ABCC6 Antibody

What is ABCC6 and why are antibodies against it important in research?

ABCC6 (ATP-binding cassette subfamily C member 6) is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. It functions as a transporter of unknown metabolites that directly or indirectly control mineralization of dermal, ocular, and cardiovascular tissues . Antibodies against ABCC6 are crucial for investigating disease mechanisms in conditions like pseudoxanthoma elasticum (PXE) and for studying the protein's localization, expression, and function in normal and pathological states .

What types of ABCC6 antibodies have been developed for research applications?

Several types of ABCC6 antibodies have been developed for research:

• Monoclonal antibodies with specificity to human ABCC6 (hABCC6), including:

  • M6II-7 antibody that recognizes an internal epitope

  • M6II-31 antibody specific to human ABCC6

  • mEChC6, a novel monoclonal antibody recognizing an extracellular epitope

• Polyclonal antibodies:

  • HB6 antibody recognizing an epitope in the N-proximal L0 region

  • K-14 antibody that recognizes both human ABCC6 and mouse Abcc6 proteins, raised against the C-terminal end of rat Abcc6

The development of mEChC6, the first monoclonal antibody recognizing an extracellular epitope of hABCC6, represents a significant advancement in the field, as generation of such antibodies has been hampered by the short extracellular segments of the protein .

How should researchers select appropriate ABCC6 antibodies for their experimental systems?

Researchers should select ABCC6 antibodies based on:

• Species specificity: Some antibodies recognize only human ABCC6 (like M6II-31), while others recognize both human and mouse proteins (like K-14) .

• Epitope location: For studying protein localization in the plasma membrane, antibodies recognizing extracellular epitopes (like mEChC6) may be preferable for live cell applications .

• Experimental application: Different antibodies perform optimally in various applications:

  • For Western blotting: M6II-7 and HB6 have been successfully used

  • For immunofluorescence: M6II-31 and K-14 work effectively on frozen sections

  • For detecting protein fragments: HB6, which recognizes N-proximal epitopes, can detect degradation products

• Detection of mutant forms: Consider whether the antibody's epitope might be affected by the mutation being studied .

What are the optimal protocols for ABCC6 detection by immunofluorescence?

For optimal immunofluorescence detection of ABCC6:

• Tissue preparation:

  • Use frozen sections of liver tissue rather than paraffin-embedded samples to preserve protein antigenicity

  • For mouse liver expressing human ABCC6, section preparation following hydrodynamic tail vein injection (HTVI) has been successful

• Antibody selection:

  • For human ABCC6 detection: use M6II-31 monoclonal antibody

  • For co-localization studies: pair with antibodies against mouse Abcc6 or membrane markers like Na,K-ATPase

• Visualization techniques:

  • Co-staining with a plasma membrane marker (Na,K-ATPase) helps confirm proper membrane localization

  • Use confocal microscopy for precise localization assessment, particularly when evaluating mutant trafficking

• Controls:

  • Include negative controls using tissue from Abcc6-knockout mice

  • For mutant studies, wild-type ABCC6 expression serves as a positive control

Researchers can expect wild-type ABCC6 to show clear basolateral plasma membrane localization in hepatocytes, while trafficking-deficient mutants will show intracellular retention patterns .

How can ABCC6 antibodies be effectively used in Western blotting applications?

For effective Western blotting with ABCC6 antibodies:

• Sample preparation:

  • Total protein extraction from liver tissue should be performed with detergent-containing buffers suitable for membrane proteins

  • For cell culture studies, Sf9 insect cells have proven effective for expressing both wild-type and mutant ABCC6

• Antibody selection:

  • M6II-7 monoclonal antibody works well for detecting full-length ABCC6 (~160 kDa)

  • For detecting degradation products, use the HB6 polyclonal antibody that recognizes N-proximal epitopes

• Technical considerations:

  • Use appropriate positive controls (wild-type ABCC6)

  • Wild-type ABCC6 should appear at approximately 160 kDa

  • The N-terminal truncated mutant (ΔABCC6) appears at approximately 130 kDa

  • Some mutants like R1339C may show degradation products (~75 kDa and ~85 kDa)

• Troubleshooting:

  • If a mutant is not detected with one antibody (e.g., R1339C with M6II-7), try an alternative antibody recognizing a different epitope (e.g., HB6)

What methods enable the study of ABCC6 expression in vivo?

To study ABCC6 expression in vivo:

• Hydrodynamic tail vein injection (HTVI):

  • Sub-clone ABCC6 cDNAs (wild-type or mutant) into pLIVE vectors under a liver-specific promoter

  • Rapidly inject the plasmid solution into the mouse tail vein (hydrodynamic injection)

  • This technique achieves transient expression in 5-10% of hepatocytes

• Adenovirus-mediated delivery:

  • Generate recombinant adenoviruses carrying ABCC6 variants

  • Inject the viral vectors into Abcc6-knockout mice

  • This method provides sustained expression for up to four weeks

• Protein detection and quantification:

  • Perform immunofluorescence on frozen liver sections using anti-human ABCC6 antibodies

  • Quantify protein levels by Western blotting of total liver extracts

  • Co-stain with plasma membrane markers to assess proper localization

• Functional assessment:

  • Measure plasma PPi levels as a functional readout of ABCC6 activity

  • Evaluate the degree of ectopic mineralization in appropriate mouse models

How can ABCC6 antibodies help characterize disease-causing mutations?

ABCC6 antibodies are invaluable for characterizing disease-causing mutations through:

• Protein expression analysis:

  • Western blotting to determine if mutants are stably expressed or degraded

  • Example: R1339C mutant was found to be degraded in Sf9 cells, showing multiple fragments (75 kDa and 85 kDa) when detected with the HB6 antibody

• Subcellular localization studies:

  • Immunofluorescence microscopy to determine if mutants are properly trafficked to the plasma membrane

  • Co-staining with organelle markers (ER, Golgi) to identify where mutants are retained

• Mutant categorization:

  • Researchers can categorize ABCC6 mutants based on their behavior:

    • Class I: Normal plasma membrane localization (e.g., V1298F)

    • Class II: Partial plasma membrane localization with intracellular accumulation (e.g., R1138Q)

    • Class III: Primarily intracellular retention (e.g., R1314W, G1321S, R1339C)

• Rescue experiments:

  • Antibodies can evaluate whether compounds like 4-phenylbutyrate (4-PBA) can rescue trafficking of mutants

  • Example: 4-PBA treatment significantly improved cellular localization of R1314W

What imaging techniques utilizing ABCC6 antibodies are most informative for studying protein trafficking defects?

For studying ABCC6 trafficking defects, the following imaging techniques are most informative:

• Co-localization immunofluorescence microscopy:

  • Co-stain with compartment-specific markers:

    • Plasma membrane: Na,K-ATPase for basolateral membrane

    • Endoplasmic reticulum: Antibodies against ER-resident proteins

    • Golgi apparatus: Golgi-specific markers

• High-resolution confocal microscopy:

  • Allows precise determination of protein localization in subcellular compartments

  • Essential for distinguishing between partial and complete trafficking defects

• Live cell imaging (with extracellular-epitope antibodies):

  • The newly developed mEChC6 antibody recognizing an extracellular epitope enables live-cell surface labeling

  • This approach can potentially monitor dynamic trafficking processes in real-time

• Comparative analysis across cell types:

  • MDCKII cells: Useful for polarized trafficking studies

  • Hepatocytes (in vivo): Most physiologically relevant for ABCC6 function

Importantly, in vivo imaging using liver-specific expression systems provides the most physiologically relevant assessment of mutant trafficking, as it reveals behavior in the proper cellular environment with appropriate chaperone and trafficking machinery .

How can ABCC6 antibodies be used to study transcriptional regulation of the gene?

ABCC6 antibodies can contribute to transcriptional regulation studies through:

• Chromatin immunoprecipitation (ChIP) assays:

  • ChIP using antibodies against transcription factors like C/EBPβ can identify binding to ABCC6 regulatory regions

  • A study demonstrated a more than 3-fold enrichment of an ABCC6 intronic region (+503/+700) in C/EBPβ immunoprecipitated fractions from HepG2 cells

• Protein expression correlation with regulatory elements:

  • After identifying regulatory elements through DNase I hypersensitivity assays, antibodies can confirm protein expression levels

  • This approach identified a primate-specific activator element in the first intron of ABCC6

• Cell-type specific expression analysis:

  • ABCC6 antibodies can determine protein expression in cells where the gene is expressed (HepG2, Caco-2) versus cells where it is not (HeLa, HEK)

  • This helps correlate expression with the presence of tissue-specific transcription factors

• Validation of gene regulation manipulations:

  • After modulating putative regulatory pathways, antibodies can confirm resulting changes in ABCC6 protein levels

  • This is critical for establishing causative relationships in gene regulation studies

What innovations in ABCC6 antibody development have overcome technical challenges?

Recent innovations overcoming technical challenges in ABCC6 antibody development include:

• Generation of antibodies against extracellular epitopes:

  • The development of mEChC6, recognizing an extracellular epitope, overcame the challenge of the protein's short extracellular segments

  • This was achieved by immunizing bovine FcRn transgenic mice (exhibiting augmented humoral immune response) with HEK293 cells expressing human ABCC6

• Epitope mapping techniques:

  • Limited proteolysis revealed that the mEChC6 epitope is within a loop in the N-terminal half of ABCC6

  • The epitope likely spans amino acids 338-347

• Validation in transgenic models:

  • mEChC6 successfully recognized hABCC6 in the liver of hABCC6 transgenic mice

  • This verified both specificity and extracellular binding to intact hepatocytes

• Cross-species reactivity considerations:

  • Development of antibodies like K-14 that recognize both human and mouse ABCC6 enables comparative studies

  • This is particularly valuable for translating findings between model systems and human disease

How can ABCC6 antibodies contribute to therapeutic development strategies?

ABCC6 antibodies can advance therapeutic development through:

• Identification of rescue-responsive mutants:

  • Antibodies helped identify that R1314W mutant trafficking could be improved by 4-phenylbutyrate (4-PBA) treatment

  • This demonstrates the feasibility of rescuing cellular maturation of some ABCC6 mutants in physiological conditions

• Screening of therapeutic compounds:

  • Antibodies enable high-throughput screening of compounds that may rescue trafficking-defective mutants

  • This approach identifies candidates for potential chaperone therapy

• Validation of gene therapy approaches:

  • Antibodies confirm successful expression and localization of ABCC6 following adenovirus-mediated gene delivery

  • This helps evaluate the efficacy of gene therapy strategies

• Monitoring therapeutic outcomes:

  • Antibodies can assess whether treatments restore proper ABCC6 localization in the plasma membrane

  • The correlation between protein localization and functional outcomes (e.g., plasma PPi levels) can be evaluated

• Development of targeted therapeutics:

  • Antibodies recognizing extracellular epitopes (like mEChC6) could potentially be developed into therapeutic agents themselves

  • Alternatively, they could be used to deliver drugs to cells expressing ABCC6

How should researchers interpret discrepancies between different ABCC6 antibodies?

When facing discrepancies between different ABCC6 antibodies, researchers should:

• Consider epitope location:

  • Different antibodies recognize distinct epitopes that may be differentially affected by mutations

  • Example: R1339C mutant was undetectable with M6II-7 but could be detected as degradation products with HB6

• Evaluate detection sensitivity:

  • Monoclonal antibodies may have higher specificity but potentially lower sensitivity

  • Polyclonal antibodies might detect more variants but with increased background

• Assess method compatibility:

  • Some antibodies perform better in specific applications (Western blot vs. immunofluorescence)

  • Example: M6II-7 works well for Western blotting, while M6II-31 is preferred for immunofluorescence

• Use complementary approaches:

  • Combine multiple antibodies recognizing different epitopes

  • Correlate protein detection with functional assays (e.g., transport activity, ATP binding)

• Consider protein processing:

  • Discrepancies may reveal information about protein folding, degradation, or post-translational modifications

  • Example: Detection of fragments with HB6 revealed degradation patterns of R1339C

What controls are essential when working with ABCC6 antibodies?

Essential controls when working with ABCC6 antibodies include:

• Expression controls:

  • Positive control: Wild-type ABCC6 expressed in the same system

  • Negative control: Untransfected cells or tissues from Abcc6-knockout mice

• Specificity controls:

  • Secondary antibody only (no primary antibody) to assess background

  • Non-relevant primary antibody of the same isotype to evaluate non-specific binding

  • When possible, pre-absorption of the antibody with the immunizing peptide

• Localization controls:

  • Co-staining with established markers (Na,K-ATPase for plasma membrane, ER markers, etc.)

  • Known mutants with characterized localization patterns (e.g., V1298F for normal membrane localization)

• Cross-reactivity controls:

  • Testing antibodies in systems lacking ABCC6 expression

  • For human-specific antibodies, testing in non-human cells or tissues

  • For antibodies like K-14 that recognize both human and mouse proteins, appropriate species controls

• Functional controls:

  • Correlation of antibody signals with functional assays (ATP binding, vanadate trapping, transport activity)

  • Correlation with known phenotypes (e.g., plasma PPi levels, ectopic mineralization)