STARD13 Antibody, FITC conjugated

What are the key structural and functional domains of STARD13 protein?

STARD13 contains multiple functional domains that contribute to its biological activities:

• N-terminal sterile alpha motif (SAM) for protein-protein interactions

• ATP/GTP-binding motif

• GTPase-activating protein (GAP) domain

• C-terminal STAR-related lipid transfer (START) domain

These domains enable STARD13 to function as a GTPase-activating protein primarily for RhoA and potentially for Cdc42 . Through its GAP activity, STARD13 regulates cytoskeletal reorganization, suppresses cell proliferation, and inhibits cell motility . The gene is located in a region of chromosome 13 associated with loss of heterozygosity in hepatocellular carcinomas, further supporting its role as a tumor suppressor .

What are the typical applications for STARD13 antibody, FITC conjugated?

STARD13 antibody, FITC conjugated is primarily used in the following applications:

The FITC conjugation enables direct fluorescent detection without requiring secondary antibodies, which is particularly advantageous for multicolor immunofluorescence experiments . While unconjugated STARD13 antibodies are frequently used for Western Blot applications, the FITC-conjugated version is optimized for applications requiring direct fluorescent visualization .

What are the optimal sample preparation methods for using STARD13 antibody, FITC conjugated in immunofluorescence?

For optimal immunofluorescence results with STARD13 antibody, FITC conjugated:

• Cell Fixation: Fix cells using 4% paraformaldehyde for 15-20 minutes at room temperature. Avoid methanol fixation as it may disrupt the epitope recognized by the antibody.

• Permeabilization: Permeabilize cells with 0.1-0.5% Triton X-100 in PBS for 5-10 minutes.

• Blocking: Block with 1-5% BSA or normal serum (from the species not used to generate the primary antibody) for 30-60 minutes.

• Antibody Incubation: Dilute the FITC-conjugated STARD13 antibody to 1:50-1:500 in blocking buffer and incubate for 1-2 hours at room temperature or overnight at 4°C in a humidified chamber protected from light .

• Washing: Wash extensively with PBS (3-5 times, 5 minutes each) to remove unbound antibody.

• Counterstaining: Consider counterstaining nuclei with DAPI or other nuclear dyes that don't overlap with FITC emission spectrum.

• Mounting: Mount slides using an anti-fade mounting medium specifically formulated for fluorescent dyes.

Positive and negative controls should be included, with A549 or NIH/3T3 cells recommended as positive controls based on validation data .

How should researchers optimize STARD13 antibody concentration for different experimental applications?

Optimization of STARD13 antibody concentration is essential for generating reliable and reproducible results:

• Titration Experiments: Perform initial titration experiments using a range of antibody dilutions:

  • For IF/ICC: Test dilutions from 1:50 to 1:500

  • For Western Blot: Test dilutions from 1:500 to 1:2000

  • For ELISA: Begin with manufacturer's recommended dilution and adjust as needed

• Signal-to-Noise Ratio Assessment: Evaluate the signal-to-noise ratio at each concentration. The optimal concentration should provide strong specific signal with minimal background.

• Cell Type Considerations: Note that different cell types may require different antibody concentrations. For instance, MCF-7 and HeLa cells have been validated for Western Blot applications, while NIH/3T3 and A549 cells have been validated for IF/ICC applications .

• Fixation Method Influence: Consider that antibody performance may vary depending on fixation methods. If initial results are unsatisfactory, alternative fixation protocols should be tested.

• Sample-Dependent Optimization: As noted in product documentation, optimal dilution can be sample-dependent, so verification in your specific experimental system is essential .

Remember that "it is recommended that this reagent should be titrated in each testing system to obtain optimal results" .

How can STARD13 antibodies be used to investigate the relationship between STARD13 expression and cancer stemness?

STARD13 has been shown to attenuate cancer stemness properties in hepatocellular carcinoma and potentially other cancer types . Researchers can use STARD13 antibodies to:

• Comparative Expression Analysis: Compare STARD13 expression between cancer stem cell (CSC) populations (identified by markers such as ALDH1 or CD133) and non-stem cancer cells using immunofluorescence or flow cytometry with FITC-conjugated STARD13 antibody.

• Spheroid Formation Assays: Investigate the correlation between STARD13 expression and spheroid formation ability by:

  • Isolating cells from spheroids versus adherent cultures

  • Analyzing STARD13 expression levels using FITC-conjugated antibodies

  • Correlating expression with stemness markers like ALDH1 and Nanog

Research has demonstrated that STARD13 expression is significantly decreased in nonadherent spheres compared with adherent cells, and overexpression of STARD13 significantly decreases sphere size and number in hepatocellular carcinoma cells .

• Manipulation Studies: Following STARD13 overexpression or knockdown, use FITC-conjugated STARD13 antibodies to:

  • Verify manipulation efficiency

  • Track changes in cellular localization

  • Correlate with stemness markers and functional assays

• YAP/TAZ Pathway Analysis: Simultaneously analyze STARD13 expression and YAP/TAZ localization, as STARD13 regulates YAP/TAZ activity through its Rho GTPase activity .

This approach has revealed that STARD13 overexpression suppresses YAP translocation from nuclear to cytoplasm via inhibiting RhoA activity, thereby attenuating cancer stemness .

What methods can be employed to investigate the role of STARD13 in chemosensitivity using STARD13 antibodies?

STARD13 has been shown to enhance chemosensitivity in cancer cells, including sensitivity to 5-FU in hepatocellular carcinoma and doxorubicin in breast cancer . Researchers can employ the following methods using STARD13 antibodies:

• Expression Correlation Studies:

  • Use FITC-conjugated STARD13 antibodies to quantify STARD13 expression in patient samples

  • Correlate expression levels with clinical response to chemotherapy

  • Compare STARD13 expression in chemoresistant versus chemosensitive cell lines

• Manipulation and Drug Response Assays:

  • Overexpress or knock down STARD13 in cancer cell lines

  • Verify manipulation using STARD13 antibodies

  • Assess changes in chemosensitivity using cell viability assays, apoptosis assays, and drug accumulation studies

• Mechanistic Investigations:

  • Use FITC-conjugated STARD13 antibodies in combination with antibodies against:

    • MDR proteins (e.g., P-glycoprotein)

    • RhoA pathway components

    • YAP/TAZ pathway components

  • Perform co-localization studies to understand spatial relationships

Research has shown that STARD13 overexpression enhances 5-FU sensitivity characterized by decreased cell viability, reduced Ki67 expression, and increased cell apoptosis . Additionally, STARD13-correlated ceRNA network has been shown to sensitize breast cancer cells to doxorubicin .

How can researchers use STARD13 antibodies to elucidate the Rho GTPase regulatory mechanisms of STARD13?

STARD13 functions as a GTPase-activating protein (GAP) for RhoA and potentially Cdc42, regulating cytoskeletal reorganization . Researchers can use STARD13 antibodies to investigate these mechanisms through:

• Co-localization Studies:

  • Use FITC-conjugated STARD13 antibody in combination with antibodies against RhoA, Cdc42, or other cytoskeletal components

  • Analyze subcellular co-localization patterns using confocal microscopy

  • Assess changes in co-localization patterns following cellular stimulation

• F-actin Visualization Assays:

  • Combine FITC-conjugated STARD13 antibody staining with rhodamine-labeled phalloidin to visualize F-actin structures

  • Analyze how STARD13 expression levels correlate with stress fiber formation

  • Assess the impact of STARD13 overexpression or knockdown on F-actin organization

• RhoA Activity Measurements:

  • Use G-LISA RhoA activation assay in conjunction with STARD13 antibody staining

  • Correlate RhoA activity with STARD13 expression and localization

  • Investigate how manipulation of STARD13 affects RhoA activity

Research has shown that STARD13 overexpression significantly decreases basal levels of RhoA activity and markedly inhibits stress fiber formation . Additionally, phosphorylation levels of MLC-S19, a downstream effector of RhoA, are significantly decreased in STARD13 overexpressed cells, providing further evidence of STARD13's role in RhoA regulation .

What are common pitfalls in immunofluorescence experiments using FITC-conjugated antibodies and how can they be addressed?

When working with FITC-conjugated STARD13 antibodies, researchers may encounter several challenges:

• Autofluorescence Issues:

  • Problem: Cellular autofluorescence in the FITC emission spectrum may mask specific signals.

  • Solution: Include unstained controls to assess autofluorescence levels; consider using Sudan Black B (0.1-0.3%) treatment to reduce autofluorescence; or use spectral unmixing during image acquisition if available.

• Photobleaching:

  • Problem: FITC is susceptible to photobleaching during extended imaging sessions.

  • Solution: Use anti-fade mounting media containing anti-photobleaching agents; minimize exposure to excitation light; capture images quickly; consider using lower intensity illumination with longer exposure times.

• Fixation-Related Issues:

  • Problem: Certain fixation methods may alter the epitope recognized by the STARD13 antibody.

  • Solution: Compare paraformaldehyde, methanol, and acetone fixation to determine optimal preservation of the epitope; consider antigen retrieval methods if necessary.

• Non-Specific Binding:

  • Problem: FITC-conjugated antibodies may bind non-specifically to certain cell types or structures.

  • Solution: Optimize blocking conditions using higher concentrations of blocking agents; include additional washing steps; pre-absorb the antibody with cell lysates from irrelevant species; use isotype controls.

• Insufficient Signal:

  • Problem: Weak signal despite proper experimental setup.

  • Solution: Increase antibody concentration; extend incubation time; confirm that your cell type expresses STARD13 (MCF-7, HeLa, NIH/3T3, or A549 cells are recommended as positive controls) ; ensure proper storage of the antibody at -20°C to maintain activity .

• Signal Variability Between Experiments:

  • Problem: Results vary significantly between experimental replicates.

  • Solution: Standardize all protocol steps; prepare fresh fixatives and buffers; aliquot antibodies to avoid freeze-thaw cycles; standardize image acquisition settings.

How should researchers interpret variations in STARD13 localization patterns in different cell types?

STARD13 localization can vary between cell types and experimental conditions, requiring careful interpretation:

• Subcellular Distribution Patterns:

  • STARD13 may show cytoplasmic, membrane-associated, or punctate distribution patterns depending on cell type and activation state.

  • Compare patterns in your experimental system with published patterns in similar cell types.

  • Quantify distribution patterns using image analysis software for objective comparison.

• Cell Type-Specific Considerations:

  • Different cell lines may express varying levels of STARD13 interaction partners, affecting localization.

  • Cancer cell lines (e.g., MCF-7, HeLa) versus normal cell lines (e.g., NIH/3T3) may show distinct localization patterns reflecting functional differences .

  • Consider co-staining for cell type-specific markers to correlate STARD13 localization with cell identity.

• Activation State Assessment:

  • STARD13 localization may change depending on the activation state of Rho GTPases.

  • Changes in F-actin organization (assessed by phalloidin staining) may correlate with STARD13 localization shifts.

  • Consider performing time-course experiments after stimulation to capture dynamic changes.

• Physiological versus Overexpression System Differences:

  • Interpret overexpression systems cautiously, as artificially high levels may alter normal localization patterns.

  • Compare endogenous staining with overexpression systems to identify potential artifacts.

  • Validate key findings using complementary approaches such as subcellular fractionation followed by Western blotting.

• Resolution Limitations:

  • Standard epifluorescence microscopy may not resolve fine details of STARD13 localization.

  • Consider super-resolution microscopy techniques for detailed localization studies.

  • Electron microscopy with immunogold labeling can provide ultrastructural localization information.

What are the most reliable approaches for validating STARD13 antibody specificity in experimental systems?

Ensuring STARD13 antibody specificity is crucial for experimental validity. Consider these approaches:

• Genetic Validation:

  • STARD13 Knockdown/Knockout: Compare staining patterns between wild-type cells and those with STARD13 knockdown or knockout. Specific signal should be significantly reduced or absent in knockdown/knockout cells .

  • Overexpression Validation: Compare endogenous staining with cells overexpressing STARD13, which should show increased signal intensity.

• Peptide Competition Assays:

  • Pre-incubate the STARD13 antibody with excess immunizing peptide before staining.

  • Specific staining should be blocked by the peptide competition.

  • Use an irrelevant peptide as a negative control to confirm specificity.

• Multiple Antibody Validation:

  • Compare staining patterns using multiple antibodies targeting different STARD13 epitopes.

  • Consistent patterns across antibodies support specificity.

  • Consider using both monoclonal and polyclonal antibodies as complementary approaches.

• Western Blot Correlation:

  • Confirm that the STARD13 antibody detects a protein of the expected molecular weight (approximately 125 kDa) in Western blot .

  • Verify that the detection pattern in Western blot corresponds to expected expression patterns across different cell types (e.g., lower in cancer tissues compared to normal tissues) .

• Cross-Reactivity Assessment:

  • Test the antibody on cell lines or tissues from species with known sequence variations.

  • Consider testing in cells from knockout organisms if available.

  • Review product validation data showing reactivity with human and mouse samples .

• Reproducibility Testing:

  • Establish protocol reproducibility across different batches of antibody.

  • Verify consistent staining patterns across multiple experimental replicates.

  • Document all validation steps meticulously for publication and future reference.

How might STARD13 antibodies be used to explore the potential of STARD13 as a therapeutic target in cancer?

STARD13's role as a tumor suppressor makes it an interesting potential therapeutic target. Researchers can use STARD13 antibodies to explore this potential through:

• Therapeutic Response Biomarker Development:

  • Use FITC-conjugated STARD13 antibodies to analyze STARD13 expression levels before and after treatment.

  • Correlate STARD13 expression with response to various therapies, particularly those targeting Rho GTPase pathways or YAP/TAZ signaling.

  • Develop flow cytometry or immunofluorescence-based assays for rapid assessment of STARD13 status in patient samples.

• Combination Therapy Research:

  • Investigate how modulation of STARD13 expression affects sensitivity to chemotherapeutic agents.

  • Use FITC-conjugated antibodies to monitor changes in STARD13 expression and localization during drug treatment.

  • Develop rational combination strategies targeting STARD13-related pathways.

Research has shown that STARD13 enhances 5-FU sensitivity by suppressing cancer stemness in hepatocellular carcinoma cells via attenuating YAP transcriptional activity . Additionally, STARD13-correlated ceRNA network sensitizes breast cancer cells to doxorubicin .

• Drug Screening Approaches:

  • Develop high-content screening assays using FITC-conjugated STARD13 antibodies to identify compounds that upregulate STARD13 expression or enhance its activity.

  • Screen for compounds that mimic STARD13's effects on downstream pathways, especially RhoA inhibition and YAP/TAZ cytoplasmic retention.

  • Use these assays to identify potential therapeutic candidates for further development.

• Targeted Therapeutic Delivery:

  • Explore using STARD13 antibodies for targeted delivery of therapeutic agents to cells with low STARD13 expression.

  • Investigate the potential of antibody-drug conjugates or nanoparticle-based delivery systems.

  • Assess the feasibility of restoring STARD13 expression or function as a therapeutic approach.

Since "activation of STARD13 could be a potential novel therapeutic strategy to specifically target HCC stem cells and enhance chemotherapy sensitivity of HCC patients" , these research directions have significant translational potential.

What methodological innovations might enhance the utility of FITC-conjugated STARD13 antibodies in multi-parameter analyses?

Several methodological innovations could enhance the utility of FITC-conjugated STARD13 antibodies:

• Multiplexed Imaging Technologies:

  • Combine FITC-conjugated STARD13 antibodies with spectrally distinct antibodies against Rho GTPases, YAP/TAZ, and cytoskeletal markers.

  • Employ cyclic immunofluorescence or sequential imaging approaches to overcome spectral limitations.

  • Utilize advanced microscopy techniques like spectral unmixing to distinguish closely overlapping fluorophores.

• Single-Cell Analysis Integration:

  • Combine flow cytometry using FITC-conjugated STARD13 antibodies with single-cell RNA-seq to correlate protein expression with transcriptomic profiles.

  • Develop index sorting approaches to link STARD13 protein levels with subsequent single-cell genomic or transcriptomic analysis.

  • Apply computational methods to integrate these multi-omics datasets.

• Live Cell Imaging Adaptations:

  • Develop cell-permeable versions of STARD13 antibody fragments for live-cell imaging.

  • Explore alternative labeling strategies such as nanobodies conjugated to FITC for improved tissue penetration and reduced immunogenicity.

  • Combine with optogenetic tools to simultaneously visualize and manipulate STARD13 function.

• Tissue Analysis Enhancements:

  • Adapt FITC-conjugated STARD13 antibodies for tissue clearing techniques to enable 3D visualization in intact tissues.

  • Employ spatial transcriptomics in conjunction with STARD13 immunofluorescence to correlate protein localization with gene expression patterns.

  • Develop protocols for multiplexed protein detection in formalin-fixed paraffin-embedded tissues for retrospective clinical sample analysis.

• Quantitative Analysis Improvements:

  • Develop calibration standards for absolute quantification of STARD13 levels using FITC-conjugated antibodies.

  • Apply machine learning approaches to automated image analysis for objective quantification of staining patterns.

  • Implement digital pathology workflows for high-throughput analysis of STARD13 expression in clinical samples.