ABCD4 Antibody

What is ABCD4 and why is it important to study?

ABCD4 (ATP-binding cassette, sub-family D, member 4) is a lysosomal membrane protein that functions as a transporter of cobalamin (Vitamin B12) from the lysosomal lumen to the cytosol in an ATP-dependent manner . While initially thought to be peroxisomal like other ABCD family members, ABCD4 lacks the N-terminal peroxisomal targeting hydrophobic motif and is primarily localized to lysosomes when co-expressed with LMBD1 . Its importance lies in its critical role in vitamin B12 metabolism, with mutations causing methylmalonic acidemia with homocystinuria, cblJ type - a disorder characterized by developmental delay, eye defects, neurological problems, and blood abnormalities . ABCD4 has also been implicated in the peroxisomal import of fatty acids and/or fatty acyl-CoAs, potentially modifying adrenoleukodystrophy phenotypes .

What applications are ABCD4 antibodies most commonly validated for?

ABCD4 antibodies are primarily validated for the following applications:

Researchers should note that optimal dilutions may vary depending on the specific antibody and experimental conditions, and titration is recommended to obtain optimal results .

What species reactivity should be considered when selecting an ABCD4 antibody?

Most commercially available ABCD4 antibodies demonstrate reactivity with:

• Human samples - Most comprehensively validated

• Mouse samples - Well-validated with multiple antibody options

• Rat samples - Limited options compared to human and mouse

When selecting an antibody, researchers should consider the species homology of the immunogen. For example, some antibodies are generated against synthetic peptides that may have limited cross-reactivity between species. The BiCell Scientific ABCD4 antibody, for instance, uses a 16-amino acid sequence from the C-terminal region of mouse ABCD4 that is identical to rat sequence but shows only 68.8% homology to the human sequence .

How should ABCD4 antibodies be stored to maintain optimal activity?

For optimal antibody stability and performance:

• Store at -20°C for long-term storage

• Some preparations contain glycerol (typically 50%) and can be stored without aliquoting at -20°C

• Standard storage buffers include PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Antibodies are typically stable for one year after shipment when stored properly

• Smaller sizes (e.g., 20μl) may contain 0.1% BSA as a stabilizer

Researchers should avoid repeated freeze-thaw cycles which can compromise antibody performance.

What are the optimal protocols for using ABCD4 antibodies in Western blot applications?

Based on validated protocols from multiple suppliers, the following methodology is recommended:

• Sample preparation:

  • Use lysates from cells (HeLa, HepG2, Jurkat) or tissues (mouse liver, skeletal muscle)

  • Load approximately 30μg of protein per lane

• Dilution optimization:

  • Begin with 1:500-1:2000 dilution range for most polyclonal antibodies

  • Run a dilution series if working with a new antibody preparation

• Expected band size:

  • The predicted molecular weight of ABCD4 is 69-70 kDa

  • Confirm specificity by comparing to positive controls (e.g., HeLa cell lysate)

• Controls to include:

  • Positive control: HeLa cells consistently show detectable ABCD4 expression

  • Negative control: Consider using cells with ABCD4 knockdown if available

How can ABCD4 antibodies be used to study protein-protein interactions with LMBD1?

ABCD4 forms a functional complex with LMBD1, which is crucial for its correct localization and function. Research protocols for studying this interaction include:

• Co-immunoprecipitation:

  • Use ABCD4 antibodies to pull down the protein complex

  • Western blot with LMBD1 antibodies to confirm interaction

• Live-cell FRET assay:

  • This highly sensitive method has been validated for detecting ABCD4-LMBD1 interactions

  • Can demonstrate decreased interaction when ABCD4 harbors patient mutations (p.Arg432Gln, p.Asn141Lys) or when artificial mutations disrupt the ATPase domain

• Co-localization studies:

  • Use immunofluorescence with ABCD4 antibodies in combination with lysosomal markers (e.g., LAMP1)

  • Compare localization patterns between wild-type ABCD4 and mutant variants

• Complementation assays:

  • Overexpression of either ABCD4 or LMBD1 can rescue decreased Cbl cofactor levels in patient fibroblasts

  • This approach can help validate functional interactions in disease contexts

What methodological considerations are important when using ABCD4 antibodies to study its ATPase-dependent transport function?

ABCD4 transports cobalamin in an ATP-dependent manner, and studying this function requires specific experimental approaches:

• ATPase activity assays:

  • Purified ABCD4 reconstituted in liposomes shows ATPase activity that is stimulated by cobalamin inside the liposomes

  • Control experiments should include ABCD4 (K427A), a Walker A lysine mutant deficient in ATPase activity

• Transport assays using proteoliposomes:

  • Prepare ABCD4-containing liposomes loaded with cobalamin

  • Quantify the release of cobalamin by reverse-phase HPLC

  • Include time course measurements to demonstrate transport kinetics

  • Include ATP-depleted conditions as negative controls

• Orientation assessment:

  • Verify the proper insertion and orientation of ABCD4 in liposomes using trypsin treatment and immunoblot analysis

  • Approximately 70% of reconstituted ABCD4 should exist in right-side-out orientation

• Mutational analysis:

  • Include patient-derived mutations to assess their impact on transport activity

  • The K427A mutation in the Walker A motif eliminates both ATPase and transport activity

How do clinical or ATPase domain mutations in ABCD4 affect antibody binding and experimental interpretations?

Several mutations in ABCD4 have been identified in patients with methylmalonic acidemia with homocystinuria. When studying these variants:

• Epitope considerations:

  • Ensure the antibody's epitope does not overlap with the mutation site

  • For mutations in the ATPase domain (e.g., K427A, p.Arg432Gln), antibodies targeting the C-terminal region may be preferred

• Expression level assessment:

  • Some mutations may affect protein stability rather than function

  • Compare expression levels between wild-type and mutant ABCD4 using quantitative Western blot

• Structural impact analysis:

  • Mutations may cause conformational changes affecting epitope accessibility

  • Consider using multiple antibodies targeting different regions of ABCD4

• Interaction studies:

  • Clinical mutations (p.Arg432Gln, p.Asn141Lys) and ATPase domain mutations both disrupt ABCD4-LMBD1 interaction

  • This can be detected using FRET assays and co-immunoprecipitation

What are the best approaches for using ABCD4 antibodies in studies of vitamin B12 metabolism disorders?

When investigating vitamin B12-related diseases associated with ABCD4 dysfunction:

• Patient fibroblast analysis:

  • Compare ABCD4 expression and localization in control vs. patient fibroblasts

  • Assess colocalization with lysosomal markers in both conditions

• Rescue experiments:

  • Overexpression of wild-type ABCD4 can rescue Cbl cofactor deficiencies in patient fibroblasts

  • Unexpectedly, overexpression of LMBD1 can also rescue the phenotype in some cases

• Biochemical measurements:

  • Combine antibody-based detection of ABCD4 with measurements of adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) levels

  • Correlate ABCD4 expression/localization with functional outputs

• Pathway analysis:

  • Use ABCD4 antibodies alongside antibodies for other proteins in the vitamin B12 processing pathway (LMBD1, MMACHC)

  • Assess formation of the complex that transports cobalamin across the lysosomal membrane

How can researchers troubleshoot non-specific binding issues with ABCD4 antibodies?

When encountering specificity problems with ABCD4 antibodies:

• Blocking optimization:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Extend blocking time to minimize background

• Antibody validation strategies:

  • Use ABCD4 knockout or knockdown cells as negative controls

  • Compare results from multiple antibodies targeting different epitopes

  • Perform peptide competition assays with the immunizing peptide

• Sample-specific considerations:

  • The observed molecular weight of ABCD4 is approximately 70 kDa

  • Some non-specific bands at approximately 110 kDa have been reported in yeast-derived purifications

  • These non-specific proteins are not ABCD4-associated as they do not co-immunoprecipitate with ABCD4

• Application-specific troubleshooting:

  • For Western blot: Optimize antibody concentration, incubation time, and washing conditions

  • For IHC/IF: Consider antigen retrieval methods and fixation protocols

  • For IP: Use more stringent wash buffers to reduce non-specific interactions

How might ABCD4 antibodies contribute to understanding the mechanisms of lysosomal targeting and membrane protein trafficking?

ABCD4 provides a unique model for studying conditional organelle targeting since:

• Conditional lysosomal localization:

  • ABCD4 is initially localized to the endoplasmic reticulum when overexpressed alone

  • Co-expression with LMBD1 results in lysosomal targeting

  • Antibodies can be used to track this translocation process

• Mechanistic studies:

  • ABCD4-LMBD1 complex formation on the ER membrane precedes translocation to lysosomes

  • Antibodies specific to different domains of ABCD4 could help identify regions critical for this interaction

• Trafficking pathway mapping:

  • Combinations of antibodies to ABCD4, LMBD1, and markers of different cellular compartments

  • Time-course studies to follow the trafficking pathway from ER to lysosomes

• Mutational analysis:

  • Systematic mutational analysis of ABCD4 domains to identify trafficking signals

  • Correlation of localization patterns with functional readouts of vitamin B12 transport

What is the potential for using ABCD4 antibodies in studying the broader implications of vitamin B12 transport in neurological disorders?

Vitamin B12 deficiency is associated with numerous neurological manifestations, and ABCD4 antibodies could help elucidate mechanisms by:

• Brain tissue studies:

  • Analyzing ABCD4 expression and localization in brain tissue from different regions

  • Comparing expression patterns between normal and neurologically affected tissues

• Neuronal cell models:

  • Tracking ABCD4 localization in neuronal cells under various conditions

  • Assessing impact of vitamin B12 deficiency on ABCD4 expression and function

• Correlative studies:

  • Connecting ABCD4 expression/function with markers of neural health

  • Identifying potential compensatory mechanisms in neurological disorders

• Therapeutic development:

  • Screening compounds that might enhance ABCD4-mediated vitamin B12 transport

  • Identifying small molecules that could restore function of mutant ABCD4

How should researchers interpret contradictory data when comparing ABCD4 localization across different experimental systems?

When facing contradictory results regarding ABCD4 localization:

• Expression level considerations:

  • Overexpression can affect localization patterns

  • Compare endogenous vs. overexpressed ABCD4 localization

• Cell type specificity:

  • ABCD4 was initially identified as peroxisomal, then ER, and now confirmed as lysosomal when co-expressed with LMBD1

  • Different cell types may have varying levels of LMBD1, affecting ABCD4 localization

• Methodological reconciliation:

  • Fixation methods can affect membrane protein localization

  • Live-cell imaging may provide different results than fixed-cell imaging

• Experimental conditions:

  • ABCD4 shows different localization depending on the presence of LMBD1

  • Time-course experiments may reconcile apparently contradictory results by revealing dynamic trafficking processes

By systematically addressing these variables, researchers can better interpret seemingly contradictory data and develop a more comprehensive understanding of ABCD4 biology.

What controls are essential when designing experiments with ABCD4 antibodies?

For robust experimental design, include:

• Positive controls:

  • Cell lines with known ABCD4 expression: HeLa, HepG2, Jurkat cells

  • Tissues with known ABCD4 expression: mouse liver, mouse skeletal muscle

• Negative controls:

  • Primary antibody omission

  • ABCD4 knockdown/knockout samples when available

  • Isotype control antibodies

• Specificity controls:

  • ABCD4 (K427A) mutant as a non-functional control for transport studies

  • Peptide competition assays with the immunizing peptide

• Localization controls:

  • Co-staining with established markers:

    • Lysosomal markers (LAMP1) for wild-type ABCD4 with LMBD1

    • ER markers for ABCD4 expressed alone