ABCB4 Antibody

What are the validated applications for ABCB4 antibodies?

ABCB4 antibodies have been validated for several research applications, primarily Western Blot (WB) and immunohistochemistry. According to available data, ABCB4 antibodies such as the 27726-1-AP demonstrate reactivity with human samples in Western Blot applications with recommended dilution ranges of 1:1000-1:5000 . Some antibodies, like PACO24684, have also been validated for ELISA applications . When selecting an ABCB4 antibody, researchers should verify the specific applications that have been validated for their antibody of interest, as validation varies between manufacturers and clones.

What is the appropriate storage protocol for ABCB4 antibodies?

ABCB4 antibodies are typically stored at -20°C and remain stable for one year after shipment. They are generally supplied in a storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For optimal performance, unnecessary freeze-thaw cycles should be avoided. Most manufacturers indicate that aliquoting is unnecessary for -20°C storage, though this may vary by product. Some antibody preparations, particularly those in smaller sizes (20μl), may contain 0.1% BSA as a stabilizer .

How should I determine the optimal antibody concentration for my experiment?

The optimal concentration of ABCB4 antibody varies depending on the specific application and experimental conditions. While manufacturers provide recommended dilution ranges (e.g., 1:1000-1:5000 for Western Blot) , it is advisable to titrate the antibody in your specific testing system. Factors that influence optimal concentration include:

• Cell or tissue type being analyzed

• Protein expression level of ABCB4 in your samples

• Detection method employed

• Background interference in your experimental system

A titration series using positive control samples (such as HeLa, HepG2, or MCF-7 cells, which are known to express ABCB4) should be performed to determine the optimal antibody concentration that provides maximum specific signal with minimal background.

What are the appropriate positive controls for ABCB4 antibody validation?

When validating ABCB4 antibodies, several cell lines serve as reliable positive controls:

These cell lines have been tested and validated as positive controls for ABCB4 detection by Western Blot . For more stringent validation, researchers may also consider using ABCB4 knockout or knockdown models as negative controls to confirm antibody specificity .

How can I differentiate between ABCB4 and other closely related ABC transporters when using antibodies?

Distinguishing ABCB4 from other ABC transporters, particularly ABCB1 (MDR1/P-glycoprotein), represents a significant challenge due to their structural similarity. To ensure specificity:

• Select antibodies raised against unique epitopes of ABCB4 that are not conserved in other ABC transporters

• Use antibodies validated with knockout or knockdown controls

• Consider cross-reactivity data provided by manufacturers

• Employ multiple antibodies targeting different epitopes of ABCB4 for confirmation

Research has demonstrated the importance of this distinction, as seen in studies using Abcb1 knockout MDCKII-ABCB4 cell lines that completely lack Abcb1 background activity but maintain ABCB4 activity . When investigating potential drug interactions with ABCB4, such cell models help distinguish ABCB4-specific effects from those mediated by related transporters.

What are the best methods for detecting ABCB4 localization at the plasma membrane versus intracellular retention?

Detecting ABCB4 localization is crucial for studies investigating trafficking-defective ABCB4 variants. Recent research has developed sophisticated approaches including:

• Dual labeling strategy: Using antibodies against total ABCB4 (visualized in red) and plasma membrane ABCB4 (visualized in green) with confocal microscopy

• High-content screening (HCS) approach: Employing automated confocal microscopy with software-assisted image analysis for quantifying surface versus total ABCB4 expression

• Cell surface biotinylation: Labeling only surface proteins followed by pull-down and immunoblotting for ABCB4

The HCS approach is particularly valuable for drug screening studies seeking compounds that can correct trafficking-defective ABCB4 variants. This method allows quantification of the percentage of cells with surface ABCB4 expression following treatment with potential therapeutic agents .

How can I assess ABCB4 function beyond protein expression?

While antibodies primarily detect ABCB4 protein expression, assessing ABCB4 function requires additional methodologies:

• Phospholipid transport assays: Measuring the translocation of fluorescently labeled phospholipids across cell membranes

• ATP hydrolysis assays: Quantifying ATPase activity associated with ABCB4 function

• Digoxin transport assays: Particularly useful in cell lines like MDCKII-ABCB4 for measuring ABCB4-mediated transport

• In vivo rescue experiments: Evaluating functional restoration in disease models, such as PFIC3 mouse models, after ABCB4 mRNA therapy

Recent research demonstrated that functional ABCB4 protein, translated from synthetic human ABCB4 mRNA, could restore phospholipid transport in cultured cells and in PFIC3 mouse livers , highlighting the importance of functional assays alongside protein expression analysis.

What are the common issues when detecting ABCB4 by Western blot and how can they be resolved?

Detecting ABCB4 by Western blot can present several challenges:

Published protocols recommend using 50-100 μg of total protein for ABCB4 detection, separating samples on 4-12% SDS-PAGE gels, and transferring to nitrocellulose membranes using standard procedures . The Odyssey Blocking Buffer has been successfully used to reduce background, followed by probing with specific anti-ABCB4 antibodies at concentrations of 0.2-1 μg/ml .

How should I validate ABCB4 antibody specificity?

Thorough validation of ABCB4 antibody specificity is essential for reliable research outcomes. A comprehensive validation approach includes:

• Genetic controls: Testing in ABCB4 knockout or knockdown models versus wild-type samples

• Peptide competition assays: Pre-incubating the antibody with the immunizing peptide to block specific binding

• Multiple antibody comparison: Using different antibodies targeting distinct epitopes of ABCB4

• Cross-species reactivity assessment: Verifying specificity across species if working with non-human models

Published studies have employed multiple ABCB4-specific antibodies (PII26, C-219, HPA053288, LS-Bio357461) to confirm specificity . The P3-II26 antibody has been noted to be specific for human ABCB4 but not mouse ABCB4, while C219 shows cross-reactivity to both human and mouse ABCB4 , making antibody selection critical for cross-species studies.

How can ABCB4 antibodies be utilized in identifying correctors for traffic-defective ABCB4 variants?

ABCB4 antibodies play a crucial role in high-content screening (HCS) approaches for identifying pharmacological correctors of intracellularly-retained ABCB4 variants. Recent research has developed sophisticated methodologies:

• Fluorescence-based readouts: Using dual labeling with mCherry-tagged ABCB4 (total protein) and anti-FLAG antibodies (surface expression)

• Automated image analysis: Software-assisted quantification of the percentage of cells with surface ABCB4 expression after drug treatment

• Maturation assessment: Western blot analysis to evaluate the conversion of core-glycosylated to mature forms of ABCB4

This approach has successfully identified compounds like cyclosporin A (CsA), itraconazole, and posaconazole as potential correctors for certain ABCB4 variants, with varying efficacy (e.g., itraconazole yielded 49.3 ± 4.0% surface ABCB4-positive cells at 10 μM) . The methodology allows for comprehensive screening of chemical libraries to identify novel therapeutic candidates for ABCB4-related diseases.

What are the considerations for using ABCB4 antibodies in therapeutic mRNA evaluation studies?

When evaluating synthetic mRNA therapies targeting ABCB4 deficiencies, antibodies serve as critical tools for assessing protein expression and functionality:

• Translation efficiency assessment: Measuring the expression of full-length ABCB4 protein following mRNA transfection

• Intracellular localization: Determining whether the translated protein correctly localizes to the plasma membrane

• Species-specific detection: Using antibodies that can distinguish between endogenous and therapeutically introduced ABCB4

Research has demonstrated that chemically and genetically modified mRNA variants encoding human ABCB4 (hABCB4 mRNA) encapsulated in lipid nanoparticles can be evaluated using Western blotting with specific antibodies like P3-II26 (specific to human ABCB4) or C219 (cross-reactive to both human and mouse ABCB4) . These approaches confirm the expression of the expected ~140 kDa protein from the therapeutic mRNA constructs .

How do different detection systems compare for ABCB4 antibody-based assays?

Researchers have several detection systems available for ABCB4 antibody-based assays, each with distinct advantages:

For quantitative applications, IR-labeled goat anti-rabbit secondary antibodies (IRDye 800CW) combined with the Odyssey CLx imaging instrument have been successfully employed to assess ABCB4 protein expression levels , providing more reliable quantification than traditional chemiluminescence-based methods.

How can ABCB4 antibodies be used to study liver diseases associated with ABCB4 dysfunction?

ABCB4 antibodies are instrumental in investigating liver diseases associated with ABCB4 dysfunction, particularly progressive familial intrahepatic cholestasis type 3 (PFIC3) and low-phospholipid-associated cholelithiasis syndrome (LPAC):

• Expression pattern analysis: Evaluating ABCB4 expression in patient liver biopsies compared to healthy controls

• Protein mislocalization assessment: Determining whether mutant ABCB4 properly localizes to the canalicular membrane of hepatocytes

• Therapeutic response monitoring: Measuring changes in ABCB4 expression and localization following treatment

Studies using ABCB4 antibodies have demonstrated that homozygous disruption of the ABCB4 gene in mouse models recapitulates human PFIC3 phenotypes . These models serve as valuable platforms for testing potential therapeutic approaches, including mRNA therapy, which has shown promise in restoring normal liver function by promoting hepatocyte-driven liver regeneration .

What considerations are important when using ABCB4 antibodies across different species models?

When using ABCB4 antibodies across different species models, researchers should consider several factors:

• Species-specific reactivity: Verify the antibody's reactivity with the target species; many antibodies are human-specific or have limited cross-reactivity

• Epitope conservation: Check whether the antibody's target epitope is conserved across species

• Validation in the species of interest: Confirm antibody specificity in your model organism

• Selection of appropriate controls: Use positive and negative controls from the same species

Research has utilized species-specific antibodies, such as P3-II26 (specific to human ABCB4) and C219 (cross-reactive to both human and mouse ABCB4) , to distinguish between endogenous and exogenously expressed ABCB4 in mouse models. This approach is particularly valuable when studying human ABCB4 expression in mouse disease models.

How are ABCB4 antibodies being used in high-throughput screening for therapeutic discovery?

ABCB4 antibodies have become essential tools in high-throughput screening approaches for identifying potential therapeutics for ABCB4-related diseases:

• High-content screening (HCS): Using fluorescently labeled antibodies to quantify ABCB4 trafficking to the cell surface following compound treatment

• Automated image analysis: Software-assisted quantification of surface versus total ABCB4 expression

• Dose-response curve generation: Systematic evaluation of compound efficacy at varying concentrations

Recent research employed a miniaturized and automated approach for screening the Prestwick chemical library (1280 compounds) to identify correctors for ABCB4-I541F trafficking defects . This methodology successfully identified several hits, including cyclosporin A (31.6 ± 4.9% surface ABCB4-positive cells at 10 μM), demonstrating the value of antibody-based screening approaches in therapeutic discovery .

What novel research techniques incorporate ABCB4 antibodies for studying membrane transport?

Innovative research techniques incorporating ABCB4 antibodies have expanded our understanding of membrane transport mechanisms:

• In vitro transport assays: Development of Abcb1 knockout MDCKII-ABCB4 cell lines that eliminate background activity from related transporters

• Drug interaction studies: Using ABCB4 antibodies to correlate protein expression with functional transport in the presence of potential inhibitors or enhancers

• Structural biology applications: Combining antibody-based detection with structural techniques to understand ABCB4 conformational changes during transport cycles

A unique in vitro assay using the Abcb1KO MDCKII-ABCB4 cell line has been developed to investigate ABCB4 transport function specifically . This system allows researchers to identify ABCB4-specific drugs by excluding overlapping specificity from Abcb1, representing a significant advancement in the field of ABC transporter research .