RPL17 Antibody

What applications are RPL17 antibodies validated for?

RPL17 antibodies have been validated for multiple research applications with specific performance characteristics:

It is critical to note that the optimal dilution should be determined empirically for each experimental system to obtain reliable results .

What tissue and cell line reactivity has been confirmed for RPL17 antibodies?

Positive Western blot detection has been confirmed in:

• Human cell lines: HeLa, HepG2, HEK-293, Jurkat cells

• Mouse tissues: Pancreas tissue

• Rat tissues: Pancreas tissue

• Additional cell lines: HSC-T6, NIH/3T3, 4T1 cells

Positive immunohistochemistry (IHC) detection has been confirmed in human pancreatic cancer tissue, with suggested antigen retrieval using TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0 .

What are the standard storage and handling recommendations for RPL17 antibodies?

Based on manufacturer specifications, RPL17 antibodies should be stored at -20°C, where they remain stable for approximately one year after shipment . The antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. Contrary to some common practices, aliquoting is generally considered unnecessary for -20°C storage of these antibodies . Some preparations (especially smaller volumes of 20μl) may contain 0.1% BSA as a stabilizer . When working with these antibodies, avoid repeated freeze-thaw cycles and maintain cold chain integrity during experimental procedures to preserve antibody performance.

How can I optimize antigen retrieval protocols for RPL17 IHC applications in different tissue types?

For optimal RPL17 immunohistochemical detection, antigen retrieval represents a critical step that requires tissue-specific optimization. The primary recommended method involves using TE buffer at pH 9.0, with citrate buffer at pH 6.0 as an alternative approach .

When working with vascular tissue or investigating VSMC-related pathologies, a modified protocol has shown success: After deparaffinization and rehydration in Tris-buffered solution (TBS), treat sections with 3% H₂O₂ followed by TBS washing. For antigen retrieval specifically in vascular tissues, heat-induced epitope retrieval (HIER) with 10mM citrate buffer (pH 6.0) has demonstrated effective results . This should be followed by blocking with 5% normal horse serum for 30 minutes before applying the primary RPL17 antibody (recommended dilution 1:1000 in appropriate antibody diluent) .

For detection systems, biotin-based amplification methods have proven effective, such as biotinylated secondary antibodies (horse anti-goat IgG at 1:500 dilution) followed by Vectastain ABC kit reagents. DAB (3,3'-diaminobenzidine) substrate can then be used for visualization with hematoxylin counterstaining . Digital image capture at 60X magnification permits quantitative analysis.

What is the current understanding of RPL17's non-canonical functions in different disease contexts?

Research has revealed intriguing dual roles for RPL17 that appear context-dependent:

In vascular tissue: RPL17 functions as a growth inhibitor of vascular smooth muscle cells, similar to a tumor suppressor gene. Experimental reduction of RPL17 via siRNA delivered to C3H/F carotid arteries resulted in an 8-fold increase in proliferating cells, demonstrating its anti-proliferative function in this context . Following partial carotid ligation in SJL mice, RPL17 expression decreased in intima and media layers while proliferating cell numbers increased, suggesting RPL17 downregulation contributes to pathological vascular proliferation .

In colorectal cancer: Conversely, RPL17 exhibits oncogenic properties in colorectal cancer, where it is upregulated and promotes both proliferation and stemness characteristics. Mechanistically, this occurs through modulation of ERK and NEK2/β-catenin signaling pathways . This represents a striking functional divergence from its vascular role and suggests tissue-specific regulatory mechanisms.

These contrasting functions highlight the importance of cellular context in ribosomal protein biology and suggest RPL17 may interact with different molecular partners in various tissues, leading to distinct signaling outcomes and physiological effects.

What are the critical methodological considerations for measuring RPL17 protein expression quantitatively?

Quantitative assessment of RPL17 protein expression requires careful methodological consideration:

For Western blot quantification:

• Antibody selection: Both polyclonal (14121-1-AP) and monoclonal (67223-1-Ig) antibodies have been validated, with the monoclonal offering higher specificity at dilutions between 1:5000-1:50000 .

• Loading controls: Given RPL17's ribosomal association, traditional housekeeping genes may be inappropriate; non-ribosomal housekeeping proteins like GAPDH or β-actin are preferred.

• Molecular weight verification: RPL17 should be detected between 20-23 kDa, with precise band position potentially varying by tissue type .

For immunohistochemical quantification:

• Standardized image analysis: Digital image analysis using software such as ImagePro Plus with consistent pixel threshold settings for both total nuclei and RPL17-positive cells permits reliable quantification .

• Cell-specific analysis: When examining heterogeneous tissues, clearly define medial and intimal areas for separate analysis of RPL17 expression .

• Controls: Include both negative controls (normal IgG at equivalent concentration) and positive controls (tissues with confirmed expression) .

Comparative expression analysis between experimental groups requires consistency in all methodology, including sample preparation, antibody lots, and imaging parameters. In published studies, RPL17 expression has been documented as >6-fold higher at the mRNA level and approximately 2.5-fold higher at the protein level in C3H/F compared to SJL mouse aortic smooth muscle cells, providing a reference for expected fold-changes in vascular models .

How should I troubleshoot inconsistent RPL17 antibody performance across different applications?

When encountering variability in RPL17 antibody performance, a systematic troubleshooting approach is essential:

For Western blot inconsistencies:

• Verify protein extraction protocol: Different lysis buffers may affect epitope accessibility. For RPL17, RIPA buffer with protease inhibitors has proven effective across multiple cell types .

• Adjust antibody concentration: If signal is weak, try a more concentrated dilution (1:2000 for polyclonal, 1:5000 for monoclonal) .

• Extended incubation: For weak signals, overnight primary antibody incubation at 4°C may improve detection.

• Consider transfer efficiency: RPL17's relatively small size (21-23 kDa) may require adjusted transfer parameters to prevent protein loss through membranes.

For immunohistochemistry/immunofluorescence issues:

• Optimize antigen retrieval: Compare TE buffer (pH 9.0) with citrate buffer (pH 6.0) to determine optimal conditions for your specific tissue type .

• Extend blocking steps: Increase blocking time to 60 minutes if background is high.

• Validate with multiple detection methods: If IHC results are inconclusive, confirm with IF or Western blot.

• Antibody validation: Consider testing both monoclonal and polyclonal antibodies as they may perform differently depending on sample preparation and tissue type .

For immunoprecipitation challenges:

• Increase antibody amount: For difficult targets, adjust to the upper recommended range (4.0 μg for 3.0 mg of total protein) .

• Pre-clear lysates: Additional pre-clearing steps may reduce non-specific binding.

• Adjust bead volume and incubation time: Optimize protein A/G bead amount and extend binding time for improved capture.

How can RPL17 antibodies be used to investigate its role in cancer progression models?

RPL17 antibodies can be strategically deployed to investigate its emerging role in cancer progression through several approaches:

• Expression correlation studies: Using validated RPL17 antibodies (14121-1-AP or 67223-1-Ig) for immunohistochemical analysis of cancer tissue microarrays can establish correlations between RPL17 expression levels and clinical parameters including tumor stage, grade, and patient outcomes . This approach has been successfully applied in pancreatic cancer tissues .

• Mechanistic pathway investigations: Research has identified RPL17's involvement in ERK and NEK2/β-catenin signaling pathways in colorectal cancer . Co-immunoprecipitation experiments using RPL17 antibodies can help identify binding partners within these pathways. The recommended protocol involves:

  • Lysate preparation: 1.0-3.0 mg total protein

  • Antibody amount: 0.5-4.0 μg

  • Immunoprecipitation followed by Western blot for suspected interaction partners

• Functional studies in cellular models: After modulating RPL17 levels through knockdown or overexpression, RPL17 antibodies can be used to:

  • Confirm modification efficiency via Western blot

  • Examine subcellular localization changes via immunofluorescence

  • Identify altered protein-protein interactions via proximity ligation assays

• In vivo model validation: Following animal model interventions (such as xenografts with RPL17-modulated cells), immunohistochemical staining with RPL17 antibodies can confirm expression changes and correlate with proliferation markers such as PCNA, as demonstrated in vascular models .

Given the context-dependent roles of RPL17 (growth inhibitory in vascular tissue versus pro-proliferative in colorectal cancer), careful experimental design with appropriate controls is essential for accurate interpretation of results across different cancer types .

What are the current research frontiers involving RPL17 antibodies?

Current research frontiers utilizing RPL17 antibodies span several emerging areas:

• The dual functionality paradox: Investigating how RPL17 functions as both a tumor suppressor in vascular tissue and an oncogene in colorectal cancer represents a significant research opportunity . RPL17 antibodies are essential tools for mapping these context-dependent functions across different tissue and disease models.

• Therapeutic targeting potential: Research suggests RPL17 represents a potential therapeutic target for limiting carotid intima-media thickening, with implications for vascular disease treatment . Conversely, in colorectal cancer contexts, RPL17 inhibition strategies may offer therapeutic potential . Antibody-based detection methods are critical for validating these therapeutic approaches.

• Signaling pathway integration: Further delineation of how RPL17 integrates with ERK and NEK2/β-catenin signaling requires sophisticated antibody-based techniques including multiplexed immunofluorescence and proximity ligation assays .

• Extra-ribosomal functions: The expanding understanding of ribosomal proteins' non-canonical roles places RPL17 within a larger conceptual framework of ribosomal proteins as multifunctional signaling molecules. Continued application of RPL17 antibodies in varied experimental contexts will help map these functions completely.