ABCB6 Antibody

What is ABCB6 and why is it significant in cellular research?

ABCB6 is a member of the ATP-binding cassette (ABC) superfamily of transmembrane proteins that facilitate energy-dependent transport of diverse substrates across cellular membranes. This 842 amino acid protein plays a crucial role in heme synthesis and binds heme and porphyrins, assisting in their ATP-dependent uptake into mitochondria. ABCB6 is particularly significant because:

• It forms homodimers in the outer mitochondrial membrane, plasma membrane, and Golgi apparatus

• It is upregulated by cellular porphyrins

• It shows predominant expression in skeletal muscle and heart tissues

• It exists in multiple isoforms generated through alternative splicing

• It has been implicated in multidrug resistance mechanisms and various disease states

The intracellular localization of ABCB6 has been debated in the literature, with evidence supporting both mitochondrial and endo-lysosomal localization, making it an important target for subcellular transport studies .

What types of ABCB6 antibodies are commercially available for research applications?

Several types of ABCB6 antibodies are available for research applications, with varying properties:

These antibodies are available in both unconjugated forms and conjugated formats (HRP, FITC, PE, and various Alexa Fluor® conjugates), allowing flexibility for different experimental approaches .

What molecular weight should be expected when detecting ABCB6 using Western blotting?

The molecular weight of ABCB6 reported in the literature shows some variation:

This variability may be attributed to:

• Post-translational modifications

• Different isoforms resulting from alternative splicing

• Proteolytic processing during sample preparation

• Differences in expression systems and cell types

Researchers should validate the expected molecular weight in their specific experimental system using appropriate positive controls .

How should I optimize Western blotting protocols for reliable ABCB6 detection?

For optimal ABCB6 detection using Western blotting:

• Sample preparation:

  • Use fresh cell/tissue lysates with protease inhibitors

  • Include both cytosolic and membrane fractions as ABCB6 can localize to multiple cellular compartments

  • Consider enriching for the membrane fraction if detection is difficult

• Antibody selection and dilution:

  • Western Blot dilutions typically range from 1:500-1:2000 depending on the antibody

  • For Proteintech antibodies (51007-1-AP and 14996-1-AP), the recommended dilution is 1:500-1:2000

  • For GeneTex GTX48698, a 1:1,400 dilution has been reported effective

• Positive controls:

  • HeLa cells and U-251 cells have shown positive WB results with certain antibodies

  • Mouse brain tissue has been used successfully as a positive control

  • KB cells transfected with ABCB6 have been used as a positive control versus vector control

• Protocol specifics:

  • Follow vendor-specific protocols; for example, Proteintech provides specific WB protocols for their ABCB6 antibodies

  • Pay attention to transfer conditions, as ABCB6 is a membrane protein that may require optimization

What are the recommended applications and dilutions for ABCB6 antibodies?

It is recommended that each antibody be titrated in the specific testing system to obtain optimal results, as performance can be sample-dependent .

How can I validate ABCB6 antibody specificity for my experimental system?

To ensure antibody specificity for ABCB6:

• Genetic validation approaches:

  • Use ABCB6 knockdown/knockout samples as negative controls

  • The use of shRNA against ABCB6 has been documented to validate antibody specificity, as observed in proximity ligation assays (PLA) where ABCB6 shRNA significantly reduced signal compared to scramble controls

• Expression system controls:

  • Compare cells with and without ABCB6 expression (e.g., KB cells transfected with ABCB6 versus vector control)

  • Use tagged versions of ABCB6 (e.g., Flag-tagged) and confirm detection with both anti-ABCB6 and anti-tag antibodies

• Multiple antibody validation:

  • Use different antibodies targeting distinct ABCB6 epitopes to confirm consistent detection patterns

  • Verify that the observed molecular weight corresponds to predicted ABCB6 size and known post-translational modifications

• Isotype controls:

  • Perform isotype control experiments to determine specificity of signals in immunoprecipitation and Western blot experiments

How does ABCB6 contribute to multidrug resistance mechanisms, and how can antibodies help investigate this?

ABCB6 has been implicated in multidrug resistance (MDR) through several research approaches:

• Expression correlation with drug resistance:

  • Quantitative real-time RT-PCR studies in the NCI-60 human cancer cell lines identified 131 inverse correlations between ABC gene expression and drug sensitivity

  • Increased ABCB6 expression correlated with decreased toxicity of specific drugs

  • In arsenite-resistant KB cells (KAS), ABCB6 mRNA levels were 2.5-fold higher than in parental cells

• Functional validation:

  • KB cells stably transfected with ABCB6 (KB-B6) showed resistance to arsenite and cisplatin

  • This indicates a causal relationship between ABCB6 expression and drug resistance

• Investigation approaches using antibodies:

  • Western blotting with anti-ABCB6 antibodies to monitor expression levels in sensitive versus resistant cell lines

  • Immunofluorescence to determine subcellular localization changes in resistant cells

  • Co-immunoprecipitation to identify interaction partners potentially involved in resistance mechanisms

  • Proximity ligation assays to detect protein-protein interactions in situ

Understanding ABCB6's role in MDR is particularly important given its expression pattern and potential as a therapeutic target in cancer drug resistance .

What is known about ABCB6 heterodimerization with other ABC transporters, and how can this be investigated?

Recent research has identified novel heterodimeric interactions of ABCB6 with other ABC transporters:

• Identified heterodimeric pairs:

  • ABCB5β-ABCB6 and ABCB5β-ABCB9 have been confirmed as specific heterodimeric pairs through multiple experimental approaches

• Detection methodologies:

  • NanoBRET assays: Used to detect protein-protein interactions in living cells

  • Donor saturation assays: Confirmed the specificity of interactions between ABCB5β-ABCB6 and ABCB5β-ABCB9 protein pairs

  • Co-immunoprecipitation: Using antibodies against ABCB5, ABCB6, or ABCB9 to precipitate the interacting partners

• Validation in endogenous systems:

  • Interactions were confirmed in melanoma cell lines (Mel JuSo and UACC-257) that constitutively express the target proteins

  • Immunoprecipitation with either anti-ABCB5, anti-ABCB6, or anti-ABCB9 antibody brought out the interacting partners

• Controls and specificity:

  • Several control experiments were performed to rule out false positives:

    • Donor saturation assays to distinguish specific interactions from bystander effects

    • Controls without antibodies or with single antibodies

    • shRNA-mediated knockdown of ABCB6 or ABCB9 significantly reduced PLA signals

This research area is particularly important for understanding the functional implications of ABC transporter heterodimers in substrate specificity and cellular localization.

How does the N-terminal transmembrane domain (TMD0) of ABCB6 influence its localization and function?

The N-terminal transmembrane domain (TMD0) of ABCB6 plays a crucial role in its subcellular localization:

• Structural organization of ABCB6:

  • In addition to the canonical nucleotide-binding domain (NBD) and transmembrane domain (TMD), ABCB6 contains a unique N-terminal TMD (TMD0)

  • TMD0 does not show sequence homology to known proteins

• Functional studies of TMD0:

  • Molecular dissection experiments revealed that the core-ABCB6 complex devoid of TMD0 maintains:

    • Proper folding

    • Dimerization capability

    • Membrane insertion

    • ATP binding/hydrolysis functions

• Role in subcellular targeting:

  • Full-length ABCB6 is internalized from the plasma membrane through endocytosis and distributed to multivesicular bodies and lysosomes

  • Core-ABCB6 without TMD0 is retained at the plasma membrane and does not appear in Rab5-positive endosomes

  • TMD0 alone is directly targeted to lysosomes without passage through the plasma membrane

• Functional significance:

  • TMD0 represents an independently folding unit

  • It is dispensable for catalytic activity

  • It has a crucial role in the lysosomal targeting of ABCB6

This research highlights the importance of protein domains in determining subcellular localization and potentially substrate specificity of ABC transporters.

What is the role of ABCB6 in blood group systems, and how can antibodies be used to study this?

ABCB6 has been identified as the molecular basis of the Langereis blood group:

• Historical context:

  • In 1962, van der Hart and colleagues identified an antibody to a common RBC antigen related to a severe and immediate transfusion reaction

  • The phenotype, termed anti-Lan, was discovered in patients (Mr. Langereis and his brother) who experienced transfusion reactions

  • Their RBCs were non-reactive to the antibody

• Clinical significance:

  • The Lan-negative phenotype is associated with:

    • Hemolytic transfusion reactions (ranging from none to severe)

    • Hemolytic disease of the fetus and newborn (with varying severity)

• Molecular identification:

  • In 2012, Helias and colleagues developed a monoclonal antibody (anti-Lan, OSK43) that established the molecular identity of this blood group system

  • ABCB6 was identified as the protein responsible for the Langereis blood group

• Research applications using antibodies:

  • ABCB6 antibodies can be used to:

    • Screen blood samples for Lan status

    • Study the expression of ABCB6 in different tissues and cell types

    • Investigate the genetic basis of Lan-negative phenotypes through correlation with ABCB6 expression

    • Examine potential functional consequences of ABCB6 variants in blood cells

This connection between ABCB6 and the Langereis blood group represents an important translational aspect of ABCB6 research with clinical implications.

What is known about ABCB6 knockout models, and how can antibodies help validate these models?

ABCB6 knockout models have provided surprising insights into its function:

• Expected versus observed phenotypes:

  • Initial hypotheses: Given ABCB6's role in porphyrin biosynthesis, its absence was expected to be lethal

  • Surprising findings: When Abcb6-null mice were generated, they appeared phenotypically normal

• Mild hematological phenotypes:

  • Elevated erythroid protoporphyrin IX (PPIX) levels

  • Modest alterations in mean corpuscular volume and hematocrit

  • Findings suggesting mild anemia

  • Elevated zinc-protoporphyrin IX (Zn-PPIX), a byproduct formed when Fe+2 is not readily available for heme synthesis

• Compensatory mechanisms:

  • Abcb6-null mice showed compensatory increases in genes positively influencing porphyrin biosynthesis and iron homeostasis (e.g., FECH and EKLF)

  • These adaptations help maintain near-normal function despite ABCB6 absence

• Validation approaches using antibodies:

  • Western blot analysis to confirm the absence of ABCB6 protein in knockout tissues

  • Immunohistochemistry to examine potential alterations in expression of related transporters

  • Co-immunoprecipitation studies to identify potential compensatory protein interactions

Like other ABC transporter knockout models (e.g., ABCB1), ABCB6-null mice appear normal until challenged, suggesting redundancy or compensatory mechanisms in normal physiological conditions .

Why might there be discrepancies in the observed molecular weight of ABCB6 in Western blot experiments?

Several factors contribute to the variability in ABCB6 molecular weight observed in Western blots:

Potential causes of these discrepancies include:

• Alternative splicing: ABCB6 exists in multiple isoforms generated through alternative splicing

• Post-translational modifications: Glycosylation, phosphorylation, or other modifications can alter apparent molecular weight

• Protein processing: Evidence of truncated forms (79 kDa) alongside full-length protein has been observed

• Dimerization: Incomplete denaturation may lead to detection of dimeric forms

• Tissue/cell-specific differences: The observed molecular weight may vary depending on the sample source

When troubleshooting unexpected molecular weights, researchers should:

• Use multiple antibodies targeting different epitopes

• Include appropriate positive controls

• Consider sample preparation methods to ensure complete denaturation

• Verify results with alternative detection methods

How can I optimize co-immunoprecipitation protocols for studying ABCB6 interactions?

For successful co-immunoprecipitation of ABCB6 and its interaction partners:

• Antibody selection:

  • Choose antibodies validated for immunoprecipitation applications

  • Consider using tagged versions of proteins and corresponding tag antibodies if native antibodies have poor IP efficiency

  • Multiple antibodies (anti-ABCB5, anti-ABCB6, or anti-ABCB9) have been successfully used in co-IP studies

• Sample preparation:

  • For membrane proteins like ABCB6, use appropriate detergents that solubilize membranes while preserving protein-protein interactions

  • Common detergents include CHAPS, digitonin, or NP-40 at optimized concentrations

  • Include protease inhibitors to prevent degradation during processing

• Controls:

  • Isotype controls are essential to determine the specificity of signals obtained in Western blots

  • Input samples (pre-IP) should be run alongside IP samples

  • For tagged proteins, compare results using both anti-tag and anti-ABCB6 antibodies

• Validation approaches:

  • Confirm interactions using reciprocal co-IP (using antibodies against the partner protein)

  • Verify results with alternative interaction detection methods like proximity ligation assays

  • Use cells with knocked down expression of interaction partners as negative controls

• Detection optimization:

  • Consider using more sensitive detection methods for Western blotting after IP

  • Try different blocking agents to reduce background

  • Optimize antibody concentrations for the Western blot detection step

Recent studies have successfully used these approaches to identify and validate interactions between ABCB6 and other ABC transporters like ABCB5β .

What are the key considerations for immunofluorescence studies of ABCB6 localization?

For accurate determination of ABCB6 subcellular localization using immunofluorescence:

• Fixation and permeabilization:

  • Optimize fixation methods (paraformaldehyde vs. methanol) as this can affect epitope accessibility

  • For membrane proteins like ABCB6, permeabilization conditions are critical and may need optimization

  • Consider dual fixation protocols for simultaneous detection of membrane and intracellular proteins

• Antibody selection and validation:

  • Choose antibodies validated for IF applications (e.g., Proteintech 51007-1-AP, 1:20-1:200 dilution)

  • Positive MCF-7 cells have shown successful staining with certain antibodies

  • Consider using multiple antibodies targeting different epitopes to confirm localization patterns

• Controls for subcellular localization:

  • Use co-staining with established organelle markers:

    • Mitochondrial markers (for potential mitochondrial localization)

    • Rab5 (for endosomal localization)

    • Lysosomal markers (for lysosomal localization)

    • Plasma membrane markers

• Addressing localization controversies:

  • The intracellular localization of ABCB6 has been debated, with evidence for both mitochondrial and endo-lysosomal localization

  • ABCB6 has been shown to be internalized from the plasma membrane through endocytosis, then distributed to multivesicular bodies and lysosomes

  • The N-terminal TMD0 domain plays a crucial role in lysosomal targeting

• Advanced imaging approaches:

  • Consider super-resolution microscopy for more precise localization

  • Live cell imaging with fluorescently tagged ABCB6 can provide insights into trafficking dynamics

  • Electron microscopy has been used successfully to confirm endo-lysosomal localization of ABCB6

Understanding ABCB6 localization is particularly important given its complex trafficking patterns and the role of its domains in determining subcellular distribution.

What are emerging applications of ABCB6 antibodies in cancer research?

Emerging applications of ABCB6 antibodies in cancer research include:

• Multidrug resistance mechanisms:

  • ABCB6 has been implicated in conferring resistance to arsenite and cisplatin

  • Antibodies can be used to monitor ABCB6 expression levels in patient samples and correlate with treatment response

  • Potential for development of predictive biomarkers for chemotherapy resistance

• Prognostic indicators:

  • Research indicates connections between ABCB6, ferroptosis, and iron metabolism signatures in hepatocellular carcinoma

  • These signatures may predict clinical diagnosis, prognosis, and immune microenvironment

  • Antibodies are essential tools for validating these signatures at the protein level

• Therapeutic targeting:

  • Antibodies can be used to screen for compounds that modulate ABCB6 function

  • Potential development of inhibitory antibodies that could restore drug sensitivity

  • Antibody-drug conjugates targeting ABCB6-overexpressing cancer cells

• Heterodimeric complexes:

  • The discovery of heterodimeric interactions between ABCB6 and other transporters opens new research avenues

  • Antibodies are crucial for investigating these complexes and their functional significance in cancer cells

  • Understanding the regulation of these heterodimers could reveal new therapeutic approaches

As ABCB6 research progresses, antibodies will continue to be essential tools for unraveling its complex roles in cancer biology and drug resistance mechanisms.

How might advances in antibody technology improve ABCB6 research in the future?

Emerging antibody technologies hold promise for advancing ABCB6 research:

• Single-domain antibodies (nanobodies):

  • Smaller size allows better access to epitopes in complex membrane proteins

  • Potential for improved detection of ABCB6 in its native conformational state

  • Applications in super-resolution microscopy for more detailed localization studies

• Recombinant antibody fragments:

  • Fab or scFv formats with improved specificity for particular ABCB6 isoforms or conformations

  • Potential for engineered antibodies that distinguish between ATP-bound and nucleotide-free states

  • Development of conformation-specific antibodies to study transport mechanisms

• Proximity-labeling antibodies:

  • Antibodies conjugated to enzymes that catalyze proximity-dependent labeling

  • Allows identification of proteins in close proximity to ABCB6 in living cells

  • Potential for mapping the ABCB6 interactome in different cellular compartments

• Multiplexed imaging approaches:

  • Antibodies compatible with multiplexed imaging technologies (e.g., CycIF, CODEX)

  • Simultaneous visualization of ABCB6 alongside multiple markers in the same sample

  • Applications in studying ABCB6 in the context of the tumor microenvironment

• Intrabodies:

  • Antibodies designed for intracellular expression

  • Potential for disrupting specific protein-protein interactions involving ABCB6

  • Applications in studying ABCB6 function through targeted perturbation

These technological advances will facilitate more detailed investigations into ABCB6 structure, function, and role in disease processes.