RPL28 Antibody

What is RPL28 and why is it significant in cellular research?

RPL28 (ribosomal protein L28) is an essential component of the 60S ribosomal subunit involved in protein synthesis. It belongs to the L28E family of ribosomal proteins and is primarily localized in the cytoplasm . Its significance stems from its critical role in cellular processes including protein synthesis, cell growth, and proliferation. Recent research has highlighted RPL28's potential role in cancer biology, particularly in colorectal cancers where variable expression compared to adjacent normal tissues has been observed . Additionally, RPL28 has been implicated in sorafenib resistance in hepatocellular carcinoma (HCC), making it a protein of interest in drug resistance studies .

Most commercially available RPL28 antibodies demonstrate cross-reactivity with multiple species:

This cross-reactivity is advantageous for comparative studies across different model organisms .

How should I optimize RPL28 antibody conditions for Western blot analysis?

For optimal Western blot results with RPL28 antibodies:

• Sample Preparation: Use standard SDS-PAGE with 12-15% gels as RPL28 has a relatively low molecular weight (approximately 16 kDa) .

• Antibody Dilution: Start with a 1:1000 dilution for polyclonal antibodies and adjust based on signal intensity. For example, Proteintech's 16649-1-AP has been successfully used at 1:1000 dilution with incubation at room temperature for 1.5 hours .

• Blocking Conditions: Use 5% non-fat milk in TBST for 1 hour at room temperature to minimize background .

• Positive Controls: Include HeLa or NIH/3T3 cell lysates as positive controls, which have been validated for RPL28 detection .

• Expected Band Size: Look for a band at approximately 16 kDa, which corresponds to the observed molecular weight of RPL28 .

• Sample Loading: Load 20-30 μg of total protein per lane for optimal detection of endogenous RPL28 .

What are the key considerations for immunofluorescence experiments using RPL28 antibodies?

For successful immunofluorescence/immunocytochemistry with RPL28 antibodies:

• Fixation Method: Use 4% paraformaldehyde for 10-15 minutes at room temperature, which preserves the cytoplasmic structure where RPL28 is localized .

• Permeabilization: Treat with 0.1-0.3% Triton X-100 for 5-10 minutes to allow antibody access to cytoplasmic proteins .

• Antibody Dilution: Begin with a 1:100 dilution for polyclonal antibodies and optimize as needed. The recommended range for most RPL28 antibodies is 1:50-1:200 .

• Subcellular Localization: Expect cytoplasmic staining pattern with potential enrichment in cytoplasmic ribonucleoprotein granules and cytosol .

• Co-staining Markers: Consider co-staining with markers for ribosomes or translational machinery to confirm specificity and localization .

• Controls: Include both negative controls (secondary antibody only) and positive controls (cells known to express RPL28 such as HT-29, 293T, or DU145) .

How can RPL28 antibodies be utilized in cancer research, particularly regarding sorafenib resistance?

RPL28 has emerged as a key gene in sorafenib resistance in hepatocellular carcinoma (HCC), offering several research applications:

• Resistance Biomarker Detection: RPL28 antibodies can be used to assess protein expression levels in patient samples to potentially predict sorafenib resistance. Studies have shown that RPL28 expression is significantly increased in sorafenib-resistant HCC cells .

• Mechanistic Studies: Researchers can employ RPL28 antibodies in combination with knockdown studies to investigate the mechanisms underlying RPL28's role in drug resistance. After knocking down RPL28 in HepG2 sorafenib-resistant cells, studies showed significant inhibition of cell proliferation and increased apoptosis, suggesting RPL28 as a key contributor to resistance .

• Therapeutic Target Validation: Western blotting with RPL28 antibodies can confirm successful knockdown of RPL28 in experimental models, which is crucial when evaluating RPL28 as a potential therapeutic target for drug-resistant HCC .

• Cell Cycle Analysis: Following RPL28 knockdown, researchers observed increased cell numbers in S phase and decreased cells in G1 phase, indicating a strong association between RPL28 function and cell cycle regulation in sorafenib-resistant cells .

• In vivo Confirmation: Both RNA and protein expression of RPL28 were found to be increased in sorafenib-resistant specimens from Morris Hepatoma rats, which can be detected using appropriate RPL28 antibodies .

What is the significance of RPL28 genetic variants in cancer prognosis and how can antibodies complement genetic studies?

Research has revealed important connections between RPL28 genetic variants and cancer outcomes:

How can matched antibody pairs for RPL28 be utilized in multiplex assays?

Matched antibody pairs, such as Proteintech's 60607-1-PBS (capture) and 60607-2-PBS/60607-3-PBS (detection), enable sophisticated multiplex assays for RPL28 research:

• Cytometric Bead Arrays: These validated matched pairs can be used in cytometric bead arrays to simultaneously detect multiple proteins, including RPL28, in the same sample .

• Sandwich ELISA Development: Matched pairs provide higher specificity and sensitivity for quantitative detection of RPL28 in complex biological samples .

• Multiplex Imaging Applications: Conjugation-ready formats make these antibodies suitable for multiplex imaging, where multiple targets can be visualized simultaneously .

• Mass Cytometry: The conjugation-ready format of antibodies like 60607-1-PBS (in PBS buffer without BSA and azide) makes them ideal for mass cytometry applications, where metal isotope labeling is required .

• Optimization Requirements: While these matched pairs are validated, researchers should optimize antibody concentrations for their specific assay conditions and sample types .

How can I address common issues with RPL28 antibody specificity and validation?

Ensuring antibody specificity is crucial for reliable RPL28 research:

• Multiple Validation Methods: Confirm antibody specificity using multiple techniques (e.g., Western blot, immunofluorescence, and knockdown validation) as demonstrated in published studies .

• Positive Controls: Include validated positive controls such as:

  • HeLa or NIH/3T3 cells for Western blotting

  • HT-29, 293T, or DU145 cells for immunocytochemistry

  • Mouse kidney, mouse liver, or rat spleen tissue for tissue-based applications

• Knockdown Validation: Consider using shRNA or siRNA against RPL28 as a negative control to confirm antibody specificity. Studies have shown efficient knockdown of RPL28 using specific target sequences (e.g., RPL28-1) .

• Cross-Reactivity Testing: When working with less common species, conduct preliminary validation experiments to confirm cross-reactivity, even if the antibody is reported to react with your species of interest .

• Isoform Awareness: Be mindful that alternative splicing results in multiple transcript variants of RPL28, which might affect antibody recognition depending on the immunogen sequence used .

How should I interpret RPL28 expression data in the context of cancer research?

Interpreting RPL28 expression data requires careful consideration of several factors:

• Baseline Expression Variation: RPL28 expression varies significantly among different tissues and cell types. Compare expression levels to appropriate controls relevant to your specific research question .

• Cancer vs. Normal Tissue: RPL28 expression is significantly higher (up to 124%) in colorectal tumors compared to paired normal tissues, suggesting potential oncogenic functions .

• Prognostic Significance: High RPL28 expression correlates with poorer outcomes in metastatic cases, warranting careful interpretation of expression levels in patient samples .

• Pathway Analysis: Gene expression studies have identified that high RPL28 expression is associated with:

  • Upregulation of immunoglobulin-related pathways

  • Downregulation of extracellular matrix (ECM) and collagen-related pathways
    These associations should be considered when interpreting RPL28 expression data .

• Genetic Variant Impact: Consider the potential influence of genetic variants (such as rs4806668G>T) on RPL28 expression when interpreting expression data across different patient populations .

What are the best storage and handling practices to maintain RPL28 antibody performance over time?

To ensure optimal performance of RPL28 antibodies:

• Long-term Storage: Store antibodies at -20°C (for most antibodies) or -80°C (for certain formulations) to maintain activity .

• Working Aliquots: For antibodies frequently used, prepare small working aliquots to avoid repeated freeze-thaw cycles .

• Short-term Storage: For frequent use over short periods (up to one month), store at 4°C to minimize freeze-thaw cycles .

• Buffer Considerations: Note the specific storage buffer for your antibody:

  • Antibodies in glycerol-containing buffers (e.g., PBS with 50% glycerol) are more stable during freeze-thaw cycles

  • Antibodies without preservatives (e.g., azide-free, BSA-free formulations) may require more careful handling

• Reconstitution: Follow manufacturer-specific instructions for reconstituting lyophilized antibodies to maintain optimal activity .

• Expiration Dating: Properly stored antibodies typically maintain activity for at least one year from the date of receipt .

How can RPL28 antibodies contribute to studies on ribosome biogenesis and translational regulation?

RPL28 antibodies offer valuable tools for investigating fundamental aspects of ribosome biology:

• Ribosome Assembly Studies: RPL28 antibodies can help track the incorporation of this protein into the 60S ribosomal subunit during biogenesis, providing insights into assembly mechanisms .

• Stress Response Analysis: Under various cellular stresses, ribosomal proteins may relocalize or undergo modifications. RPL28 antibodies can help monitor these changes through techniques like immunofluorescence or Western blotting .

• Extraribosomal Functions: Beyond their role in ribosomes, ribosomal proteins like RPL28 may have extraribosomal functions. Antibodies can help identify novel protein-protein interactions through co-immunoprecipitation experiments .

• Ribosome Heterogeneity: Recent research suggests ribosomes may be heterogeneous in composition. RPL28 antibodies can contribute to studies examining the variable incorporation of this protein into different ribosome populations .

• Cancer-Specific Translation: Given the association between RPL28 and cancer outcomes, antibodies can help investigate whether RPL28-containing ribosomes preferentially translate specific mRNAs in cancer cells .

What role does RPL28 play in cellular stress response and how can antibodies help elucidate this function?

Understanding RPL28's role in stress response requires sophisticated approaches:

• Stress Granule Association: RPL28 has been localized to cytoplasmic ribonucleoprotein granules, which may include stress granules. RPL28 antibodies can be used for co-localization studies with stress granule markers under various stress conditions .

• Quantitative Analysis: Western blotting with RPL28 antibodies allows quantitative assessment of protein levels in response to different stressors, including drug treatments like sorafenib .

• Pathway Integration: Studies have shown associations between RPL28 and various cellular pathways. Antibodies can help confirm these connections through co-immunoprecipitation and immunofluorescence co-localization experiments .

• Translational Control: Under stress, cells often reprogram translation. RPL28 antibodies can be used in polysome profiling experiments to examine whether RPL28-containing ribosomes are differentially engaged in translation during stress .

• Post-translational Modifications: Cellular stress often triggers post-translational modifications of proteins. RPL28 antibodies, particularly phospho-specific ones if available, could help identify stress-induced modifications .

How can researchers leverage RPL28 antibodies in exosome and microvesicle research?

Emerging research has identified ribosomal proteins as components of extracellular vesicles:

• Cargo Identification: RPL28 antibodies can be used to detect and quantify this protein in isolated exosomes and microvesicles through Western blotting techniques .

• Differential Analysis: Publications have described ribosomal proteins, including RPL28, as "passengers" in microvesicles derived from myeloma cells and mesenchymal stem cells, suggesting a role in intercellular communication. Antibodies allow comparative analysis of RPL28 content across different vesicle populations .

• Functional Studies: After identifying RPL28 in extracellular vesicles, researchers can use antibodies to deplete or block the protein to assess its functional significance in recipient cells .

• Biomarker Development: Given RPL28's association with cancer, its presence in circulating vesicles might serve as a biomarker. Antibodies enable the development of detection methods for such applications .

• Vesicle Biogenesis: Immunofluorescence studies with RPL28 antibodies can help track the protein's journey from ribosomes to extracellular vesicles, providing insights into vesicle formation mechanisms .