ROBO1 Antibody, FITC conjugated

What is ROBO1 and why is it important in research settings?

ROBO1 (Roundabout homolog 1) is a 190-230 kDa transmembrane protein belonging to the ROBO/roundabout receptor family. It functions as a receptor for SLIT1 and SLIT2 ligands, mediating cellular responses to guidance cues in cellular migration processes. ROBO1 plays critical roles in axonal navigation at the ventral midline of the neural tube and projection of axons to different regions during neuronal development. Additionally, it regulates cell migration through interaction with MYO9B, inhibiting MYO9B-mediated stimulation of RHOA GTPase activity . The protein is expressed in commissural axons from multiple nuclei and is also found on vascular endothelium, bronchial epithelium, and syncytiotrophoblasts . ROBO1's involvement in tumor metastasis and angiogenesis makes it a valuable target in cancer research .

What is the structure of ROBO1 and how do antibodies target it?

The human ROBO1 precursor is a 1651 amino acid type I transmembrane protein containing a 25 amino acid signal sequence, followed by an 872 amino acid extracellular region (aa 26-897). The extracellular domain possesses five C2-type Ig-like domains (aa 68-541) and three fibronectin type III domains (aa 561-864) . ROBO1 exhibits multiple isoform variants, including ROBO1b/DUTT1 (with specific substitutions and deletions compared to the canonical sequence) and ROBO1a . Anti-ROBO1 antibodies are typically designed to recognize specific epitopes within the extracellular domain, such as amino acid residues 491-506 in the case of certain commercial antibodies . This strategic targeting allows researchers to investigate ROBO1 expression and function without interfering with its natural ligand interactions.

What are the advantages of using FITC-conjugated ROBO1 antibodies compared to unconjugated alternatives?

FITC-conjugated ROBO1 antibodies offer several methodological advantages over unconjugated alternatives:

• Direct detection without secondary antibodies, reducing background and cross-reactivity issues

• Simplified experimental workflows with fewer washing steps

• Enhanced suitability for live cell applications where membrane permeabilization is undesirable

• Compatibility with multicolor flow cytometry when combined with other fluorophore-conjugated antibodies

• Efficient visualization in immunofluorescence microscopy without additional amplification steps

The primary amines in the side chains of lysine residues and the N-terminus of the antibody are conjugated with FITC using standard chemical labeling methods, resulting in a stable fluorescent signal suitable for a variety of research applications .

How should ROBO1 antibody, FITC conjugated be stored to maintain optimal activity?

To maintain optimal activity of FITC-conjugated ROBO1 antibodies, researchers should adhere to these storage recommendations:

• Store at 2-8°C and protect from prolonged exposure to light to prevent photobleaching of the FITC fluorophore

• Avoid repeated freeze-thaw cycles which can damage both the antibody structure and the conjugated fluorophore

• For long-term storage, aliquot the antibody solution to minimize freeze-thaw cycles

• Use sterile techniques when handling to prevent microbial contamination

• Monitor for signs of degradation such as precipitation or loss of binding activity in control experiments

Additionally, researchers should consult manufacturer-specific recommendations, as stabilizers and preservatives may vary between commercial preparations.

What are the optimal concentrations for using ROBO1 antibody, FITC conjugated in flow cytometry applications?

For flow cytometry applications, optimal concentrations of FITC-conjugated ROBO1 antibody typically range from 5-10 μg per 1×10^6 cells. Experimental evidence from studies using various cell lines provides insight into effective concentrations:

• For mouse J774 macrophage cell lines, 5 μg of Anti-ROBO1 (extracellular)-FITC antibody has been shown to produce clear separation between positive and negative populations

• For human THP-1 monocytic leukemia cell lines, similar concentrations (5 μg) have demonstrated effective cell surface detection

• For anti-ROBO1 CAR-293 cells, concentrations of 10 μg/mL in 100 μL volumes have provided robust binding activity

What are the essential controls required for experiments using ROBO1 antibody, FITC conjugated?

Proper experimental controls are essential for accurate interpretation of results when using FITC-conjugated ROBO1 antibodies:

• Isotype control: Rabbit IgG Isotype Control-FITC conjugated at the same concentration as the ROBO1 antibody to assess non-specific binding

• Unstained cells control: Cells without any antibody to establish autofluorescence baseline

• Negative control cells: Cell lines known not to express ROBO1 or ROBO1-knockout cells to confirm specificity

• Positive control cells: Cell lines with validated ROBO1 expression such as J774 macrophage or THP-1 monocytic leukemia cells

• Blocking control: Pre-incubation with unconjugated ROBO1 antibody to demonstrate specific epitope binding

• Single-stain controls: Required for compensation when performing multicolor flow cytometry

These controls help distinguish specific ROBO1 detection from technical artifacts and establish the validity of experimental findings.

How can researchers validate the specificity of ROBO1 antibody, FITC conjugated?

Validating antibody specificity is crucial for reliable research outcomes. For FITC-conjugated ROBO1 antibodies, multiple complementary approaches should be employed:

• Western blot analysis: Using unconjugated version of the same ROBO1 antibody clone to confirm single band at expected molecular weight (190-230 kDa for full-length ROBO1)

• Peptide competition assays: Pre-incubation with the immunizing peptide (e.g., peptide corresponding to amino acids 491-506 of human ROBO1) should abolish specific staining

• Genetic validation: Testing on ROBO1 knockout models or cells treated with ROBO1-specific siRNA

• Cross-reactivity assessment: Testing on cell lines from multiple species to confirm expected reactivity pattern (e.g., human and mouse ROBO1 share 97% amino acid identity over residues 20-861)

• Comparison with alternative antibody clones: Using different antibodies targeting distinct ROBO1 epitopes to confirm expression patterns

Researchers should document these validation steps thoroughly to strengthen confidence in their experimental findings.

How can ROBO1 antibody, FITC conjugated be utilized for studying cancer progression and metastasis?

The application of FITC-conjugated ROBO1 antibodies in cancer research leverages the protein's role in tumor metastasis and angiogenesis. Several methodological approaches include:

• Flow cytometric profiling: Quantifying ROBO1 expression levels across patient-derived cancer cells or established cancer cell lines to correlate with invasive phenotypes

• Fluorescence microscopy: Visualizing ROBO1 localization during cancer cell migration and invasion in in vitro models

• Xenograft model imaging: Using FITC-conjugated antibodies for ex vivo analysis of tumor tissue sections to assess ROBO1 distribution

• Cancer stem cell identification: Combining ROBO1-FITC with other cancer stem cell markers to identify and isolate potential metastasis-initiating cell populations

• Therapeutic targeting validation: Monitoring ROBO1 expression changes in response to experimental therapies targeting the SLIT/ROBO pathway

These applications are particularly relevant for small cell lung cancer (SCLC), breast cancer, and colorectal cancer, where ROBO1 has been identified as having oncogenic roles .

What considerations are important when designing multiplex immunofluorescence studies including ROBO1 antibody, FITC conjugated?

When designing multiplex immunofluorescence studies incorporating FITC-conjugated ROBO1 antibodies, researchers should consider several technical aspects:

• Spectral compatibility: FITC (excitation ~495 nm, emission ~519 nm) should be paired with fluorophores having minimal spectral overlap, such as Alexa Fluor 647 (used in other available ROBO1 antibody conjugates)

• Expression level balancing: Adjust antibody concentrations based on relative expression levels of target proteins to achieve balanced signal intensities

• Sequential staining approach: If cross-reactivity is observed, consider sequential staining protocols with appropriate blocking steps between antibody applications

• Pixel shift correction: When imaging multiple fluorophores, account for potential pixel shifts between channels through proper alignment calibration

• Antibody species compatibility: When combining multiple primary antibodies, ensure they are raised in different host species or use directly conjugated antibodies

• Fixation method optimization: Different fixatives may affect epitope accessibility and fluorophore stability, requiring protocol adjustments

A titration matrix examining various concentrations of each antibody in the multiplex panel is recommended to determine optimal staining conditions.

How can researchers distinguish between ROBO1 isoforms using immunofluorescence techniques?

Distinguishing between ROBO1 isoforms requires careful experimental design and antibody selection based on isoform-specific epitopes:

• Epitope mapping: Select antibodies recognizing regions that differ between isoforms. For example, antibodies targeting regions affected by the 18 amino acid substitution for residues 1-47 in ROBO1a/b or the deletion of residues 939-947 in ROBO1b

• Size differentiation: Combine immunofluorescence with Western blot analysis to correlate observed signals with isoform-specific molecular weights

• Co-localization studies: Perform dual staining with antibodies recognizing common and isoform-specific regions of ROBO1

• RT-PCR validation: Confirm isoform expression at the mRNA level to support protein detection findings

• Recombinant protein controls: Use cells transfected with specific ROBO1 isoforms as positive controls

The search results indicate that ROBO1 exhibits multiple isoform variants including ROBO1a, ROBO1b/DUTT1, and other variants with additional deletions or alternative start sites , making isoform-specific detection an important consideration for comprehensive research.

What are common causes of weak or absent signal when using ROBO1 antibody, FITC conjugated, and how can they be addressed?

When encountering weak or absent signals with FITC-conjugated ROBO1 antibodies, researchers should systematically evaluate these potential causes and solutions:

Systematic adjustment of variables and inclusion of appropriate controls will help identify and resolve technical issues affecting signal quality.

How does fixation affect ROBO1 antibody, FITC conjugated performance, and what are the recommended protocols?

Fixation methods significantly impact the performance of FITC-conjugated ROBO1 antibodies through effects on epitope accessibility and fluorophore stability:

For FITC-conjugated antibodies specifically targeting the extracellular epitopes of ROBO1, live cell staining protocols or gentle fixation methods are generally recommended to maintain both antibody binding and fluorophore activity.

What strategies can address signal variability in flow cytometry using ROBO1 antibody, FITC conjugated?

Signal variability in flow cytometry experiments using FITC-conjugated ROBO1 antibodies can be addressed through several methodological refinements:

• Standardized cell handling: Process all samples with consistent dissociation methods, as excessive enzymatic treatment may cleave extracellular epitopes

• Time-controlled staining: Standardize antibody incubation time and temperature across experiments

• Fluorescence calibration: Use calibration beads with defined fluorescence intensities to normalize results between experiments

• Consistent gating strategy: Establish and adhere to well-defined gating protocols based on appropriate controls

• Instrument calibration: Regularly perform quality control on flow cytometers using standard beads

• Sample viability: Include viability dyes to exclude dead cells, which may bind antibodies non-specifically

• Batch processing: When possible, process and analyze samples in a single batch to minimize technical variation

Implementing these standardization measures can significantly reduce experimental variability and increase confidence in quantitative flow cytometry data using ROBO1-FITC antibodies.

How can ROBO1 antibody, FITC conjugated be used to investigate interactions between ROBO1 and its binding partners?

Investigating ROBO1 interactions with binding partners using FITC-conjugated antibodies requires sophisticated methodological approaches:

• Proximity ligation assays (PLA): Combining ROBO1-FITC with unconjugated antibodies against potential binding partners (such as SLIT1, SLIT2, DCC, or FLRT3) to visualize protein-protein interactions at single-molecule resolution

• FRET analysis: Pairing FITC-conjugated ROBO1 antibodies with acceptor fluorophore-conjugated antibodies against interacting proteins to measure Förster resonance energy transfer

• Co-immunoprecipitation validation: Using fluorescence microscopy with ROBO1-FITC to visualize co-localization of proteins identified through co-immunoprecipitation experiments

• Live cell imaging: Tracking dynamic interactions between ROBO1 and its partners during processes such as axon guidance or cell migration

• Competitive binding assays: Using FITC-conjugated ROBO1 antibodies to monitor displacement by potential binding partners

These approaches are particularly valuable for studying interactions such as the ROBO1-DCC complex formation, which may be required for the silencing of the attractive effect of NTN1 by SLIT2 in axon growth cones .

What are the considerations for using ROBO1 antibody, FITC conjugated in conjunction with radioimmunotherapy approaches?

When considering FITC-conjugated ROBO1 antibodies as complementary tools in radioimmunotherapy research, several specialized considerations apply:

• Biodistribution correlation: FITC-labeled antibodies can be used for ex vivo validation of radioisotope-labeled antibody localization in tissue sections

• Target expression confirmation: Flow cytometry with FITC-ROBO1 antibodies verifies target expression levels prior to radioimmunotherapy studies

• Competitive binding studies: Assessing whether FITC-conjugated and radioisotope-conjugated antibodies compete for the same epitope, which may affect therapeutic efficacy

• Treatment monitoring: Using FITC-ROBO1 antibodies to track changes in ROBO1 expression during and after radioimmunotherapy

• Dual-modality imaging validation: Correlating fluorescence microscopy results with radionuclide imaging to improve targeting strategies

Research has demonstrated that 90Y-labeled anti-ROBO1 monoclonal antibodies show antitumor effects against ROBO1-positive tumors, including small cell lung cancer models . FITC-conjugated versions of the same antibody clones provide valuable complementary tools for target validation and mechanism studies.

How can quantitative analysis of ROBO1 expression be performed using FITC-conjugated antibodies?

Quantitative analysis of ROBO1 expression using FITC-conjugated antibodies can be achieved through several methodological approaches:

• Quantitative flow cytometry: Using calibration beads with known quantities of fluorophore to convert mean fluorescence intensity (MFI) values to antibody binding capacity or molecules of equivalent soluble fluorochrome (MESF)

• Imaging cytometry: Combining flow cytometry with microscopy to correlate fluorescence intensity with cellular localization patterns

• High-content imaging: Automated image acquisition and analysis for quantifying ROBO1 expression levels across large populations of cells

• Fluorescence standardization: Including internal standards such as FITC-labeled beads in microscopy samples to normalize between experiments

• Receptor quantification assay: Using saturation binding with increasing concentrations of FITC-ROBO1 antibody to determine receptor density

For ROBO1 specifically, quantitative analysis has been demonstrated using flow cytometry with 5 μg of FITC-conjugated anti-ROBO1 antibody in cell lines such as J774 macrophages and THP-1 monocytic leukemia cells . Standardized protocols ensure reproducible quantification of ROBO1 expression levels across different experimental contexts.