ROBO1 Antibody, HRP conjugated

What is ROBO1 and what is its biological significance in research applications?

ROBO1 (Roundabout, axon guidance receptor, homolog 1) is a membrane protein and member of the immunoglobulin superfamily that functions in axon guidance. Originally identified in Drosophila, ROBO1 is highly conserved across species from fruit flies to mammals . The protein plays critical roles in:

• Angiogenesis and organ development processes

• Tumor metastasis and progression

• Axon navigation at the ventral midline of the neural tube

ROBO1 is particularly significant in cancer research as it is upregulated in 84.7% of hepatocellular carcinoma cases and shows differential expression patterns in prostate cancer models between racial groups . The protein has a calculated molecular weight of 181 kDa but is typically observed at 200-250 kDa in Western blot applications due to glycosylation .

How do different anti-ROBO1 antibody formats compare in research applications?

For optimal results, researchers should consider:

• Polyclonal antibodies offer broader epitope recognition but may show batch-to-batch variability

• Monoclonal antibodies provide higher specificity but may be affected by epitope masking

• Directly HRP-conjugated primaries eliminate secondary antibody cross-reactivity concerns but may offer lower signal amplification compared to secondary detection systems

How can ROBO1 antibodies be leveraged for targeted cancer therapeutics?

ROBO1 antibodies have demonstrated significant potential for targeted cancer therapy, particularly for hepatocellular carcinoma (HCC). Research has shown several promising therapeutic approaches:

• Radioimmunotherapy (RIT):

  • 90Y-labeled anti-ROBO1 monoclonal antibodies exhibited significant antitumor effects against HCC xenografts

  • Biodistribution studies with 111In-labeled anti-ROBO1 confirmed high accumulation in HCC models

  • Tumor volume reductions were observed with minimal effects on normal tissues

• Complement-Dependent Cytotoxicity (CDC):

  • The monoclonal antibody B2318C induced complement-dependent cytotoxicity in ROBO1-expressing cell lines

  • Effective CDC was demonstrated in the liver cancer cell line PLC/PRF/5

• Targeting Considerations:

  • ROBO1 shows limited distribution in normal tissues, making it an ideal target for selective therapy

  • The ectodomain of ROBO1 is shed into serum of HCC patients, potentially interfering with antibody-based therapies

These approaches highlight ROBO1's potential as both a therapeutic target and a serological marker for hepatocellular carcinoma .

What structural and conformational changes in ROBO1 affect antibody binding and experimental outcomes?

Recent NMR analyses reveal important insights about ROBO1 structural dynamics that researchers should consider when designing experiments with anti-ROBO1 antibodies:

• Conformational Changes Upon Ligand Binding:

  • The hRobo1-Ig1-2 structure undergoes subtle but significant conformational changes when binding to heparan sulfate (HS)

  • The protein becomes more rigid upon HS binding, which may affect antibody accessibility to certain epitopes

  • These structural changes have implications for Robo-Slit signaling mechanisms

• Domain-Specific Considerations:

  • Most commercial antibodies target specific domains of ROBO1

  • Researchers should verify which domain their antibody recognizes, as accessibility may vary based on experimental conditions

  • The immunoglobulin-like (Ig) domains of ROBO1, particularly Ig1 and Ig2, are involved in key protein interactions

• Post-Translational Modifications:

  • ROBO1 is heavily glycosylated, resulting in observed molecular weights (200-250 kDa) significantly higher than calculated (181 kDa)

  • Glycosylation patterns may mask epitopes and affect antibody binding efficiency

  • Deglycosylation treatments prior to Western blotting may be necessary for certain applications

Understanding these structural nuances is critical for proper experimental design and accurate interpretation of results when using ROBO1 antibodies.

How do expression patterns of ROBO1 differ across cancer types and ethnic populations, and what are the implications for antibody-based detection?

Research has revealed significant variations in ROBO1 expression patterns with important implications for experimental design:

• Cancer Type Variations:

  • Hepatocellular Carcinoma (HCC): 84.7% of cases (83/98) show ROBO1 overexpression

  • Prostate Cancer: Significant loss of ROBO1 in primary and metastatic tumors

• Ethnic Differences in Expression:

  • In prostate cancer, African-Americans show significant differences in ROBO1 levels between primary and metastatic phenotypes

  • Caucasians exhibit similar ROBO1 levels across primary and metastatic prostate tumors

  • Promoter methylation of ROBO1 was identified specifically in African-American metastatic prostate cancer cells

• Subcellular Localization:

  • ROBO1 expression is primarily localized to cell membranes in normal tissues

  • In cancer tissues, altered localization patterns may occur

  • ROBO1:DOCK1 complexes are observed at the membrane of prostate epithelial cells

• Experimental Implications:

  • Sample selection should account for potential ethnic variations

  • Antibody validation should include diverse cell line panels

  • Detection methods should be optimized for capturing both membrane-bound and potentially shed forms of ROBO1

These findings suggest researchers should carefully consider demographic factors when designing experiments and interpreting results with ROBO1 antibodies.

What are the optimal protocols for Western blot detection of ROBO1 using HRP-conjugated antibody systems?

Based on successful research protocols, the following optimized Western blot procedure is recommended for ROBO1 detection:

• Sample Preparation:

  • Use 15-30 μg of whole cell lysate for optimal detection

  • Tested cell lines with consistent ROBO1 detection include: HeLa, HepG2, SH-SY5Y, MDA-Mb-361, and HCC1937

• Gel Electrophoresis:

  • Use 5% SDS-PAGE to properly resolve the high molecular weight ROBO1 protein (200-250 kDa)

  • PVDF membrane is preferred for transfer over nitrocellulose

• Antibody Dilutions and Incubation:

  • Primary anti-ROBO1 antibody:

    • GTX114103: 1:1000 dilution

    • 25181-1-AP: 1:1000-1:4000 dilution

    • AF7118: 1 μg/mL concentration

  • Secondary HRP-conjugated antibody:

    • Anti-rabbit IgG (GTX213110-01) when using rabbit primaries

    • Anti-sheep IgG (HAF016) when using sheep primaries

• Detection Conditions:

  • Use reducing conditions for all ROBO1 Western blot applications

  • Conduct experiments using Immunoblot Buffer Group 1 for optimal results

• Expected Results:

  • A specific band should be detected at approximately 200-250 kDa

  • Some cell lines may show variable expression levels requiring optimization of exposure times

What controls and validation steps are essential when using ROBO1 antibodies in immunohistochemistry and immunofluorescence applications?

Proper validation is critical when using anti-ROBO1 antibodies for tissue and cellular imaging applications:

• Positive Control Tissues/Cells:

  • Hepatocellular carcinoma tissue (84.7% positive for ROBO1)

  • HeLa cells show consistent ROBO1 expression in Golgi apparatus

  • HepG2 cells demonstrate strong cell surface and cytoplasmic staining

  • Brain tissue from human and mouse models

• Negative Controls:

  • Include secondary-only controls to assess background

  • Use ROBO1-negative cell lines or ROBO1-knockdown samples for specificity verification

  • Compare with normal adjacent tissue in cancer specimens

• Antigen Retrieval Optimization:

  • For IHC applications, test both:

    • TE buffer pH 9.0 (preferred method)

    • Citrate buffer pH 6.0 (alternative method)

• Recommended Dilutions:

  • For IHC: 1:50-1:500

  • For IF/ICC: 1:50-1:500

  • For IF-P: 1:50-1:500

• Subcellular Localization Verification:

  • In normal cells: primarily membrane localization

  • In HeLa cells: Golgi apparatus localization

  • Use appropriate compartment markers (e.g., DAPI for nuclei) to confirm localization patterns

• Specificity Validation:

  • Competitive binding assays

  • Correlation with mRNA expression data

  • Comparison with other validated anti-ROBO1 antibodies

How can researchers troubleshoot common issues when working with ROBO1 antibodies in mechanistic studies?

When investigating ROBO1's role in molecular mechanisms such as the ROBO1:DOCK1:Rac1 pathway, researchers may encounter several challenges:

• Protein-Protein Interaction Detection:

  • Challenge: Detecting transient ROBO1:DOCK1 interactions

  • Solution:

    • Use chemical crosslinking prior to immunoprecipitation

    • Employ immunofluorescence-confocal microscopy to visualize co-localization

    • ROBO1 (green fluorescence) and DOCK1 (red fluorescence) co-localization can be observed at membrane regions of prostate epithelial cells

• Signaling Pathway Activation Assessment:

  • Challenge: Determining if ROBO1 affects Rac1 activation

  • Solution:

    • Use Rac1-GTP pull-down assays following ROBO1 manipulation

    • Visualize Rac1-GTP using immunofluorescence-confocal microscopy

    • Suppression of ROBO1 leads to increased Rac1-GTP formation in the cytoplasm

• Functional Validation:

  • Challenge: Confirming ROBO1's role in cell migration

  • Solution:

    • Compare migration in ROBO1-expressing vs. ROBO1-suppressed cells

    • Use ROBO1-expressing/ROBO1-C2C3-mutant constructs to identify functional domains

    • Monitor E-Cadherin/β-catenin cytoskeleton destabilization as a readout

• Technical Considerations:

  • Challenge: High background in IF imaging

  • Solution:

    • Optimize blocking conditions (1% BSA and 0.1mM EDTA in PBS)

    • For intracellular epitopes, use 0.02% saponin for permeabilization

    • Wash cells thoroughly with dilution buffer before secondary antibody application

How are ROBO1 antibodies being applied in multimodal imaging and theranostic approaches?

Innovative research is expanding the utility of anti-ROBO1 antibodies beyond traditional applications:

• Radioisotope Labeling for Dual Diagnosis and Therapy:

  • 111In-labeled anti-ROBO1 antibodies serve as excellent imaging agents for tumor localization

  • 90Y-labeled anti-ROBO1 antibodies deliver targeted radiotherapy to ROBO1-positive tumors

  • Biodistribution studies confirm high accumulation in HCC xenografts with minimal impact on normal tissues

• Serum Biomarker Development:

  • The ectodomain of ROBO1 is shed into serum of HCC patients (detected in 6 of 11 patients)

  • Anti-ROBO1 antibodies can be used to develop ELISA or other immunoassays for serum ROBO1 detection

  • This approach may enable non-invasive monitoring of ROBO1-positive cancers

• Potential Applications in Development:

  • Antibody-drug conjugates targeting ROBO1-expressing tumors

  • Bispecific antibodies linking ROBO1-positive cells to immune effectors

  • ROBO1-targeted nanoparticles for drug delivery or imaging applications

The versatility of anti-ROBO1 antibodies in these applications stems from the high specificity of these reagents and the elevated expression of ROBO1 in multiple cancer types compared to limited distribution in normal tissues .

What is the relationship between ROBO1 epitope selection and antibody performance in different experimental systems?

Understanding epitope selection is crucial for optimal antibody performance across various experimental platforms:

• Domain-Specific Targeting:

  • The extracellular domain of ROBO1 contains multiple immunoglobulin-like domains

  • Antibodies targeting the Ig1-2 domains are effective for detecting ROBO1:Slit2 interactions

  • NMR experiments show that hRobo1-Ig1-2 structure changes slightly upon heparan sulfate binding, potentially affecting antibody access to certain epitopes

• Epitope Accessibility Considerations:

  • Membrane-embedded vs. shed forms of ROBO1 may present different epitope accessibility profiles

  • Antibodies against the ectodomain (e.g., residues Ser20-Pro861) perform well in detecting both cell-surface and soluble ROBO1

  • Some commercial antibodies target specific regions, such as the Val310-Ser312 deletion variant

• Performance Correlation Table:

• Validation Approaches:

  • Competitive ELISA with soluble ROBO1 protein can confirm antibody specificity

  • IC50 values for anti-ROBO1, DOTA-anti-ROBO1, and labeled versions should be comparable (0.41-0.60 μg/mL range) if conjugation hasn't affected binding

  • Correlation with mRNA expression data provides additional validation of antibody specificity

This detailed understanding helps researchers select the most appropriate anti-ROBO1 antibody for their specific experimental question and system.