RPL24 Antibody

What is RPL24 and what cellular functions does it perform?

RPL24 (60S ribosomal protein L24) is a component of the large ribosomal subunit in eukaryotes, belonging to the eukaryotic ribosomal protein eL24 family. It plays a critical role in protein synthesis as part of the ribosomal translation machinery. The protein has a calculated molecular weight of approximately 18 kDa, although it is typically observed at 21-23 kDa in experimental conditions due to post-translational modifications . Beyond its canonical role in translation, emerging evidence suggests that RPL24 may have extraribosomal functions that influence cellular processes including proliferation and oncogenic signaling .

In which experimental applications can RPL24 antibodies be utilized?

RPL24 antibodies have demonstrated effectiveness across multiple laboratory techniques:

The versatility of these antibodies enables comprehensive characterization of RPL24 in various experimental contexts, from protein expression to protein-RNA interactions.

What species reactivity is typically observed with commercial RPL24 antibodies?

Most commercially available RPL24 antibodies demonstrate reactivity with human samples as the primary target. Many antibodies also cross-react with mouse and rat samples due to the high sequence conservation of ribosomal proteins across species . Some antibodies have documented cross-reactivity with additional species including pig, zebrafish, bovine, sheep, rabbit, dog, chicken, and xenopus, though validation studies may vary between manufacturers . When selecting an antibody for multi-species applications, it's advisable to check the manufacturer's validation data for species-specific reactivity.

How should researchers optimize Western blot protocols for RPL24 detection?

Optimizing Western blot conditions for RPL24 detection requires attention to several critical parameters:

• Sample preparation: Extract proteins using RIPA lysis buffer with protease inhibitors

• Protein loading: 50 μg of protein per lane is typically sufficient

• Gel selection: 10% SDS-PAGE gels effectively resolve RPL24

• Transfer: Standard electrophoretic transfer to PVDF membranes

• Blocking: 5% non-fat dry milk in PBST (0.1% Tween 20) for 1 hour at room temperature

• Primary antibody incubation: Apply at manufacturer-recommended dilution (typically 1:500 to 1:50000) at 4°C overnight or room temperature for 1.5 hours

• Detection: Use enhanced chemiluminescence reagents for visualization

• Quantification: Employ ImageJ or similar software, normalizing to housekeeping proteins (β-actin is commonly used)

Researchers should note the difference between calculated (18 kDa) and observed (21-23 kDa) molecular weights to avoid misidentification of the target band .

What approaches are recommended for establishing RPL24 overexpression or knockdown models?

Creating reliable experimental models for studying RPL24 function requires careful consideration of expression systems:

For overexpression models:

• Clone full-length human RPL24 sequences (NM_000986.4) into appropriate expression vectors such as pcDNA3.0

• Transfect target cells using standard reagents like Lipofectamine 2000

• Select positive clones using G418-containing media for transient expression

• Alternatively, use lentiviral vectors (e.g., Pez-Lv105) for stable expression

• Verify expression levels via Western blot using validated RPL24 antibodies

For in vivo validation:

• Inject stable cell lines subcutaneously into immunodeficient mice (e.g., NOD/SCID)

• Monitor tumor formation and growth over time (typically measurable 7-9 days post-injection)

• Calculate tumor volume using the formula: Volume = (length × width²) × 1/2

• Analyze effects on tumor development, growth kinetics, and treatment response

These models are essential for investigating RPL24's functional roles in normal and pathological contexts.

How does RPL24 expression relate to cancer biology and potential therapeutic applications?

Research has revealed significant associations between RPL24 and cancer:

These findings suggest that RPL24 may represent both a biomarker and potential therapeutic target in certain cancer contexts. Targeted inhibition of RPL24 or related pathways might offer novel treatment strategies, particularly for cancers driven by Akt or Myc.

What validation steps should be implemented to ensure RPL24 antibody specificity?

Comprehensive validation is essential for generating reliable data with RPL24 antibodies:

• Positive controls: Include lysates from cells known to express RPL24 (A549, HEK-293, Jurkat cells) in experimental workflows

• Negative controls:

  • Primary antibody omission controls

  • Ideally, include RPL24 knockdown/knockout samples to confirm specificity

• Cross-validation approaches:

  • Compare results across multiple antibody clones or from different manufacturers

  • Test across multiple applications (WB, IHC, IF) to ensure consistent patterns

  • Verify appropriate molecular weight (observed at 21-23 kDa despite calculated 18 kDa)

• Application-specific validation:

  • For IHC: Include tissue sections known to express RPL24 (placenta, kidney, liver)

  • For IF: Include appropriate subcellular localization controls

These validation steps help minimize artifacts and ensure that observed signals genuinely reflect RPL24 expression or localization.

What are the critical factors in optimizing immunohistochemistry protocols for RPL24?

Successful IHC staining for RPL24 requires careful optimization:

• Fixation and processing: Standard formalin fixation and paraffin embedding protocols are typically suitable

• Antigen retrieval: Most protocols recommend TE buffer pH 9.0, though citrate buffer pH 6.0 may also be effective

• Blocking: Employ appropriate blocking reagents to minimize non-specific binding

• Antibody concentration: Titrate between 1:50-1:500 dilutions to determine optimal signal-to-noise ratio for your specific tissue type

• Detection system: Standard avidin-biotin or polymer-based detection systems are suitable

• Counterstaining: Hematoxylin provides effective nuclear contrast

• Controls: Include known positive tissues such as human placenta, kidney, liver, spleen, or ovary tissue

• Evaluation: Assess both staining intensity and percentage of positive cells for comprehensive analysis

How can researchers address discrepancies between expected and observed molecular weight of RPL24?

The observed molecular weight of RPL24 in Western blot (21-23 kDa) frequently differs from its calculated mass (18 kDa) , which can create confusion in data interpretation. Researchers should:

• Understand potential causes:

  • Post-translational modifications (phosphorylation, ubiquitination)

  • Altered gel migration due to protein structure or composition

  • Incomplete denaturation affecting migration

• Implementation strategies:

  • Run positive control samples with confirmed RPL24 expression

  • Consider using reducing agents like DTT to ensure complete denaturation

  • Include molecular weight markers spanning 15-25 kDa range

  • Document laboratory-specific migration patterns for reference

• Validation approaches:

  • Perform additional experiments using antibodies targeting different epitopes

  • If necessary, confirm identity through mass spectrometry

  • Consider running 2D gels to better characterize RPL24 isoforms

How might RPL24 function as a biomarker in clinical oncology?

Recent research indicates promising applications for RPL24 as a cancer biomarker:

• Differential expression: Two-thirds of breast cancers show upregulated RPL24 expression compared to matched normal tissue, suggesting diagnostic potential

• Predictive value: RPL24 has been identified as a potential biomarker for predicting cervical cancer patient prognosis and assessing concurrent chemoradiotherapy (CCRT) efficacy

• Mechanistic relevance: RPL24 haploinsufficiency protects against Akt or Myc-driven cancers, suggesting biological significance beyond correlation

Implementation considerations for biomarker development include:

• Standardization of detection methods across clinical laboratories

• Establishment of reference ranges in healthy and diseased states

• Prospective validation in diverse patient cohorts

• Integration with existing biomarker panels for improved predictive power

What experimental approaches are recommended for investigating RPL24's role in chemotherapy response?

To explore the relationship between RPL24 and treatment response:

• In vitro approaches:

  • Compare RPL24 expression in sensitive versus resistant cell lines

  • Assess expression changes before and after drug treatment (e.g., cisplatin)

  • Manipulate RPL24 expression (overexpression/knockdown) and measure impact on drug sensitivity

  • Investigate interactions with known resistance pathways

• In vivo strategies:

  • Develop xenograft models with modulated RPL24 expression

  • Evaluate tumor growth kinetics and response to treatment regimens

  • Analyze tumor samples for RPL24 expression correlation with treatment response

• Analytical considerations:

  • Combine protein (Western blot, IHC) and transcript (qPCR, RNA-seq) analyses

  • Evaluate temporal dynamics of expression changes during treatment

  • Consider correlation with established markers of treatment response

These approaches can help elucidate whether RPL24 is merely a marker or a functional mediator of treatment response in cancer.

What is the significance of RPL24 in translational regulation beyond its structural role?

Emerging evidence suggests RPL24 may influence translation in ways extending beyond structural support:

• Selective mRNA translation: RPL24 may differentially affect translation of specific mRNA subsets, potentially influencing cellular phenotypes

• Signaling pathway crosstalk: The relationship between RPL24 and oncogenic pathways (Akt, Myc) suggests it may integrate signaling and translational regulation

• Extraribosomal functions: Like many ribosomal proteins, RPL24 may perform roles independent of the ribosome

• Stress response: RPL24 might participate in translational reprogramming during cellular stress

Experimental approaches to investigate these functions include ribosome profiling, RNA immunoprecipitation, and proteomics analysis of RPL24-associated complexes. Understanding these non-canonical roles may reveal new therapeutic opportunities in diseases characterized by dysregulated translation.

What are the best practices for quantifying RPL24 expression in experimental samples?

Accurate quantification requires appropriate methodological approaches:

For all quantification methods, researchers should:

• Include appropriate technical and biological replicates

• Apply statistical tests suitable for the experimental design

• Consider the relationship between transcript and protein levels, which may not always correlate

How should researchers design experiments to investigate RPL24's contribution to oncogenic pathways?

Experimental design for exploring RPL24's role in cancer requires:

• Expression manipulation strategies:

  • CRISPR-based knockout or knockdown approaches

  • Overexpression of wild-type or mutant forms

  • Inducible expression systems for temporal control

• Pathway analysis frameworks:

  • Assess effects on Akt and Myc signaling components

  • Determine impact on downstream effectors using phospho-specific antibodies

  • Evaluate consequences for cellular phenotypes (proliferation, survival, migration)

• Translation-specific investigations:

  • Polysome profiling to assess global translation effects

  • Ribosome profiling to identify differentially translated mRNAs

  • Metabolic labeling to measure protein synthesis rates

• Clinical relevance:

  • Correlate experimental findings with patient data

  • Stratify analyses based on cancer subtypes and stages

  • Consider potential therapeutic implications

This multi-faceted approach can help delineate whether RPL24 represents a passenger or driver in cancer development and progression.