RBBP9 Antibody

What is RBBP9 and why is it significant in cellular research?

RBBP9 (Retinoblastoma-binding protein 9) is a multifunctional protein involved in cell cycle regulation, DNA damage repair, chromatin remodeling, and transcriptional regulation. It plays a critical role in maintaining the expression of both pluripotency and cell cycle genes in human pluripotent stem cells (hPSCs) by influencing cell cycle progression through the RB/E2F pathway. Additionally, RBBP9 possesses serine hydrolase (SH) activity in non-pluripotent cells, acting on currently undefined target proteins. The significance of RBBP9 extends to cancer biology, as its dysregulation has been implicated in the development and progression of multiple cancer types including pancreatic, ovarian, colon, lung, and breast cancer . Due to these diverse functions, RBBP9 antibodies are valuable tools for investigating cellular mechanisms related to development, pluripotency, and oncogenesis.

What types of RBBP9 antibodies are available for research applications?

Researchers have access to both monoclonal and polyclonal RBBP9 antibodies, each with specific applications and advantages. Available options include:

When selecting an antibody, consider the specific experimental application (WB, IHC, IF), species reactivity requirements, and whether a monoclonal (higher specificity) or polyclonal (broader epitope recognition) antibody would be more appropriate for your research question .

How should I validate a newly acquired RBBP9 antibody?

Proper validation of RBBP9 antibodies is essential to ensure experimental reliability. A comprehensive validation protocol should include:

• Positive and negative control samples: Use cell lines or tissues with known RBBP9 expression levels. For negative controls, consider RBBP9 knockdown cells using siRNA or CRISPR/Cas9 methods.

• Western blot validation: Verify a single band at the expected molecular weight (~21 kDa for human RBBP9).

• Immunoprecipitation followed by mass spectrometry: This confirms the antibody's ability to specifically capture RBBP9 from complex protein mixtures.

• Cross-reactivity testing: Especially important when working with antibodies reacting with multiple species to ensure specificity.

• Application-specific controls: For IHC/IF applications, include peptide competition assays and isotype control antibodies to confirm staining specificity.

Validation parameters should be documented and reported in research publications to enhance reproducibility of findings across different laboratories .

What are the optimal conditions for Western blot detection of RBBP9?

Successful Western blot detection of RBBP9 requires attention to several methodological details:

• Sample preparation: Extract total protein using RIPA buffer supplemented with protease inhibitors. For nuclear localization studies, consider using nuclear extraction protocols.

• Protein loading: Load 20-30 μg of total protein lysate per lane.

• Gel percentage: Use 12-15% SDS-PAGE gels to optimally resolve RBBP9 (~21 kDa).

• Transfer conditions: Semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 60 minutes using PVDF membrane (preferred over nitrocellulose for small proteins).

• Blocking: 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute RBBP9 antibodies according to manufacturer recommendations (typically 1:500-1:2000) and incubate overnight at 4°C.

• Detection: Enhanced chemiluminescence (ECL) systems are generally sufficient for detection, though more sensitive methods may be needed for low abundance samples.

These conditions may require optimization based on the specific antibody and sample type being analyzed .

How should immunohistochemistry protocols be optimized for RBBP9 detection?

Optimizing IHC protocols for RBBP9 detection requires careful consideration of several variables:

• Fixation method: 10% neutral buffered formalin for 24-48 hours provides good results. Overfixation can mask epitopes.

• Antigen retrieval: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is generally effective. For some tissues, EDTA buffer (pH 9.0) may yield better results.

• Blocking endogenous peroxidase: 3% hydrogen peroxide for 10 minutes before primary antibody incubation.

• Antibody dilution: Start with 1:100-1:200 dilution and optimize based on signal-to-noise ratio.

• Incubation conditions: Overnight at 4°C generally provides optimal staining.

• Detection system: Polymer-based detection systems often provide cleaner backgrounds than avidin-biotin methods.

• Counterstaining: Light hematoxylin counterstaining to avoid obscuring specific RBBP9 signals.

Always include positive control tissues (e.g., pluripotent stem cells or cancer tissues known to express RBBP9) and isotype-matched negative controls .

What strategies can be used to simultaneously detect RBBP9 and its interaction partners?

Investigating RBBP9's interactions with other proteins is critical for understanding its functional roles. Several approaches are recommended:

• Co-immunoprecipitation (Co-IP): Use RBBP9 antibodies to pull down the protein complex, followed by Western blot detection of potential binding partners (particularly components of the RB/E2F pathway).

• Proximity ligation assay (PLA): This technique can visualize protein-protein interactions in situ with high sensitivity. Use RBBP9 antibody alongside antibodies against suspected interaction partners.

• Immunofluorescence co-localization: Dual staining with RBBP9 and potential binding partners using different fluorophores, followed by confocal microscopy and colocalization analysis.

• FRET (Fluorescence Resonance Energy Transfer): For detecting close molecular interactions (<10 nm).

• ChIP-seq combined with RBBP9 immunoprecipitation: To identify genomic regions where RBBP9 may be functioning in transcriptional regulation.

When designing these experiments, consider using antibodies raised in different host species to avoid cross-reactivity issues .

How can RBBP9 antibodies be used to investigate its role in pluripotent stem cells?

RBBP9 plays a critical role in maintaining pluripotency and regulating cell cycle progression in human pluripotent stem cells (hPSCs). Several advanced research approaches using RBBP9 antibodies can elucidate its mechanisms:

• Chromatin immunoprecipitation (ChIP): Use RBBP9 antibodies to identify genomic regions where RBBP9 binds, potentially regulating pluripotency genes.

• Cell cycle phase-specific analyses: Combine RBBP9 immunostaining with cell cycle markers (e.g., EdU incorporation, cyclin antibodies) to assess changes in RBBP9 localization or abundance throughout the cell cycle.

• Phospho-specific analysis: Investigate potential post-translational modifications of RBBP9 during differentiation using phospho-specific antibodies.

• Quantitative immunofluorescence: Measure RBBP9 protein levels during differentiation protocols to correlate with expression of pluripotency factors.

• Proximity labeling techniques: Use BioID or APEX2 fusions with RBBP9 to identify proximal proteins in the pluripotent state versus during differentiation.

These approaches can reveal how RBBP9 connects cell cycle regulation with pluripotency maintenance, as suggested by studies showing RBBP9 influences the RB/E2F pathway in hPSCs .

What methodological approaches can distinguish between RBBP9's serine hydrolase activity and RB-binding functions?

Distinguishing between RBBP9's dual functions requires sophisticated experimental designs:

• Comparative inhibitor studies: Use ML114 (a selective chemical inhibitor of RBBP9 serine hydrolase activity) alongside RBBP9 knockdown approaches. ML114 treatment (cell-free IC50 = 0.63 μM) specifically inhibits the serine hydrolase activity without affecting protein expression, allowing researchers to separate effects due to enzyme activity versus protein-protein interactions .

• Structure-function analysis: Use antibodies specific to different RBBP9 domains in combination with mutant RBBP9 constructs (particularly active site serine mutants) to determine which domain mediates specific cellular effects.

• Activity-based protein profiling (ABPP): Combine with RBBP9 immunoprecipitation to directly measure serine hydrolase activity in different cellular contexts.

• Substrate identification: Use proteomics approaches with RBBP9 antibodies to identify proteins that might be substrates for its serine hydrolase activity.

Research indicates that RBBP9 serine hydrolase activity may specifically promote hPSC proliferation, while its RB-binding activity impacts differentiation - an important distinction when designing experiments to study either function .

How can RBBP9 antibodies be used to investigate its roles in cancer progression?

RBBP9 has been implicated in multiple cancer types, including pancreatic, ovarian, colon, lung, and breast cancer. Advanced applications of RBBP9 antibodies in cancer research include:

• Tissue microarray (TMA) analysis: Use IHC with RBBP9 antibodies on cancer TMAs to correlate expression with clinical outcomes, stage, and other molecular markers.

• Cell cycle checkpoint analysis: Combine RBBP9 immunostaining with markers of cell cycle checkpoints to understand how its dysregulation may contribute to aberrant proliferation.

• Invasion and migration assays: Analyze RBBP9 expression and localization during cancer cell invasion processes.

• RBBP9 interactome in cancer cells: Compare RBBP9 protein interaction networks between normal and cancer cells using immunoprecipitation combined with mass spectrometry.

• Drug resistance mechanisms: Investigate whether RBBP9 expression or activity changes in response to chemotherapy or targeted therapies.

• Cancer stem cell analysis: Examine RBBP9 expression in putative cancer stem cell populations versus differentiated tumor cells.

These approaches can help determine whether RBBP9 might serve as a potential diagnostic marker or therapeutic target for specific cancer types .

What are common causes of false negative results when detecting RBBP9?

False negative results when working with RBBP9 antibodies may stem from several factors:

• Epitope masking: RBBP9 may undergo post-translational modifications or form protein complexes that mask antibody binding sites. Try multiple antibodies targeting different epitopes.

• Fixation and processing issues: Overfixation can destroy epitopes. For formalin-fixed samples, optimize antigen retrieval methods (try both citrate and EDTA buffers at different pH values).

• Low expression levels: RBBP9 may be expressed at levels below detection limits in some cell types. Consider using more sensitive detection methods or signal amplification techniques.

• Protein degradation: RBBP9 may be susceptible to proteolytic degradation. Ensure samples are processed rapidly and with appropriate protease inhibitors.

• Incorrect antibody application: Verify the antibody is validated for your specific application (WB, IHC, IF) and species of interest.

• Buffer compatibility issues: Some lysis buffers may interfere with epitope recognition. Test alternative extraction methods if standard protocols fail.

Implementing a systematic troubleshooting approach addressing each of these potential issues will help resolve false negative results .

How can contradictory results from different RBBP9 antibodies be reconciled?

When different RBBP9 antibodies yield contradictory results, consider these analytical approaches:

• Epitope mapping: Determine the exact epitopes recognized by each antibody. Antibodies targeting different domains may detect distinct conformations or isoforms of RBBP9.

• Validation hierarchy: Prioritize results from antibodies with more extensive validation documentation (knockout controls, multiple application validations).

• Cross-validation with non-antibody methods: Confirm key findings using orthogonal techniques such as mass spectrometry or mRNA analysis.

• Biological context consideration: Some antibodies may perform differently depending on cell/tissue type or experimental conditions. Document these context-dependent differences.

• Isoform specificity: Check whether antibodies recognize specific RBBP9 isoforms or post-translationally modified variants.

• Quantitative comparison: Use quantitative methods like ELISA or quantitative Western blotting to compare relative binding affinities of different antibodies.

• Competitive binding assays: Determine if antibodies compete for the same epitope or can bind simultaneously.

These approaches can help distinguish between technical artifacts and genuine biological complexity in RBBP9 expression or function .

What are best practices for interpreting RBBP9 subcellular localization studies?

RBBP9 can function in multiple cellular compartments, making localization studies particularly informative but challenging. Consider these practices:

• Multiple fixation and permeabilization protocols: Different methods may reveal distinct aspects of RBBP9 localization. Compare cross-linking fixatives (paraformaldehyde) with precipitating fixatives (methanol).

• Super-resolution microscopy: Standard confocal microscopy may not resolve fine details of RBBP9 localization. Consider techniques like STED, STORM, or PALM for higher resolution analysis.

• Co-localization controls: Include markers for specific subcellular compartments (nuclear lamina, nucleoli, chromatin regions, ER, Golgi).

• Dynamic localization studies: Assess RBBP9 localization throughout the cell cycle or during differentiation processes using time-lapse imaging or synchronized cell populations.

• Biochemical fractionation validation: Complement imaging with subcellular fractionation followed by Western blotting to confirm compartment-specific localization.

• Perturbation analysis: Examine how RBBP9 localization changes in response to cell cycle inhibitors, differentiation signals, or ML114 treatment.

• Functional correlation: Relate localization patterns to specific RBBP9 functions (serine hydrolase activity versus RB-binding).

These approaches can provide mechanistic insights into how RBBP9 localization correlates with its diverse cellular functions .

How might RBBP9 antibodies be used to identify novel therapeutic targets in cancer?

RBBP9 antibodies can facilitate several cutting-edge approaches to cancer therapeutic discovery:

• Spatial proteomics: Combine RBBP9 immunoprecipitation with proximity labeling techniques to map the complete protein interaction network in cancer versus normal cells.

• Functional screening: Use RBBP9 antibodies to monitor protein levels during CRISPR or small molecule screens to identify synthetic lethal interactions in RBBP9-overexpressing cancers.

• Post-translational modification profiling: Develop modification-specific antibodies to determine if cancer-specific RBBP9 modifications could be targeted therapeutically.

• Patient-derived xenograft (PDX) models: Use RBBP9 antibodies to stratify PDX models based on expression patterns, then correlate with treatment responses.

• Liquid biopsy development: Investigate whether RBBP9 or its modified forms can be detected in circulation as potential biomarkers.

• Immune interaction studies: Examine how RBBP9 expression in cancer cells influences tumor immune microenvironment using multiplexed immunofluorescence approaches.

These strategies may reveal whether targeting RBBP9 directly, or indirectly through its interaction partners or enzymatic substrates, could provide therapeutic opportunities for multiple cancer types .

What experimental designs can elucidate the relationship between RBBP9 and NFYA in pluripotent stem cells?

Recent studies suggest nuclear transcription factor Y subunit A (NFYA) may be a candidate effector of RBBP9 serine hydrolase activity in human pluripotent stem cells. To investigate this relationship:

• Sequential ChIP (Re-ChIP): Use sequential immunoprecipitation with RBBP9 and NFYA antibodies to identify genomic regions where both proteins co-localize.

• Proximity-dependent biotinylation: Use BioID or APEX2 fusions with RBBP9 to determine if NFYA is in close proximity in live cells.

• Activity-dependent studies: Compare NFYA post-translational modifications in wild-type cells versus ML114-treated cells or cells expressing catalytically inactive RBBP9 mutants.

• Transcriptional reporter assays: Use NFYA-dependent reporters to assess how RBBP9 inhibition or knockdown affects NFYA transcriptional activity.

• Rescue experiments: Test whether NFYA overexpression can rescue proliferation defects caused by ML114 treatment or RBBP9 knockdown.

• Phospho-proteomics: Compare the phosphorylation status of NFYA in control versus ML114-treated cells to identify potential regulatory mechanisms.

These approaches could establish whether NFYA is a direct substrate of RBBP9 serine hydrolase activity or functions through an indirect mechanism in the regulation of stem cell proliferation .

How can RBBP9 antibodies contribute to understanding the coupling between cell cycle regulation and differentiation?

RBBP9 studies present a unique opportunity to investigate the mechanistic coupling between proliferation and differentiation decisions in stem cells. Advanced experimental designs using RBBP9 antibodies include:

• Single-cell correlation analyses: Combine RBBP9 immunostaining with markers of cell cycle phases and differentiation status at the single-cell level to identify transitional states.

• Chromatin landscape mapping: Use RBBP9 ChIP-seq alongside histone modification profiling to determine how RBBP9 influences the epigenetic landscape during cell fate decisions.

• Live-cell imaging: Develop fluorescent RBBP9 reporter systems to track protein dynamics during differentiation in real-time.

• Microfluidic approaches: Use microfluidic devices to precisely control exposure to ML114 or differentiation factors while monitoring RBBP9 levels and localization.

• Synthetic biology circuits: Design genetic circuits to modulate RBBP9 levels or activity at specific cell cycle phases to test causality in proliferation-differentiation coupling.

• Comparative systems analysis: Apply RBBP9 antibodies across multiple stem cell types to determine if its role in uncoupling proliferation from differentiation is conserved or context-dependent.

These approaches could help resolve the intriguing observation that ML114 treatment reduces hPSC proliferation without inducing differentiation - a phenomenon that challenges traditional models of stem cell regulation .