcellular localization, combine multiple approaches:

• Immunofluorescence microscopy:

  • Fix cells with 4% paraformaldehyde for 15 minutes

  • Permeabilize with 0.1% Triton X-100 for 5-10 minutes

  • Block with 5% BSA for 1 hour

  • Incubate with ABCC4 primary antibody overnight at 4°C

  • Use fluorophore-conjugated secondary antibodies

  • Co-stain with established subcellular markers:

    • Plasma membrane: Na⁺/K⁺-ATPase or wheat germ agglutinin-lectin

    • ER: Calreticulin

    • Golgi: GM130

  • Analyze using confocal microscopy with Z-stack imaging for 3D localization

• Subcellular fractionation and Western blotting:

  • Separate cellular components (membrane, cytosol, organelles)

  • Perform Western blotting on each fraction

  • Use fractionation markers to confirm purity

• Surface biotinylation:

  • Incubate live cells with membrane-impermeable NHS-SS-biotin

  • Isolate biotinylated proteins with streptavidin beads

  • Detect ABCC4 by Western blotting in biotinylated (surface) fraction

Research has demonstrated ABCC4 primarily localizes to the plasma membrane, with potential additional locations in extracellular vesicles (EVs) derived from colorectal cancer cells .

Why might I detect multiple bands or unexpected molecular weights with my ABCC4 antibody?

Multiple bands or unexpected molecular weights in ABCC4 Western blots could result from:

• Post-translational modifications: ABCC4 undergoes glycosylation, which can be confirmed by PNGase F treatment. Both human ABCC4 and zebrafish Abcc4 show band shifts after PNGase F treatment .

• Protein degradation: Incomplete protease inhibition during sample preparation may cause degradation products. Ensure fresh protease inhibitors are used in all buffers.

• Isoforms: ABCC4 can exist in multiple isoforms with different molecular weights. Up to 4 different isoforms have been reported .

• Cross-reactivity: The antibody may recognize related ABC transporters. Validate using ABCC4 knockout/knockdown samples.

• Protein aggregation: Incomplete sample denaturation may cause aggregates. Try more stringent denaturation conditions (higher SDS concentration, longer heating).

To address these issues:

• Include PNGase F-treated samples to identify glycosylation effects

• Use fresh samples with complete protease inhibitor cocktails

• Compare with recombinant ABCC4 protein standards

• Validate with genetic controls (ABCC4 knockout/knockdown)

How can I improve sensitivity when ABCC4 is expressed at low levels?

When ABCC4 is expressed at low levels, consider these approaches to improve detection:

• Sample enrichment techniques:

  • Perform membrane fractionation to concentrate ABCC4 (as a membrane protein)

  • Use immunoprecipitation to concentrate ABCC4 before Western blotting

• Signal amplification methods:

  • Use more sensitive detection reagents (e.g., SuperSignal West Femto)

  • Consider tyramide signal amplification for IHC/IF applications

  • Try biotin-streptavidin amplification systems

• Antibody optimization:

  • Test different antibody concentrations and incubation conditions

  • Try polyclonal antibodies which may offer higher sensitivity by recognizing multiple epitopes

  • Consider using antibodies targeting different epitopes of ABCC4

• Protein loading:

  • Increase total protein loading for Western blots (up to 100 μg if necessary)

  • Load larger volumes for immunoprecipitation

• Detection systems:

  • Use digital imaging systems with adjustable exposure times

  • Consider fluorescent secondary antibodies for quantitative detection

Research shows that ABCC4 expression varies significantly between tissues and cell types, with particularly high levels in prostate and low levels in liver , requiring different detection strategies.

How can I minimize background when using ABCC4 antibodies in immunostaining?

To reduce background in ABCC4 immunostaining:

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Increase blocking time (2-3 hours or overnight)

  • Include 0.1-0.3% Triton X-100 in blocking buffer to reduce non-specific binding

• Antibody dilution:

  • Titrate primary antibody to find optimal concentration

  • Use antibody diluent containing 0.05-0.1% Tween-20

• Washing steps:

  • Increase number and duration of washes

  • Use PBS-T (PBS with 0.1% Tween-20) for more effective washing

• Endogenous enzyme blocking:

  • For IHC, block endogenous peroxidase with 3% H₂O₂

  • For IF, consider treating with 0.1-1% sodium borohydride to reduce autofluorescence

• Antibody specificity:

  • Use affinity-purified antibodies

  • Pre-absorb antibody with related proteins if cross-reactivity is suspected

• Controls:

  • Include ABCC4-negative tissues/cells

  • Include no-primary antibody controls

  • Consider peptide competition controls

How can ABCC4 antibodies be used to study drug resistance mechanisms?

ABCC4 antibodies are valuable tools for investigating drug resistance mechanisms:

• Expression correlation studies:

  • Quantify ABCC4 protein levels in drug-sensitive vs. resistant cell lines

  • Correlate ABCC4 expression with drug response in patient samples

  • Research has shown ABCC4 expression is associated with resistance to nucleoside-based drugs and certain anticancer agents

• Functional studies:

  • Monitor ABCC4 expression changes during development of drug resistance

  • Use ABCC4 antibodies in combination with inhibitors (e.g., MK571) to study functional consequences

  • Track ABCC4 localization changes in response to drug treatment

• Mechanistic investigations:

  • Immunoprecipitate ABCC4 to identify interaction partners involved in resistance

  • Use ABCC4 antibodies in ChIP assays to study transcriptional regulation during resistance development

  • Perform proximity ligation assays to visualize ABCC4 interactions with other proteins

• Clinical correlations:

  • Use ABCC4 immunohistochemistry on patient samples to correlate with treatment outcomes

  • Develop ABCC4-based biomarker panels for predicting therapy response

Research has demonstrated that ABCC4 gene amplification occurs during step-wise selection for ciprofloxacin resistance in macrophages, with progressive increases in ABCC4 protein levels detected by antibodies .

How can I use ABCC4 antibodies to study its role in cyclic nucleotide transport?

ABCC4 functions as an important cyclic nucleotide exporter, particularly for cAMP. To study this role:

• Localization in signaling microdomains:

  • Use high-resolution microscopy (STED, STORM) with ABCC4 antibodies to visualize its distribution in membrane microdomains

  • Co-localize ABCC4 with adenylyl cyclases, phosphodiesterases, and cAMP effectors

  • Research has shown ABCC4 membrane localization affects cAMP signaling involved in cell migration

• Transport assays with antibody validation:

  • Measure intracellular cAMP levels after ABCC4 inhibition (e.g., with MK571)

  • Correlate cAMP export with ABCC4 protein levels determined by immunoblotting

  • Studies have demonstrated that ABCC4 inhibition increases intracellular cAMP in colorectal cancer cells

• Stimulus-induced trafficking:

  • Monitor ABCC4 redistribution after cAMP-elevating stimuli using antibody-based imaging

  • Quantify surface vs. intracellular ABCC4 pools using biotinylation combined with antibody detection

• Structure-function analysis:

  • Use antibodies to verify expression of ABCC4 mutants with altered cyclic nucleotide transport

  • Research has identified cytoplasmic loop 3 as critical for ABCC4 expression and function

• Physiological consequences:

  • Correlate ABCC4 expression with functional readouts of cAMP signaling

  • For example, PEL-negative individuals (lacking ABCC4) show impaired platelet aggregation

What approaches can I use to study ABCC4 in extracellular vesicles (EVs)?

ABCC4 has been detected in extracellular vesicles , and studying this localization requires specialized techniques:

• EV isolation and characterization:

  • Isolate EVs using differential ultracentrifugation, size exclusion chromatography, or commercial kits

  • Verify EV purity with markers (positive: CD63, CD9, CD81; negative: cytochrome c)

  • Quantify ABCC4 in EVs using antibody-based Western blotting

• Immunoelectron microscopy:

  • Visualize ABCC4 on the surface of individual EVs using immunogold labeling with ABCC4 antibodies

  • Determine the percentage of ABCC4-positive EVs in different conditions

• Functional studies:

  • Compare ABCC4 levels in EVs from drug-sensitive vs. resistant cells

  • Investigate whether ABCC4-containing EVs can transfer drug resistance to recipient cells

  • Research has shown higher levels of ABCC4 in EVs from cells with mesenchymal characteristics

• EV proteomics:

  • Use ABCC4 antibodies for immunoprecipitation from EV lysates to identify interacting partners

  • Compare the interactome of ABCC4 in cellular membranes vs. EVs

• Physiological relevance:

  • Track the transfer of ABCC4-containing EVs between cells using antibody labeling

  • Correlate EV ABCC4 levels with disease progression or treatment response

How can I quantitatively analyze ABCC4 expression in different experimental conditions?

For rigorous quantitative analysis of ABCC4 expression:

• Western blot quantification:

  • Use digital imaging systems with linear dynamic range

  • Include standard curves with recombinant ABCC4 protein

  • Normalize to appropriate loading controls (β-actin for total lysates; Na⁺/K⁺-ATPase for membrane fractions)

  • Use at least three biological replicates

• Flow cytometry:

  • Optimize fixation and permeabilization for intracellular vs. surface ABCC4

  • Use median fluorescence intensity (MFI) for quantification

  • Include isotype controls and ABCC4-negative cells

  • Consider using calibration beads to standardize between experiments

• Immunofluorescence quantification:

  • Use confocal microscopy with identical acquisition settings

  • Measure mean fluorescence intensity in defined regions of interest

  • Analyze multiple cells (>30) across different fields

  • Use automated image analysis software for unbiased quantification

• qRT-PCR correlation:

  • Pair protein quantification with mRNA analysis

  • Research has shown that ABCC4 mRNA levels can be suppressed up to 80% in knockdown cells, with corresponding protein reduction

• Statistical analysis:

  • Apply appropriate statistical tests (t-test, ANOVA)

  • Report mean ± standard deviation or standard error

  • Consider using non-parametric tests if data is not normally distributed

How do I interpret conflicting results between different ABCC4 antibodies?

When faced with conflicting results from different ABCC4 antibodies:

• Consider epitope differences:

  • Antibodies targeting different regions may yield different results if:

    • The protein is truncated or alternatively spliced

    • Post-translational modifications mask certain epitopes

    • Protein interactions shield specific regions

• Evaluate antibody validation:

  • Check if each antibody was validated using genetic controls

  • Review published literature for antibody-specific caveats

  • Consider validating in your experimental system using siRNA knockdown

• Resolution approaches:

  • Use multiple antibodies targeting different epitopes and look for consensus

  • Complement antibody-based detection with genetic approaches (e.g., tagged ABCC4)

  • Consider orthogonal techniques (mass spectrometry, functional assays)

• Context-specific factors:

  • Cell/tissue-specific post-translational modifications

  • Experimental conditions affecting ABCC4 conformation

  • Species differences in epitope conservation

• Technical considerations:

  • Optimization requirements may differ between antibodies

  • Some antibodies may work better for specific applications (WB vs. IHC)

What are the implications of altered ABCC4 expression in disease states, and how can antibodies help elucidate these?

ABCC4 expression alterations have been implicated in several diseases, and antibodies are crucial for investigating these associations:

• Cancer:

  • ABCC4 overexpression has been detected in non-small cell lung cancer patients using antibody-based techniques

  • Antibodies have revealed that ABCC4 silencing inhibits lung cancer cell growth by affecting cell cycle progression

  • Immunohistochemistry with ABCC4 antibodies can help stratify patients and predict treatment responses

• Drug resistance:

  • Antibody detection has shown ABCC4 gene amplification correlates with increased protein expression during development of ciprofloxacin resistance

  • ABCC4 antibodies help monitor expression changes in response to chemotherapy

• Platelet function disorders:

  • ABCC4 absence defines the PEL-negative blood group and is associated with impaired platelet aggregation

  • Antibodies have confirmed ABCC4's membrane localization in platelets where it regulates cAMP export

• Metabolic disorders:

  • Research utilizing ABCC4 antibodies has demonstrated crosstalk between ABCC4 and ABCC5 in adipocytes, potentially affecting adipogenesis

  • Antibody-based detection has shown that ABCC4 knockdown alters lipid status and adipogenic gene expression

• Research approaches:

  • Tissue microarrays with ABCC4 antibodies for screening multiple patient samples

  • Correlation of ABCC4 expression with clinical parameters and outcomes

  • Longitudinal monitoring of ABCC4 changes during disease progression or treatment

Understanding ABCC4's role in these conditions opens avenues for targeted therapies and potential biomarker development, with antibodies serving as essential tools throughout the research pipeline.

How can ABCC4 antibodies be utilized in studying the structural biology of this transporter?

ABCC4 structural biology investigations with antibodies include:

• Conformational-specific antibodies:

  • Generate antibodies recognizing distinct conformational states (ATP-bound, substrate-bound, etc.)

  • Use these to trap and study specific states of the transport cycle

  • Research has highlighted the importance of cytoplasmic loop 3 (CL3) in ABCC4 structure and function

• Antibody-based protein purification:

  • Immunoaffinity purification for structural studies (cryo-EM, X-ray crystallography)

  • Isolate native ABCC4 complexes for interaction studies

• Epitope mapping:

  • Determine accessibility of various ABCC4 domains using antibody panels

  • Correlate with structural predictions and models

  • Studies have used antibodies to confirm the importance of specific amino acids (e.g., Thr-804 in zebrafish Abcc4; Thr-796 in human ABCC4) in protein folding and stability

• Fab fragments for co-crystallization:

  • Generate Fab fragments from ABCC4 antibodies

  • Use these as crystallization chaperones to stabilize specific conformations

• Localization of functional domains:

  • Map functional regions using domain-specific antibodies

  • Correlate structural features with transport function

What are the latest techniques for combining ABCC4 antibodies with other detection methods?

Advanced multi-modal detection approaches include:

• Proximity ligation assays (PLA):

  • Detect protein-protein interactions involving ABCC4

  • Requires antibodies from different species targeting ABCC4 and potential interacting partners

  • Provides spatial information about interactions at single-molecule resolution

• CRISPR-based tagging with antibody validation:

  • Endogenously tag ABCC4 with small epitopes (FLAG, HA)

  • Use well-characterized tag antibodies in combination with ABCC4 antibodies

  • Research has used CRISPR-Cas9 to validate ABCC4 antibody specificity

• Super-resolution microscopy:

  • Apply STORM, PALM, or STED microscopy with ABCC4 antibodies

  • Achieve nanometer-scale resolution of ABCC4 distribution

  • Combine with organelle markers for precise localization

• Live-cell antibody approaches:

  • Use membrane-impermeable antibodies to track surface ABCC4 dynamics

  • Develop cell-permeable miniature antibodies (nanobodies) for intracellular ABCC4 tracking

• Mass cytometry (CyTOF):

  • Label ABCC4 antibodies with metal isotopes

  • Simultaneously detect ABCC4 and dozens of other proteins

  • Ideal for heterogeneous samples and clinical specimens

These emerging techniques expand our ability to study ABCC4 biology with unprecedented precision and contextual information.

How might ABCC4 antibodies contribute to personalized medicine approaches?

ABCC4 antibodies show potential for advancing personalized medicine through:

• Predictive biomarker development:

  • Immunohistochemical assessment of ABCC4 in tumor biopsies to predict drug resistance

  • Research has shown ABCC4 expression correlates with response to nucleoside-based therapeutics

• Companion diagnostics:

  • Development of standardized ABCC4 antibody-based assays to guide treatment decisions

  • Integration into clinical laboratory workflows

• Monitoring treatment response:

  • Serial assessment of ABCC4 expression during therapy

  • Detection of emerging resistance mechanisms

• Stratification for clinical trials:

  • Selection of patients based on ABCC4 expression profiles

  • Identification of likely responders to ABCC4-targeted therapies

• Therapeutic antibody development:

  • Anti-ABCC4 antibodies as potential therapeutics to overcome drug resistance

  • Antibody-drug conjugates targeting ABCC4-expressing cells