RPS7 Antibody

What is RPS7 and why is it important to study?

Ribosomal protein S7 (RPS7) is a structural component of the ribosome involved in protein synthesis. Beyond its canonical role in translation, RPS7 has several non-ribosomal functions that make it an important research target. It interacts with and inhibits mdm2-mediated p53 degradation, highlighting its role in cell cycle regulation and cancer biology. Additionally, RPS7 plays an essential role in early development and in the maturation of ribosomal RNAs in the production pathways of both large and small ribosomal subunits . Recent research has revealed its involvement in cancer progression, particularly in hepatocellular carcinoma (HCC) where it promotes metastasis by stabilizing specific target mRNAs .

What applications can RPS7 antibodies be used for?

RPS7 antibodies can be utilized for multiple experimental applications in molecular and cell biology research:

It is essential to optimize these dilutions for your specific experimental system to obtain optimal results .

What is the molecular weight of RPS7 and how is it detected in Western blots?

The calculated molecular weight of RPS7 is 22 kDa, although it is typically observed at 22-25 kDa in Western blot applications . This slight variation between the calculated and observed molecular weight may be due to post-translational modifications or the nature of the protein's structure. When performing Western blots for RPS7, researchers typically detect positive signals in cell lines such as NIH/3T3 and HepG2 . The antibody should be used at dilutions of 1:500-1:1000 for optimal detection in Western blot applications .

What species reactivity do RPS7 antibodies demonstrate?

The reactivity profile of RPS7 antibodies varies by product. Based on the search results:

• The Proteintech RPS7 antibody (14491-1-AP) has been tested and confirmed to react with human, mouse, and rat samples .

• Published research has cited the use of this antibody with human and mouse samples .

• The Novus Biologicals RPS7 antibody is specifically validated for human samples .

When selecting an RPS7 antibody for your research, it's critical to verify the species reactivity to ensure compatibility with your experimental model system.

How is RPS7 implicated in cancer progression, particularly in hepatocellular carcinoma?

RPS7 has emerged as a significant RNA-binding protein (RBP) in hepatocellular carcinoma (HCC) progression. Recent research reveals that RPS7 expression is markedly increased in HCC tissues and strongly correlates with poor survival outcomes . Mechanistically, RPS7 promotes HCC metastasis through several pathways:

• RPS7 stabilizes Lysyl oxidase-like 2 (LOXL2) mRNA by binding to AUUUA motifs in the 3155-3375 region of the 3'UTR of LOXL2 mRNA, thereby increasing LOXL2 expression .

• LOXL2, in turn, accelerates focal adhesion formation by maintaining protein stability of ITGB1 and activating ITGB1-mediated FAK/SRC signaling pathway .

• This cascade ultimately enhances cell adhesion, migration, and invasion capabilities of HCC cells, promoting metastasis .

The clinical significance of RPS7 is underscored by its correlation with histological grades and clinical stages in HCC patients. Interestingly, RPS7 expression appears to be specifically elevated in HCC compared to several other common malignancies, including lung, colon, breast, and prostate cancers .

What methodological approaches can be used to study RPS7's RNA-binding properties?

Research into RPS7's function as an RNA-binding protein employs several sophisticated methodological approaches:

• RNA Immunoprecipitation (RIP): This technique has been used to demonstrate that RPS7 directly binds to specific mRNAs. In published research, RIP assays revealed that LOXL2 mRNA was remarkably enriched in RPS7-immunoprecipitates compared to control GAPDH mRNA .

• RNA Pull-down Assays: Using biotin-labeled mRNA fragments, researchers have mapped the specific binding regions of RPS7 on target mRNAs. For example, RPS7 was found to interact with the 3'UTR of LOXL2 mRNA, but not with the 5'UTR or CDS regions .

• mRNA Stability Assays: Treatment with actinomycin D (an inhibitor of RNA synthesis) followed by qRT-PCR at various time points can reveal how RPS7 affects mRNA half-life. This approach demonstrated that RPS7 silencing decreased the half-life of LOXL2 mRNA, while RPS7 overexpression increased it .

• Nascent RNA Capture Assays: This method helps determine whether changes in mRNA levels are due to altered synthesis or stability. Research showed that RPS7 manipulation did not affect the rate of LOXL2 mRNA synthesis .

• Luciferase Reporter Assays: These assays can assess whether RPS7 affects gene promoter activity. Studies found that RPS7 silencing or overexpression did not affect the promoter activity of the LOXL2 gene .

How can CRISPR-Cas9 gene editing be utilized to study RPS7 function?

CRISPR-Cas9 gene editing represents a powerful approach for interrogating RPS7 function in cellular models. In published research, CRISPR-Cas9 has been successfully employed to generate RPS7-knockout cell lines from highly aggressive HCC cell lines (MHCC97H and HLE) . This genetic manipulation enables several key experimental approaches:

When implementing CRISPR-Cas9 for RPS7 studies, researchers should carefully validate knockout efficiency using both Western blot and qRT-PCR to confirm complete loss of RPS7 expression before proceeding with functional assays.

What are the optimal conditions for immunohistochemistry using RPS7 antibodies?

For successful immunohistochemistry (IHC) applications with RPS7 antibodies, several methodological considerations are critical:

For researchers interested in clinical correlations, IHC studies have demonstrated that RPS7 expression is significantly associated with histological grades and clinical stages in HCC patients .

What considerations should be made when designing Western blot experiments to detect RPS7?

When designing Western blot experiments to detect RPS7, researchers should consider the following technical aspects:

• Sample preparation:

  • Cell lysates from NIH/3T3 and HepG2 cells have been validated as positive controls for RPS7 Western blot detection .

  • Standard RIPA buffer with protease inhibitors is suitable for protein extraction.

• Gel percentage and transfer conditions:

  • As RPS7 has a molecular weight of 22-25 kDa , 12-15% SDS-PAGE gels are recommended for optimal resolution.

  • Standard semi-dry or wet transfer methods are suitable, with PVDF or nitrocellulose membranes.

• Antibody selection and dilution:

  • The recommended dilution range for Western blot is 1:500-1:1000 for Proteintech antibody and 1:100-1:2000 for Novus Biologicals antibody .

  • Primary antibody incubation can be performed at room temperature for 1.5 hours or at 4°C overnight .

• Detection system:

  • Standard HRP-conjugated secondary antibodies against rabbit IgG are appropriate.

  • Both chemiluminescence and fluorescence-based detection systems are compatible.

• Loading controls:

  • For ribosomal proteins like RPS7, traditional housekeeping genes like GAPDH or β-actin are appropriate loading controls.

  • When studying RPS7 in cancer contexts, validating results with multiple loading controls is advisable due to potential variability in housekeeping gene expression.

How can RNA-binding studies of RPS7 be optimized for reproducibility?

To optimize RNA-binding studies of RPS7 for reproducibility, several methodological considerations should be implemented:

• Cross-linking conditions:

  • UV cross-linking at 254 nm or chemical cross-linking with formaldehyde can be used to stabilize RNA-protein interactions.

  • Optimization of cross-linking time and intensity is critical to balance between capturing genuine interactions and minimizing artificial associations.

• Immunoprecipitation protocol optimization:

  • Using validated antibodies with confirmed specificity for RPS7 is essential.

  • Including appropriate negative controls (IgG or isotype control) and positive controls (known RPS7-binding RNAs) in each experiment.

  • Stringent washing conditions should be empirically determined to remove non-specific interactions while maintaining genuine RPS7-RNA complexes.

• RNA extraction and analysis:

  • TRIzol-based extraction methods followed by DNase treatment are recommended for RNA isolation from immunoprecipitates.

  • Both targeted (qRT-PCR) and unbiased (RNA-seq) approaches can be used to identify and quantify RPS7-bound RNAs.

• Validation of binding sites:

  • After identifying RPS7-bound RNAs, validation of specific binding regions can be performed using RNA pull-down assays with biotin-labeled RNA fragments, as demonstrated in the LOXL2 mRNA study .

  • Mutation analysis of predicted binding motifs (e.g., AUUUA motifs in the 3'UTR of LOXL2) can confirm the specificity of the interaction .

• Functional validation:

  • Complementary approaches such as mRNA stability assays, nascent RNA synthesis assays, and luciferase reporter assays should be employed to determine the functional consequences of RPS7-RNA interactions .

What are common issues in RPS7 Western blot analysis and how can they be resolved?

When performing Western blot analysis for RPS7, researchers may encounter several common issues. Here are troubleshooting strategies for each:

• Multiple bands or unexpected molecular weight:

  • RPS7 should appear at 22-25 kDa . Multiple bands may indicate degradation or post-translational modifications.

  • Solution: Use fresh samples with protease inhibitors, optimize sample preparation conditions, and ensure appropriate gel percentage (12-15% SDS-PAGE) for optimal resolution of proteins in this molecular weight range.

• Weak or no signal:

  • May result from insufficient antibody concentration, inadequate protein loading, or poor transfer efficiency.

  • Solution: Increase antibody concentration within the recommended range (1:500-1:1000) , optimize protein loading (20-40 μg total protein), and verify transfer efficiency with reversible protein staining before blocking.

• High background:

  • Often caused by insufficient blocking, inadequate washing, or excessive antibody concentration.

  • Solution: Extend blocking time (1-2 hours at room temperature or overnight at 4°C), increase washing duration and volume, and dilute primary antibody appropriately.

• Inconsistent results between experiments:

  • May stem from variations in sample preparation, transfer efficiency, or detection methods.

  • Solution: Standardize all protocol steps, use consistent positive controls (NIH/3T3 or HepG2 cell lysates) , and implement quantitative analysis with appropriate normalization to loading controls.

• Cross-reactivity issues:

  • The antibody may detect proteins other than RPS7 in some contexts.

  • Solution: Validate antibody specificity using RPS7 knockout controls generated by CRISPR-Cas9 , or RPS7 overexpression systems as positive controls.

How can researchers validate the specificity of RPS7 antibodies in their experimental systems?

Validating antibody specificity is critical for ensuring reliable experimental results. For RPS7 antibodies, several validation approaches are recommended:

• Genetic knockout controls:

  • CRISPR-Cas9-mediated knockout of RPS7 provides the gold standard for antibody validation .

  • Western blot, IHC, or IF signals should be absent or dramatically reduced in knockout cells/tissues compared to wild-type controls.

• RNAi-mediated knockdown:

  • siRNA or shRNA against RPS7 should reduce signal intensity in proportion to the knockdown efficiency.

  • Quantify both mRNA (by qRT-PCR) and protein levels to confirm correlation between transcript reduction and protein signal decrease.

• Overexpression systems:

  • Cells transfected with RPS7 expression vectors should show increased signal intensity.

  • Tagged versions (His, FLAG, or GFP-tagged RPS7) can be detected with tag-specific antibodies to confirm localization and expression patterns match those detected by the RPS7 antibody.

• Peptide competition assays:

  • Pre-incubation of the antibody with the immunizing peptide or recombinant RPS7 protein should abolish specific signals in Western blot or immunostaining.

  • The immunogen information (e.g., "RPS7 fusion protein Ag5892" for Proteintech antibody or the peptide sequence for Novus antibody ) can guide the design of these assays.

• Multiple antibody validation:

  • Using different antibodies targeting distinct epitopes of RPS7 should yield similar results.

  • Comparison between polyclonal (e.g., Proteintech 14491-1-AP ) and monoclonal antibodies can provide complementary validation.

What approaches can be used to correlate RPS7 expression with clinical outcomes in cancer research?

Researchers investigating the clinical relevance of RPS7 in cancer can employ several methodological approaches to correlate its expression with clinical outcomes:

What are emerging roles of RPS7 beyond its canonical ribosomal function?

Research is increasingly revealing non-canonical functions of RPS7 beyond its established role in ribosome assembly and protein translation:

• Regulation of p53 pathway:

  • RPS7 interacts with and inhibits mdm2-mediated p53 degradation, positioning it as a potential tumor suppressor in certain contexts .

  • This function places RPS7 among other ribosomal proteins (like RPL5, RPL11) that link ribosomal stress to cell cycle regulation through the p53 pathway.

• RNA-binding protein (RBP) activity:

  • Recent research has identified RPS7 as an RNA-binding protein that can directly interact with specific mRNAs and regulate their stability .

  • This function appears particularly important in cancer progression, where RPS7 stabilizes LOXL2 mRNA by binding to AUUUA motifs in the 3'UTR .

• Role in development:

  • RPS7 has an essential role in early development, suggesting functions beyond basic translation .

  • It participates in the maturation of ribosomal RNAs in both large and small ribosomal subunit production pathways .

• Cancer progression and metastasis:

  • Emerging evidence suggests RPS7 promotes metastasis in hepatocellular carcinoma by enhancing cell adhesion, migration, and invasion capabilities .

  • Unlike its potential tumor-suppressive role through p53 regulation, this function suggests context-dependent oncogenic activity.

• Tissue-specific functions:

  • Research indicates that RPS7's expression and function may be tissue-specific, with particularly notable roles in liver cancer compared to other malignancies .

  • This suggests potential tissue-specific regulatory mechanisms governing RPS7 expression and activity.

How do recent findings on RPS7 in hepatocellular carcinoma inform potential therapeutic strategies?

Recent mechanistic insights into RPS7's role in hepatocellular carcinoma suggest several potential therapeutic approaches: