SRGAP3 Antibody, FITC conjugated

What are the primary applications for SRGAP3 Antibody, FITC conjugated?

SRGAP3 Antibody, FITC conjugated can be utilized in multiple experimental techniques including immunofluorescence (particularly in fixed tissues and cells), flow cytometry, and fluorescence microscopy. The antibody allows direct visualization of SRGAP3 protein localization without requiring secondary antibody incubation, streamlining experimental workflows. Testing data confirms applications in Western Blot (WB), immunohistochemistry (IHC), immunofluorescence on paraffin-embedded sections (IF-P), and ELISA, with demonstrated reactivity in human, mouse, and rat samples .

What is the recommended storage condition for maintaining antibody activity?

For optimal preservation of fluorescence signal and antibody activity, store the SRGAP3 Antibody, FITC at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles should be avoided as they can compromise both antibody binding capacity and fluorescence intensity. The antibody is typically supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . Working aliquots should be prepared to minimize freeze-thaw cycles.

What dilution ranges are recommended for different applications?

The optimal dilution varies by application type and must be empirically determined for each experimental system. General recommendations include:

• Western Blot: 1:2000-1:12000

• Immunohistochemistry: 1:500-1:2000

• Immunofluorescence: 1:50-1:500

These ranges serve as starting points, and titration is recommended to optimize signal-to-noise ratios in your specific experimental context.

How can I verify the specificity of SRGAP3 antibody staining in my samples?

Verification of antibody specificity should employ multiple approaches:

• Molecular weight confirmation: SRGAP3 has an expected molecular weight of approximately 124 kDa, with observed migration at approximately 125 kDa in SDS-PAGE .

• Positive controls: Include tissues known to express SRGAP3, such as brain tissue from mouse or rat where SRGAP3 is abundantly expressed .

• Negative controls: Employ isotype controls (rabbit IgG-FITC) at equivalent concentrations to evaluate non-specific binding.

• Peptide competition assay: Pre-incubate the antibody with immunizing peptide to confirm signal specificity.

• siRNA knockdown: Compare staining patterns in cells with and without SRGAP3 knockdown to confirm signal specificity.

What sample preparation methods are optimal for immunofluorescence with FITC-conjugated SRGAP3 antibody?

For optimal immunofluorescence results:

• Fixation: 4% paraformaldehyde for 15-20 minutes at room temperature preserves cellular morphology while maintaining epitope accessibility.

• Permeabilization: 0.1-0.3% Triton X-100 in PBS for 5-10 minutes facilitates antibody access to intracellular SRGAP3.

• Blocking: Use 5-10% normal serum (from species unrelated to primary antibody) with 1% BSA to minimize non-specific binding.

• Antigen retrieval: For formalin-fixed paraffin-embedded tissues, TE buffer (pH 9.0) is recommended, though citrate buffer (pH 6.0) may be used as an alternative .

• Counterstaining: DAPI (blue) provides optimal nuclear contrast with FITC (green) without spectral overlap.

• Mounting: Use anti-fade mounting medium to preserve FITC fluorescence during imaging and storage.

How can I troubleshoot weak or absent SRGAP3-FITC signal in immunofluorescence?

Several factors may contribute to suboptimal signal:

• Epitope masking: Try different antigen retrieval methods, including heat-induced epitope retrieval or enzymatic retrieval.

• Insufficient permeabilization: Increase Triton X-100 concentration or incubation time for accessing intracellular epitopes.

• Excessive washings: Reduce wash stringency to prevent antibody loss.

• Antibody concentration: Try higher concentrations (1:50-1:100) for challenging samples.

• Photobleaching: Minimize sample exposure to light during processing and use anti-fade mounting medium.

• Expression levels: SRGAP3 expression may be naturally low in your samples; consider signal amplification methods.

• Buffer composition: Ensure pH is optimal (generally 7.2-7.4) for antibody-epitope interaction.

How can I use SRGAP3 Antibody, FITC to investigate interactions with other srGAP family members?

Investigation of SRGAP3 interactions with other srGAP family members requires careful experimental design:

• Co-immunoprecipitation: Use SRGAP3 antibody to pull down protein complexes, followed by Western blot analysis for srGAP1 or srGAP2. Research demonstrates that srGAP3 interacts with all three full-length srGAP proteins through their respective IF-BAR domains .

• Co-localization studies: Combine SRGAP3-FITC antibody with differently labeled antibodies against srGAP1 or srGAP2. Confocal microscopy reveals that when co-expressed, srGAP2 and srGAP3 show distinct distribution along filopodia and co-localize mainly in punctate cytoplasmic structures, plasma membrane, and some protrusions .

• Proximity ligation assay (PLA): This technique can detect protein-protein interactions with high sensitivity and specificity when proteins are within 40nm proximity.

• FRET (Förster Resonance Energy Transfer): For examining real-time interactions in living cells, combine FITC-labeled SRGAP3 antibody with appropriately labeled antibodies against other srGAP proteins.

What role does SRGAP3 play in neuronal development, and how can I study it?

SRGAP3 functions in neuronal differentiation through Rho GTPase signaling pathways. To study its role:

• Neuronal morphology analysis: Use SRGAP3-FITC antibody to visualize protein localization during neuronal differentiation. Track changes in neurite formation, branching patterns, and filopodia/lamellipodia dynamics.

• RhoGTPase activity assays: Combine SRGAP3 visualization with pull-down assays to correlate SRGAP3 localization with GTP-bound states of Rac1 and Cdc42. SRGAP3 functions as a GTPase-activating protein for RAC1 and potentially Cdc42, but not for RhoA small GTPase .

• Time-lapse imaging: Monitor SRGAP3 dynamics during neuronal differentiation using FITC-conjugated antibody in live cell imaging (for cell lines) or fixed time points (for primary neurons).

• Knockdown/overexpression studies: Compare neuronal morphology in cells with altered SRGAP3 expression levels. Research indicates that srGAP proteins negatively regulate neuronal differentiation through synergistic interactions .

How can I analyze SRGAP3's role in modulating cytoskeletal dynamics?

To investigate SRGAP3's influence on cytoskeletal organization:

• Co-staining approaches: Combine SRGAP3-FITC antibody with phalloidin (F-actin marker) to visualize correlation between SRGAP3 localization and actin cytoskeleton organization. Similar to SRGAP1 knockdown effects, SRGAP3 modulation may influence F-actin distribution and cell protrusion formation .

• Live-cell imaging: Track SRGAP3 and cytoskeletal dynamics simultaneously using SRGAP3-FITC antibody and fluorescently labeled cytoskeletal markers.

• Super-resolution microscopy: Techniques like STED or STORM provide nanoscale resolution of SRGAP3 distribution relative to cytoskeletal elements.

• Rho GTPase activity measurements: Combine visualization with biochemical assays to correlate SRGAP3 localization with local Rho GTPase activities, particularly focusing on RAC1 and Cdc42 .

How should I design experiments to distinguish between SRGAP3 and other srGAP family proteins?

Due to sequence homology between srGAP family members, careful experimental design is essential:

• Antibody selection: Verify that the SRGAP3-FITC antibody was raised against unique epitopes not conserved in srGAP1 or srGAP2. Check immunogen information - some SRGAP3 antibodies are generated against recombinant SLIT-ROBO Rho GTPase-activating protein 3 protein (709-955AA) .

• Western blot validation: Confirm antibody specificity using samples expressing only one srGAP family member.

• siRNA validation: Perform knockdown experiments targeting SRGAP3 specifically to confirm signal reduction with the FITC-conjugated antibody.

• Cross-reactivity testing: Some commercial antibodies may cross-react with multiple srGAP proteins, as noted in product datasheets where SRGAP3 antibodies list "srGAP2" among aliases , possibly reflecting sequence similarity or historical naming conventions.

• Molecular weight differentiation: While similar, the different srGAP proteins have slightly different molecular weights that can help distinguish them in Western blot analysis.

What controls should be included when studying SRGAP3 in cancer-associated fibroblasts?

When investigating SRGAP3 in cancer contexts such as cancer-associated fibroblasts (CAFs):

• Tissue compartment controls: Include both stromal and epithelial compartments in analyses, as lymphocyte accumulation patterns differ between these regions in high-grade serous ovarian carcinoma (HGSOC) .

• Fibroblast subtype markers: Co-stain with markers for different CAF subtypes (particularly CAF-S1) to correlate SRGAP3 expression with specific fibroblast populations.

• Immune cell infiltration analysis: Include markers for T lymphocytes (CD3, CD8, FOXP3) to examine correlation between SRGAP3 expression and immune cell recruitment .

• Normal fibroblast controls: Compare SRGAP3 expression and localization between normal fibroblasts and CAFs from the same patient.

• Functional readouts: Assess migration, invasion, and cytokine production to correlate SRGAP3 expression with functional properties of CAFs.

How can I evaluate SRGAP3 protein interactions with Rho GTPases using the FITC-conjugated antibody?

To study SRGAP3-Rho GTPase interactions:

• Co-immunoprecipitation followed by Western blot: Pull down SRGAP3 and probe for associated Rho GTPases, or perform the reverse experiment.

• GST-pulldown assays: Use GST-tagged constitutively active (CA) Rho GTPases to pull down SRGAP3. Research shows that SRGAP family proteins interact differently with Rho GTPases - full-length srGAP1 and srGAP3 strongly interact with GST-CA Rac1 and Cdc42, while srGAP2 shows weaker interaction with Rac1 .

• Microscopy-based approaches: Combine SRGAP3-FITC antibody with differently labeled antibodies against Rho GTPases to assess co-localization in specific cellular compartments.

• FRET-based assays: For detecting direct protein interactions in intact cells.

• Functional validation: Correlate SRGAP3-Rho GTPase interactions with cellular phenotypes like changes in actin organization, cell morphology, or migration capacity.

How can I optimize quantitative analysis of SRGAP3 expression using the FITC-conjugated antibody?

For reliable quantitative analysis:

• Standard curve generation: Prepare samples with known quantities of recombinant SRGAP3 protein to establish fluorescence intensity-to-concentration relationship.

• Image acquisition standardization: Use consistent exposure settings, gain, and offset parameters across all experimental conditions.

• Background subtraction: Implement rigorous background correction using isotype controls and image processing algorithms.

• Internal references: Include housekeeping protein controls for normalization.

• Batch processing: Process all experimental conditions in parallel to minimize technical variation.

• Automated analysis: Develop or utilize image analysis scripts that objectively quantify SRGAP3-FITC signal intensity and distribution patterns.

• Flow cytometry optimization: For single-cell analysis, establish gates based on negative controls and use mean fluorescence intensity (MFI) for quantitative comparisons.

What methodological approaches can resolve contradictory findings regarding SRGAP3 function?

When facing contradictory results:

• Cell type considerations: Different cell types may express different SRGAP3 binding partners or regulatory proteins. Systematically test multiple relevant cell lines and primary cells.

• Domain-specific analysis: SRGAP3 contains multiple functional domains (IF-BAR, RhoGAP, SH3). Use domain-specific antibodies or truncated constructs to dissect domain-specific functions.

• Post-translational modification assessment: Phosphorylation status may alter SRGAP3 function. Combine SRGAP3-FITC staining with phospho-specific antibodies.

• Activity state discrimination: Develop assays to distinguish between active and inactive SRGAP3 pools within cells.

• Context-dependent analysis: Systematic variation of experimental conditions (serum levels, substrate stiffness, cell density) may reveal context-dependent functions.

• Temporal resolution: Implement time-course experiments to capture dynamic changes in SRGAP3 function during cellular processes.

How can advanced imaging techniques enhance SRGAP3-FITC signal detection and analysis?

To maximize information from SRGAP3-FITC labeling:

• Structured illumination microscopy (SIM): Provides 2x resolution improvement over conventional microscopy without specialized fluorophores.

• Stimulated emission depletion (STED) microscopy: Achieves super-resolution imaging with FITC-conjugated antibodies, revealing nanoscale distribution patterns.

• Deconvolution: Computational approach to improve signal-to-noise ratio and resolution of standard widefield microscopy.

• Airyscan detection: Enhances resolution and sensitivity without requiring high laser power, preserving FITC fluorescence.

• Light sheet microscopy: For 3D imaging of SRGAP3 distribution in tissue samples with minimal photobleaching.

• Quantitative image analysis: Apply advanced algorithms for colocalization analysis, intensity correlation, and morphological feature extraction.

• Spectral unmixing: Particularly useful when combining FITC with other fluorophores that have spectral overlap.