FABP9 Antibody, HRP conjugated

What is FABP9 and why is it significant in cancer research?

FABP9 belongs to the fatty acid-binding protein family involved in fatty acid transport and lipid metabolism. In prostate cancer research, FABP9 has gained attention as its expression is significantly higher in carcinomas than in benign tissues, with increased staining intensity correlating with reduced patient survival times. Additionally, FABP9 expression shows significant association with increased joint Gleason scores and androgen receptor index, suggesting its potential value as a prognostic marker for prostate cancer outcomes . Beyond cancer, FABP9 is also known under alternative names including Perf15, Tlbp, 15 kDa perforatorial protein, Testis lipid-binding protein, and Testis-type fatty acid-binding protein, indicating its presence in normal testicular tissue .

What are the key characteristics of FABP9 protein expression in different cell lines?

FABP9 protein expression varies significantly across different prostate cell lines. Research has shown that FABP9 (running as a single band at 14 kDa) is highly expressed in highly malignant prostate cancer cell lines PC-3 and PC3-M, while its expression remains undetectable in benign PNT-2 cells and other malignant cell lines. Quantitative analysis demonstrated that FABP9 expression in PC3-M cells was approximately 20% higher than in PC-3 cells (p=0.006), suggesting a correlation between FABP9 expression levels and the degree of malignancy . The protein has a predicted band size of 15 kDa when analyzed using Western blotting techniques .

How does HRP conjugation of antibodies work and what advantages does it offer in FABP9 detection?

HRP conjugation involves chemically linking Horseradish Peroxidase enzyme to antibodies targeting FABP9. The process typically follows these steps: mixing the antibody with a modifier reagent, adding this mixture to lyophilized HRP material, incubating for at least 3 hours, and finally adding a quencher reagent. This conjugation creates ready-to-use antibodies that don't require secondary antibody steps in detection assays . The primary advantage of HRP-conjugated antibodies in FABP9 detection is direct visualization without secondary antibody requirements, reducing background noise and false positives while streamlining experimental protocols. This becomes particularly valuable when working with complex tissue samples where multiple antibody binding events might create confounding results .

What is the optimal protocol for conjugating anti-FABP9 antibodies with HRP?

The optimal protocol for HRP conjugation to anti-FABP9 antibodies follows these methodological steps:

• Preparation: Ensure antibody is in a buffer free of sodium azide, BSA, gelatin, and other proteins.

• Modification: Add 1 μl of Modifier reagent to each 10 μl of antibody solution and mix gently.

• Conjugation: Remove the cap from the vial containing lyophilized HRP and pipette the antibody-modifier mixture directly onto it. Gently resuspend by withdrawing and re-dispensing once or twice.

• Incubation: Replace the cap and leave at room temperature (20-25°C) in the dark for at least 3 hours (overnight incubation is also acceptable).

• Quenching: Add 1 μl of Quencher reagent for every 10 μl of antibody used and mix gently. The conjugate will be ready to use after 30 minutes .

This protocol is specifically designed to maintain antibody functionality while providing efficient HRP conjugation. No purification step is required, making the process straightforward and resulting in ready-to-use conjugates .

What sample preparation techniques ensure optimal detection of FABP9 in prostate tissue specimens?

For optimal detection of FABP9 in prostate tissue specimens, researchers should follow these methodological steps:

• Tissue fixation: Fix tissues in neutral-buffered formalin for 24-48 hours to preserve protein structure while maintaining tissue architecture.

• Paraffin embedding and sectioning: Process tissues through graded alcohols and embed in paraffin. Cut sections at 4-5 μm thickness.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) to unmask epitopes potentially hidden during fixation.

• Blocking: Block endogenous peroxidase activity using hydrogen peroxide (3%) and prevent non-specific binding with normal serum.

• Primary antibody application: Apply anti-FABP9 antibody at an optimized concentration (typically around 20 μg/ml for immunohistochemical analysis, as demonstrated in previous studies) .

• Detection: Develop using an appropriate substrate for HRP (DAB is commonly used).

• Counterstaining: Use hematoxylin for nuclear visualization.

This comprehensive approach ensures consistent and specific staining patterns, allowing accurate assessment of FABP9 expression levels across different prostate tissue samples .

What controls should be included when using FABP9 antibodies in experimental setups?

When designing experiments using FABP9 antibodies, researchers should incorporate these essential controls:

• Positive tissue controls: Include testis tissue samples that naturally express FABP9, as confirmed by previous studies showing specific staining patterns in rat and mouse testis lysates .

• Negative tissue controls: Include tissues known not to express FABP9 or use benign prostate tissues as comparative controls, as FABP9 expression is significantly lower in benign cases compared to carcinomas .

• Technical negative controls: Omit primary antibody while maintaining all other steps to assess non-specific binding of detection reagents.

• siRNA knockdown controls: When studying FABP9 function, include samples where FABP9 expression has been suppressed using targeted siRNAs (previous studies have achieved up to 60% suppression using specific siRNAs) .

• Recombinant protein standards: Include purified recombinant FABP9 protein as a standard for calibration and antibody validation .

These methodologically sound controls help validate experimental findings and distinguish true FABP9 expression from technical artifacts, ensuring reliable research outcomes.

How should researchers interpret varying levels of FABP9 expression in relation to prostate cancer progression?

When interpreting FABP9 expression data in prostate cancer studies, researchers should consider the following methodological framework:

• Staining intensity classification: Categorize FABP9 staining into discrete intensity levels (0, +, ++, +++) as shown in previous studies.

• Correlation with Gleason scores: Analyze FABP9 expression in relation to combined Gleason scores (GS), expecting significantly higher expression in cases with high GS (8-10) compared to low GS (≤5) .

• Association with AR index: Assess correlation between FABP9 staining intensity and androgen receptor index, noting that AR index levels are typically significantly higher in cases with strong FABP9 staining .

• Patient survival analysis: Plot Kaplan-Meier survival curves stratified by FABP9 expression levels to evaluate prognostic significance.

The table below demonstrates the relationship between FABP9 staining intensity and Gleason scores from previous research:

This data demonstrates that higher Gleason scores correlate with increased FABP9 expression, providing a valuable interpretive framework for researchers .

What are the common technical challenges when using FABP9 antibodies and how can they be addressed?

Researchers commonly encounter these technical challenges when working with FABP9 antibodies:

• Non-specific binding: Address by optimizing antibody concentration through titration experiments (start with manufacturer's recommended 3 μg/mL for Western blot and 20 μg/ml for IHC) and implementing rigorous blocking steps with appropriate blocking buffers .

• Variability in staining intensity: Standardize by including positive controls (rat testis lysate) and reference standards. Normalize expression levels to housekeeping proteins for Western blot applications .

• Epitope masking in fixed tissues: Implement systematic comparison of different antigen retrieval methods (heat-induced vs. enzymatic) to determine optimal protocol for FABP9 detection.

• False negatives in benign tissues: Be aware that FABP9 expression may be below detection limits in benign tissues, requiring sensitive detection methods and longer exposure times .

• Multiple band detection: Account for potential post-translational modifications or splice variants when Western blots show additional bands besides the expected 15 kDa band (mouse testis lysates have shown bands at 15 kDa, 22 kDa, 241 kDa, and 28 kDa) .

These methodological solutions ensure consistent and reliable detection of FABP9 across different experimental conditions.

How can researchers validate the specificity of their FABP9 antibody, particularly after HRP conjugation?

To validate FABP9 antibody specificity after HRP conjugation, researchers should implement this comprehensive validation protocol:

• Western blot analysis with recombinant protein: Test the conjugated antibody against purified recombinant FABP9 protein to confirm detection at the expected molecular weight (15 kDa) .

• Comparative tissue analysis: Perform parallel experiments using the conjugated antibody on tissues known to express FABP9 (testis) and those with low or no expression (benign prostate hyperplasia) .

• siRNA knockdown validation: Apply the antibody to samples where FABP9 has been silenced using siRNA technology, expecting reduced signal intensity (previous studies achieved up to 60% reduction in FABP9 expression using specific siRNAs) .

• Peptide competition assay: Pre-incubate the antibody with excess FABP9 peptide before application to samples, expecting signal abolishment if the antibody is specific.

• Cross-reactivity assessment: Test the antibody against related FABP family members (particularly FABP5 and FABP6) to ensure selective detection of FABP9.

This methodological approach ensures that research findings truly reflect FABP9 expression patterns rather than non-specific antibody interactions .

How can FABP9 expression analysis be integrated with other biomarkers for improved prostate cancer prognosis?

For integrating FABP9 with other biomarkers in prostate cancer prognosis, researchers should implement this methodological framework:

• Multiplex immunohistochemistry: Develop protocols for simultaneous detection of FABP9 with established markers including androgen receptor (AR), PSA, and other FABP family members using differentially labeled antibodies or sequential staining approaches.

• Correlation analysis: Implement statistical methods to analyze relationships between FABP9 expression and other markers. Previous research has already established correlations between FABP9 staining intensity and both Gleason scores (p<0.001) and AR index (p=0.03) .

• Multivariate survival analysis: Perform Cox regression analysis incorporating FABP9 expression alongside clinical parameters (PSA levels, tumor stage) and other molecular markers to develop comprehensive prognostic models.

• Machine learning integration: Develop algorithms that incorporate quantitative FABP9 expression data with other biomarkers to predict patient outcomes, treatment response, and risk stratification.

This integrated approach moves beyond single-marker analysis to develop clinically relevant prognostic tools that may improve treatment decision-making for prostate cancer patients .

What functional studies can be performed to investigate the role of FABP9 in cancer cell invasiveness?

To investigate FABP9's role in cancer cell invasiveness, researchers can implement these advanced functional approaches:

• RNA interference techniques: Use siRNA targeting FABP9 (as demonstrated in previous studies where siRNA-3 achieved 60% suppression) to knock down expression in highly malignant cell lines like PC3-M .

• Matrigel invasion assays: Quantitatively assess the invasive potential of control cells versus FABP9-silenced cells through Matrigel-coated transwell chambers, measuring the number of invading cells after specific time periods.

• Live cell imaging: Employ time-lapse microscopy to analyze differences in migratory behavior and morphological changes between FABP9-expressing and FABP9-suppressed cells.

• 3D spheroid invasion models: Develop three-dimensional tumor spheroids to evaluate invasion patterns in a more physiologically relevant context than traditional 2D cultures.

• In vivo metastasis models: Utilize animal models injected with FABP9-expressing or FABP9-suppressed cancer cells to monitor metastatic potential and colonization capabilities.

Previous research has already established that suppression of FABP9 expression in highly malignant PC3-M cells inhibits their invasive potential, providing a foundation for these more detailed functional investigations .

What is the relationship between FABP9 and immune function, and how might this impact cancer immunotherapy approaches?

The relationship between FABP9 and immune function represents an emerging research area with implications for cancer immunotherapy:

• Fatty acid signaling in immune modulation: FABP9, like other fatty acid binding proteins, may facilitate fatty acid transport and signaling that regulates inflammatory responses in the tumor microenvironment. In vertebrates, FABPs are involved in inflammation regulated by fatty acids through interaction with peroxidase proliferator activate receptors (PPARs) .

• Bacterial binding and agglutination: Studies of FABP9 homologs (Es-FABP9) in invertebrates have demonstrated bacterial binding activity and bacterial agglutination against pathogens like Escherichia coli and Staphylococcus aureus .

• Growth inhibition properties: Recombinant FABP9-like proteins have shown growth inhibition activity against specific bacteria including Vibrio parahaemolyticus and Staphylococcus aureus .

• Immune cell recruitment and activation: Researchers should investigate whether FABP9 in cancer cells affects tumor-infiltrating immune cell populations, potentially through fatty acid-mediated signaling pathways.

While most FABP9 cancer research has focused on its prognostic value, these immune-related functions suggest potential applications in immunotherapy, particularly in understanding how lipid metabolism in cancer cells might influence immune surveillance and response to immunotherapeutic interventions .

How can FABP9 antibodies be utilized in developing targeted therapeutic approaches for prostate cancer?

For developing FABP9-targeted therapeutic approaches, researchers should consider these methodological strategies:

• Antibody-drug conjugates (ADCs): Conjugate anti-FABP9 antibodies with cytotoxic payloads to specifically target FABP9-expressing cancer cells. The established HRP conjugation protocols can serve as a methodological foundation for other conjugation approaches .

• FABP9-targeting nanoparticles: Develop nanoparticles functionalized with anti-FABP9 antibodies to deliver therapeutic agents specifically to prostate cancer cells with high FABP9 expression.

• CAR-T cell therapy development: Engineer chimeric antigen receptor T cells targeting FABP9, particularly for advanced prostate cancers where FABP9 expression correlates with higher Gleason scores .

• Bispecific antibodies: Design bispecific antibodies that simultaneously target FABP9 and immune checkpoint molecules to enhance anti-tumor immune responses.

• Small molecule inhibitors: Screen for compounds that disrupt FABP9's fatty acid binding function, potentially interfering with its role in promoting cancer cell invasiveness.

The differential expression pattern of FABP9 between malignant and benign prostate tissues provides a therapeutic window for targeted approaches, potentially minimizing off-target effects on normal tissues with low or undetectable FABP9 expression .