BICD2 Antibody

What is BICD2 and what cellular functions does it perform?

BICD2 functions as an adapter protein that links the dynein motor complex to various cellular cargos and converts dynein from a non-processive to a highly processive motor in the presence of dynactin . It facilitates and stabilizes the interaction between dynein and dynactin, activating dynein processivity - the ability to move along a microtubule for long distances without detaching . BICD2 regulates coat complex coatomer protein I (COPI)-independent Golgi-endoplasmic reticulum transport through its interaction with RAB6A and recruitment of the dynein-dynactin motor complex . Additionally, it contributes to nuclear and centrosomal positioning prior to mitotic entry by associating with RANBP2 at the nuclear pores during G2 phase of the cell cycle .

What types of BICD2 antibodies are available for research applications?

Researchers can access several types of BICD2 antibodies for experimental applications:

• Rabbit polyclonal antibodies: These recognize synthetic peptide regions within human BICD2 and are validated for Western blotting applications with human samples .

• Mouse monoclonal antibodies: Available as IgG2b kappa light chain antibodies that detect BICD2 protein from multiple species (mouse, rat, and human) and are validated for multiple applications including Western blotting, immunoprecipitation, immunofluorescence, immunohistochemistry, and ELISA .

• Conjugated antibodies: BICD2 antibodies are available in various conjugated forms including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates for specialized applications .

What is the molecular structure and localization of BICD2?

BICD2 consists of 824 amino acids and is expressed ubiquitously throughout the body . The protein primarily localizes to the Golgi apparatus, cytoplasm, and cytoskeleton . It undergoes post-translational modifications, particularly phosphorylation by NEK9, which can influence its function and protein interactions . BICD2 exists in two isoforms resulting from alternative splicing and is encoded by a gene located on human chromosome 9 . The protein's structure includes distinct N-terminal and C-terminal domains that serve different functions in cargo binding and motor protein recruitment .

What are the validated applications for BICD2 antibodies?

BICD2 antibodies have been validated for multiple experimental techniques:

How should I optimize immunohistochemistry protocols for BICD2 detection in tissue samples?

For optimal immunohistochemical detection of BICD2 in tissue samples, follow this validated protocol:

• Fix tissue with 4% paraformaldehyde, dehydrate, embed, and section according to standard procedures .

• Dewax sections and place in 3% methanolic hydrogen peroxide at room temperature for 10 minutes to block endogenous peroxidase activity .

• Wash three times with PBS .

• Perform antigen retrieval by immersing sections in 0.01M citrate buffer (pH 6.0), heating to boiling, and repeating this process after a 5-minute interval .

• After cooling, wash twice with PBS and apply normal goat serum as blocking solution .

• Incubate with primary BICD2 antibody at 4°C overnight .

• Apply biotinylated secondary antibody and incubate at 37°C for 30 minutes .

• Wash three times with PBS and develop using DAB Color Development Kit reagents for approximately 2 minutes .

• Counterstain lightly with hematoxylin, dehydrate, clear, and mount with neutral mounting medium .

This protocol has been validated for detecting BICD2 in heart tissue sections and can be adapted for other tissue types with appropriate optimization of antibody dilutions.

What controls should I include when working with BICD2 antibodies?

When working with BICD2 antibodies, include the following controls to ensure reliable and interpretable results:

• Positive control: Use tissue or cell lysates known to express BICD2 (BICD2 is ubiquitously expressed, but particularly abundant in Golgi apparatus) .

• Negative control:

  • Primary antibody omission: Perform the experiment without the primary antibody to detect non-specific binding of the secondary antibody.

  • Blocking peptide competition: Pre-incubate the antibody with the immunizing peptide to confirm specificity.

• Knockout/knockdown validation: When possible, use CRISPR/Cas9-generated BICD2 knockout samples (as demonstrated in zebrafish models) to validate antibody specificity .

• Loading control: For Western blot applications, include housekeeping proteins (e.g., GAPDH, β-actin) to normalize BICD2 expression levels across samples.

How can BICD2 antibodies be utilized in studying the dynein-dynactin motor complex?

BICD2 antibodies can be instrumental in investigating dynein-dynactin motor complexes through several sophisticated approaches:

• Co-immunoprecipitation studies: Use BICD2 antibodies to pull down protein complexes, followed by Western blotting for dynein and dynactin components to analyze complex formation and stability .

• Proximity ligation assays: Combine BICD2 antibodies with antibodies against dynein/dynactin components to visualize and quantify molecular interactions in situ.

• Structure-function analysis: Use domain-specific BICD2 antibodies to study how different regions (particularly the N-terminal domain) recruit and activate dynein . The N-terminal portion of BICD2 has been demonstrated to induce microtubule minus end-directed movement independently of molecular context, making it a powerful tool for studying dynein function .

• Dynein processivity studies: Examine how BICD2 converts dynein from a non-processive to a highly processive motor in the presence of dynactin, using immunodepletion and reconstitution approaches with BICD2 antibodies .

• Regulatory mechanism investigation: Study how the C-terminal domain of BICD2 might regulate interaction between BICD2 and the motor complex, as research suggests the dynein-recruiting activity of the BICD2 N-terminal domain is reduced within the full-length molecule .

What role do BICD2 antibodies play in understanding disease mechanisms?

BICD2 antibodies have emerging applications in studying disease mechanisms:

• Autoimmune disorders: BICD2 has been identified as a novel autoantibody target in systemic sclerosis (SSc) . Research shows that 25.7% (116/451) of SSc sera tested positive for anti-BICD2 antibodies, with 19.0% showing single specificity anti-BICD2 and 81.0% having other autoantibodies . BICD2 antibodies can help identify patients who lack other SSc-specific autoantibodies, serving as a potential new biomarker .

• Inflammatory myopathy and interstitial lung disease: Patients with single specificity anti-BICD2 showed higher likelihood of inflammatory myopathy (31.8% vs. 9.6%, p=.004) and interstitial lung disease (52.4% vs. 29.0%, p=.024) compared to anti-BICD2 negative subjects .

• Dilated cardiomyopathy (DCM): BICD2 has been identified as a novel candidate gene associated with familial DCM . BICD2 antibodies can be used in immunohistochemical studies to examine expression patterns in cardiac tissue samples and investigate pathogenic mechanisms .

How can BICD2 antibodies facilitate the study of intracellular transport mechanisms?

BICD2 antibodies enable detailed investigation of intracellular transport through several approaches:

• Golgi-ER transport studies: BICD2 regulates coat complex coatomer protein I (COPI)-independent Golgi-endoplasmic reticulum transport through interaction with RAB6A . Antibodies can be used to track this process through immunofluorescence co-localization studies.

• Chimeric protein experiments: The N-terminal portion of BICD2 has been used as a chimera with mitochondria and peroxisome-anchoring sequences to demonstrate rapid dynein-mediated transport of selected organelles . BICD2 antibodies can track these processes and validate experimental models.

• Nuclear positioning studies: During G2 phase, BICD2 associates with RANBP2 at nuclear pores to recruit dynein and dynactin to the nuclear envelope . Immunofluorescence with BICD2 antibodies can visualize this process during cell cycle progression.

• Analysis of regulatory mechanisms: The interaction between N- and C-terminal domains of BICD2 can be studied using domain-specific antibodies to understand how BICD2 activity is regulated during vesicular transport .

How do I address weak or non-specific signals when using BICD2 antibodies?

When encountering weak or non-specific signals with BICD2 antibodies, consider these troubleshooting approaches:

• Optimize antibody concentration: Titrate the primary antibody to determine the optimal working dilution that provides the best signal-to-noise ratio.

• Enhance antigen retrieval: For immunohistochemistry, test different antigen retrieval methods beyond the recommended citrate buffer (pH 6.0) protocol , such as EDTA buffer (pH 9.0) or enzymatic retrieval.

• Adjust blocking conditions: Increase blocking time or try alternative blocking reagents (BSA, casein, commercial blockers) to reduce background.

• Validate antibody specificity: Use CRISPR/Cas9-generated knockout models as negative controls, as demonstrated with BICD2 in zebrafish models .

• Cross-validate with multiple antibodies: Use both polyclonal and monoclonal antibodies targeting different epitopes of BICD2 to confirm results.

• Optimize detection systems: For Western blotting, try more sensitive detection reagents or longer exposure times if signals are weak.

How do I analyze BICD2 expression in correlation with dynein-dependent transport?

To analyze the relationship between BICD2 expression and dynein-dependent transport:

• Quantitative co-localization: Perform dual-labeling immunofluorescence with BICD2 antibodies and markers for dynein/dynactin components, calculating Pearson's or Mander's coefficients to measure the degree of co-localization.

• Live-cell imaging: Use fluorescently tagged BICD2 constructs alongside BICD2 antibody validation to track dynein-dependent movement of cellular components in real-time.

• Domain-specific analysis: Utilize the N-terminal domain of BICD2 as a tool to induce microtubule minus end-directed movement independently of molecular context . Compare this with full-length BICD2 behavior to understand regulatory mechanisms.

• Transport inhibition studies: Correlate BICD2 expression levels (quantified by Western blot) with transport efficiency in the presence of dynein inhibitors or after dynactin subunit dynamitin overexpression .

• Structure-function correlation: The N-terminal part of BICD2 is a potent recruitment factor for dynein, while the full-length molecule shows reduced dynein-recruiting activity, suggesting regulation by the C-terminal domain . Use appropriate antibodies to study these domain-specific functions.

What methodological considerations are important when using BICD2 antibodies in genetic studies?

When incorporating BICD2 antibodies in genetic research, consider these methodological approaches:

• Variant validation: In studies identifying BICD2 variants (as in DCM research), use antibodies to assess expression levels and localization patterns of wild-type versus variant proteins .

• Epitope mapping: When studying autoantibody responses to BICD2 (as in systemic sclerosis), use epitope mapping on solid phase matrices to identify specific binding regions, such as the serine- and proline-rich nonapeptide SPSPGSSLP (amino acids 606-614) shared with CENP-A but not CENP-B .

• CRISPR/Cas9 knockout models: When generating BICD2 knockout models (as demonstrated in zebrafish), use antibodies to validate knockout efficiency at the protein level before phenotypic analysis .

• Mutation screening: When screening for BICD2 variants in patient cohorts, combine Sanger sequencing with antibody-based protein analysis to establish genotype-phenotype correlations .

• Cross-species validation: Consider species homology when selecting antibodies for genetic studies across different model organisms, as BICD2 function is conserved from Drosophila to mammals .

How might BICD2 antibodies contribute to the study of autoimmune conditions?

BICD2 antibodies hold significant potential for advancing autoimmune research:

• Novel biomarker detection: Anti-BICD2 autoantibodies represent a new biomarker in systemic sclerosis, especially valuable for patients lacking other SSc-specific autoantibodies . Commercial BICD2 antibodies can be used to develop standardized assays for detecting these autoantibodies.

• Epitope characterization: The identified cross-reactive epitope between BICD2 and CENP-A (but not CENP-B) suggests specific molecular targeting in autoimmune responses . Further epitope mapping using domain-specific antibodies can clarify these mechanisms.

• Clinical correlation studies: Single specificity anti-BICD2 antibodies associate with inflammatory myopathy and interstitial lung disease, unlike anti-CENP antibodies . BICD2 antibodies can help stratify patients and predict disease manifestations.

• Mechanistic studies: Investigating how autoantibodies against BICD2 affect its normal cellular functions could reveal pathogenic mechanisms in systemic sclerosis and related disorders.

What novel approaches combine BICD2 antibodies with advanced imaging techniques?

Integration of BICD2 antibodies with cutting-edge imaging offers new research capabilities:

• Super-resolution microscopy: Using BICD2 antibodies with techniques like STORM or PALM can reveal nanoscale organization of dynein-dynactin complexes at the Golgi, nuclear envelope, and other cellular structures.

• Live-cell imaging with nanobodies: Developing smaller antibody fragments (nanobodies) against BICD2 for live-cell imaging can overcome limitations of conventional antibodies.

• Correlative light and electron microscopy (CLEM): Combining immunofluorescence using BICD2 antibodies with electron microscopy can provide both molecular specificity and ultrastructural context.

• Expansion microscopy: This technique physically expands specimens to improve resolution, potentially revealing previously undetectable details of BICD2-mediated transport processes when combined with specific antibodies.

• Intravital imaging: Adapting BICD2 antibodies or derived imaging tools for in vivo imaging could extend research from cell culture to living organisms.

How can BICD2 antibodies facilitate therapeutic development for transport-related disorders?

BICD2 antibodies may contribute to therapeutic development through:

• Target validation: Confirming BICD2's role in disease processes like dilated cardiomyopathy using antibody-based approaches can validate it as a therapeutic target.

• Functional rescue assessment: In BICD2 variant models, antibodies can assess whether therapeutic interventions restore normal protein expression and localization.

• Drug screening platforms: Developing high-content screening assays using BICD2 antibodies to monitor protein interactions or localization patterns in response to compound libraries.

• Biomarker development: Standardizing antibody-based detection of anti-BICD2 autoantibodies for patient stratification in clinical trials for systemic sclerosis .

• Gene therapy validation: Using antibodies to verify expression levels and cellular distribution of BICD2 following genetic therapeutic approaches in models of BICD2-associated disorders.