RPS9 Antibody

What is RPS9 and what experimental applications is the antibody suitable for?

RPS9 (Ribosomal Protein S9) is a 23 kDa protein (though typically observed at 25 kDa in gel electrophoresis) that functions as a component of the 40S ribosomal subunit . RPS9 antibody has been validated for multiple experimental applications including:

When designing experiments, it's critical to validate these dilutions in your specific experimental system as optimal concentrations may vary based on sample type and preparation method .

What are the optimal storage conditions for maintaining RPS9 antibody activity?

For maximum antibody stability and performance, researchers should follow these evidence-based storage practices:

• Store RPS9 antibody at -20°C in aliquots containing PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• The antibody remains stable for one year after shipment when stored properly

• For smaller size preparations (20μl), the commercial formulation may contain 0.1% BSA as a stabilizer

• Aliquoting is unnecessary for -20°C storage, but is recommended if multiple freeze-thaw cycles are anticipated

• Avoid repeated freeze-thaw cycles as this can diminish antibody performance

These conditions maintain epitope recognition capabilities and prevent degradation that could compromise experimental results.

How should researchers validate RPS9 antibody specificity for their experimental system?

Methodological approach to antibody validation should include:

• Positive control testing: Use known positive samples such as HeLa cells for Western blot and immunofluorescence applications

• Molecular weight verification: Confirm detection at the expected molecular weight (calculated: 23 kDa; observed: 25 kDa)

• Knockdown validation: Compare staining between wild-type and RPS9-silenced samples, as demonstrated in published knockdown studies

• Cross-reactivity assessment: Test antibody performance with samples from different species if cross-species applications are intended

• Experimental replication: Ensure consistent results across multiple experimental replicates

Evidence shows that proper validation is critical, as demonstrated in multiple publications utilizing RPS9 antibody for Western blot (7 publications), IHC (2 publications), and IF (1 publication) .

What methodological approaches should be employed when using RPS9 antibody in cancer research?

When investigating RPS9's role in cancer progression, researchers should implement these evidence-based methodological considerations:

• Expression analysis: RPS9 is significantly upregulated in NSCLC tissues compared to normal tissues (demonstrated in 68.1% of cases)

• Correlation with clinical parameters: RPS9 expression levels positively correlate with advanced TNM stages in NSCLC

• Functional studies: Implement RPS9 knockdown and overexpression experiments to assess:

  • Proliferation capacity (via CCK-8 assay)

  • Colony formation ability

  • Migration and invasion potential (via transwell assays)

  • Cell cycle distribution (via flow cytometry)

• Signaling pathway investigation: Examine the effect of RPS9 modulation on Stat3 and Erk signaling pathways, which have been identified as downstream effectors

Researchers should maintain appropriate controls and validate antibody performance in each specific cancer model system being studied.

What are the technical considerations for RPS9 antibody-based antigen retrieval in tissue samples?

For optimal antigen retrieval when using RPS9 antibody in immunohistochemistry applications:

• Primary recommendation: Use TE buffer at pH 9.0 for antigen retrieval in paraffin-embedded tissues

• Alternative method: Citrate buffer at pH 6.0 can be used as an alternative when TE buffer produces suboptimal results

• Tissue-specific optimization: Different tissues may require adjusted protocols:

  • Human tissues (lung, ovary, skin, testis) have been successfully stained following proper antigen retrieval

  • Mouse tissues (lung, pancreas, stomach) may require optimization of retrieval conditions

• Incubation parameters: After antigen retrieval, optimize primary antibody dilution (1:50-1:500) and incubation time based on signal intensity and background levels

• Detection system selection: Choose appropriate secondary detection systems compatible with rabbit IgG primary antibodies

This approach ensures maximum epitope exposure while preserving tissue morphology for accurate localization of RPS9 protein.

How should researchers design RPS9 knockdown experiments to study its function in signaling pathways?

Evidence-based design considerations for RPS9 knockdown studies include:

• Silencing method selection:

  • siRNA transfection has been successfully employed in multiple cell lines (PC9, A549, H1299)

  • Consider stable shRNA approaches for long-term studies

• Validation of knockdown efficiency:

  • Confirm RPS9 reduction at both mRNA level (qRT-PCR) and protein level (Western blot)

  • Published data shows significant reduction in RPS9 expression can be achieved in lung cancer cell lines

• Functional assays:

  • Cell proliferation: CCK-8 assay revealed RPS9 knockdown significantly inhibits proliferation

  • Colony formation: Assess clonogenic ability post-knockdown

  • Migration/invasion: Transwell assays demonstrate reduced metastatic capacity following RPS9 silencing

• Pathway analysis:

  • Examine Stat3 phosphorylation status by Western blot

  • Assess Erk signaling pathway activation

  • Consider phospho-specific antibodies to detect activation states

• Rescue experiments:

  • Perform overexpression of RPS9 in knockdown cells to confirm phenotype specificity

  • H292 cells have been successfully used for overexpression studies

This experimental approach provides comprehensive insights into RPS9's functional role in cellular signaling networks.

What approaches should be employed when studying post-translational modifications of RPS9?

When investigating post-translational modifications (PTMs) of RPS9, researchers should implement the following methodological strategies:

• PTM site identification: Multiple modification sites have been identified on RPS9, including:

This comprehensive approach enables detailed characterization of RPS9 PTMs and their functional significance.

How can researchers troubleshoot inconsistent RPS9 antibody results across different applications?

When encountering variability in RPS9 antibody performance, implement these evidence-based troubleshooting approaches:

• Western blot inconsistencies:

  • Verify protein extraction method is appropriate for ribosomal proteins

  • Adjust lysis buffer composition to ensure complete extraction

  • Optimize primary antibody concentration (1:500-1:1000)

  • Consider longer transfer times for ribosomal proteins

  • Implement validated positive controls (HeLa cells, mouse uterus tissue)

• IHC signal variability:

  • Compare antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Adjust antibody dilution across a wider range (1:50-1:500)

  • Extend primary antibody incubation time or temperature

  • Test different detection systems compatible with rabbit polyclonal antibodies

  • Validate with known positive tissues (human lung, ovary, skin)

• Immunofluorescence optimization:

  • Modify fixation method (paraformaldehyde vs. methanol)

  • Adjust permeabilization conditions

  • Test various blocking reagents to reduce background

  • Optimize antibody dilution (1:200-1:800)

  • Use confocal microscopy for improved signal-to-noise ratio

These systematic approaches address common technical challenges while maintaining experimental rigor.

What cell cycle analysis considerations should be implemented when studying RPS9 in cancer models?

Published research demonstrates that RPS9 knockdown results in G2/M phase arrest in multiple NSCLC cell lines . When designing flow cytometry-based cell cycle studies:

• Sample preparation optimization:

  • Ensure single-cell suspensions without aggregates

  • Standardize fixation methods (70% ethanol, -20°C, overnight)

  • Use RNase treatment to eliminate RNA-PI binding interference

  • Optimize propidium iodide concentration for DNA content analysis

• Gating strategy development:

  • Exclude doublets using FSC-H vs. FSC-A plots

  • Remove debris with appropriate FSC/SSC gating

  • Analyze minimum of 10,000 events for statistical significance

• Data interpretation considerations:

  • Compare cell cycle distribution between RPS9-knockdown and control cells

  • Focus on G2/M phase changes as demonstrated in PC9, A549, and H1299 cell lines

  • Supplement flow cytometry data with cyclin expression analysis (Western blot)

  • Correlate cell cycle findings with proliferation assay results

• Experimental design elements:

  • Include time-course analysis (24h, 48h, 72h post-treatment)

  • Implement synchronization protocols for improved resolution

  • Consider dual-parameter analysis (e.g., BrdU incorporation with PI staining)

This methodological approach provides comprehensive insights into RPS9's role in cell cycle regulation within cancer models.

How does RPS9 contribute to cancer progression mechanisms, and what experimental approaches reveal these functions?

Research evidence demonstrates RPS9's significant role in cancer progression, particularly in NSCLC . Key experimental findings include:

• Expression correlation with disease state:

  • RPS9 is significantly upregulated in NSCLC tissues compared to normal tissues

  • Overexpression correlates with advanced TNM stages

  • Analysis of TCGA database confirms elevated RPS9 mRNA levels in primary adenocarcinoma samples

  • Kaplan-Meier analysis from GEO database demonstrates association between RPS9 overexpression and poor patient outcomes

• Functional impact on cancer hallmarks:

  • Proliferation: RPS9 knockdown inhibits cancer cell proliferation in multiple cell lines

  • Colony formation: Decreased clonogenic ability following RPS9 silencing

  • Metastatic potential: Reduced migration and invasion capacity in transwell assays

  • Cell cycle regulation: G2/M phase arrest observed after RPS9 knockdown

• Signaling pathway involvement:

  • RPS9 modulates Stat3 and Erk signaling pathways in NSCLC

  • These pathways are critical mediators of cancer progression, connecting RPS9 to established oncogenic mechanisms

• Experimental validation through complementary approaches:

  • Loss-of-function (siRNA) in multiple cell lines (PC9, A549, H1299)

  • Gain-of-function (overexpression) in H292 cells

  • Consistent phenotypic effects across different experimental models

These findings highlight RPS9 as a potential therapeutic target and diagnostic biomarker in NSCLC, warranting further investigation of its mechanisms in other cancer types.

What methodological considerations are important when studying RPS9 in different tissue and cell types?

When investigating RPS9 across diverse experimental systems, researchers should implement these tissue/cell-specific methodological considerations:

• Species-specific reactivity optimization:

  • Antibody has confirmed reactivity with human and mouse samples

  • Predicted cross-reactivity with multiple model organisms (pig, zebrafish, bovine, horse, sheep, rabbit, dog, Xenopus)

  • Validation required when applying to predicted but untested species

• Tissue-type considerations:

  • Human tissues: Successfully detected in lung, ovary, skin, and testis tissues

  • Mouse tissues: Validated in lung, pancreas, stomach, uterus, and ovary tissues

  • Tissue-specific protocols may require optimization based on protein abundance and accessibility

• Cell line applications:

  • Positive control cell lines: HeLa cells provide reliable positive controls for multiple applications

  • Cancer model systems: Various lung cancer cell lines (PC9, A549, H1299, H292) have been successfully used

  • Consider cell-type specific extraction methods to maximize protein recovery

• Application-specific protocol adjustments:

  • For IHC: Optimize antigen retrieval method based on tissue type (TE buffer pH 9.0 primary recommendation, citrate buffer pH 6.0 as alternative)

  • For WB: Adjust lysis conditions based on cell/tissue type to ensure complete extraction

  • For IF/ICC: Modify fixation and permeabilization based on cell type

Implementation of these methodological considerations ensures reliable and reproducible results across diverse experimental systems.

What emerging research areas could benefit from RPS9 antibody applications?

Based on current findings and technological advancements, several promising research directions utilizing RPS9 antibody include:

• Cancer biomarker development:

  • RPS9 overexpression correlates with poor prognosis in NSCLC

  • Investigate potential diagnostic/prognostic applications in other cancer types

  • Develop tissue microarray studies across diverse cancer cohorts

  • Correlate expression with treatment response and survival outcomes

• Ribosomal stress response mechanisms:

  • Examine RPS9's role in nucleolar stress signaling

  • Investigate interaction with p53 pathway components

  • Study potential extra-ribosomal functions using co-immunoprecipitation with RPS9 antibody

  • Apply proximity labeling approaches to identify novel interaction partners

• Therapeutic targeting strategies:

  • Identify compounds that modulate RPS9 expression or function

  • Develop combination strategies targeting RPS9-dependent signaling pathways (Stat3, Erk)

  • Evaluate synthetic lethality approaches in RPS9-overexpressing tumors

  • Assess immune response to RPS9 in various pathological conditions

• Post-translational modification landscape:

  • Characterize dynamic changes in RPS9 PTMs during cellular stress

  • Investigate enzyme-substrate relationships for RPS9 modifications

  • Develop modification-specific antibodies for known PTM sites (methylation at R5/K11, ubiquitination at K11, phosphorylation at T12/Y13/T15)

These emerging research areas represent opportunities for impactful investigations utilizing RPS9 antibody technologies in conjunction with other advanced methodologies.

How can researchers integrate RPS9 antibody-based techniques with other emerging technologies?

Methodological approaches for integrating RPS9 antibody applications with cutting-edge technologies include:

• Single-cell analysis integration:

  • Combine RPS9 antibody with CyTOF mass cytometry for multi-parameter single-cell profiling

  • Implement imaging mass cytometry for spatial resolution of RPS9 expression in tissue contexts

  • Correlate with single-cell transcriptomics to connect protein expression with transcriptional states

• Advanced microscopy applications:

  • Apply super-resolution microscopy techniques (STORM, PALM) for nanoscale localization of RPS9

  • Implement live-cell imaging approaches using cell-permeable RPS9 antibody fragments

  • Develop FRET-based approaches to study RPS9 interactions in living cells

• Multi-omics experimental design:

  • Integrate RPS9 ChIP-seq to identify potential chromatin association patterns

  • Combine with ribosome profiling to correlate RPS9 levels with translation efficiency

  • Implement spatial transcriptomics with RPS9 IHC on sequential sections for regional correlation

• Therapeutic development platforms:

  • Screen for RPS9-modulating compounds using high-content imaging with RPS9 antibody

  • Develop antibody-drug conjugates targeting RPS9 in cancer contexts

  • Implement CRISPR screens to identify synthetic lethal interactions with RPS9 overexpression/knockdown

These integrative approaches leverage RPS9 antibody specificity within emerging technological frameworks to address complex biological questions with unprecedented resolution and insight.