RPL23A Antibody, Biotin conjugated

What is RPL23A and what cellular functions does it perform?

RPL23A (60S ribosomal protein L23a) is a critical component of the large ribosomal subunit. It functions as an integral part of the large ribonucleoprotein complex responsible for protein synthesis in the cell . Research has demonstrated that RPL23A specifically binds to a defined region on the 26S rRNA, contributing to ribosomal structural integrity and function . Additionally, RPL23A may play an extraribosomal role in promoting p53/TP53 degradation, possibly through the stimulation of MDM2-mediated TP53 polyubiquitination . This suggests that beyond its canonical role in translation, RPL23A participates in cellular regulatory pathways affecting cell cycle and apoptosis.

How do biotin-conjugated RPL23A antibodies differ from unconjugated versions?

Biotin-conjugated RPL23A antibodies contain covalently linked biotin molecules that enable strong binding to streptavidin and avidin proteins. This conjugation provides several methodological advantages over unconjugated antibodies:

• Enhanced detection sensitivity through signal amplification via the biotin-streptavidin system

• Increased flexibility in experimental design through multi-step detection protocols

• Compatibility with a wider range of detection systems, including flow cytometry, ELISA, and immunohistochemistry

The biotin-streptavidin interaction has one of the strongest non-covalent binding affinities in nature (Kd ≈ 10^-15 M), which contributes to improved detection sensitivity when compared to conventional antibody detection systems.

What sample types have been validated for RPL23A antibody applications?

Based on validated testing data, RPL23A antibodies have demonstrated consistent reactivity across multiple sample types:

When working with new sample types, researchers should perform optimization steps to establish appropriate antibody concentrations and assay conditions.

What are the optimal conditions for Western blot applications using biotin-conjugated RPL23A antibodies?

For Western blot applications using biotin-conjugated RPL23A antibodies, the following protocol parameters should be optimized:

• Sample Preparation:

  • Lyse cells in RIPA buffer containing protease inhibitors

  • Load 15-50 μg of total protein per lane (validated across HeLa, 293T, and Jurkat cell lysates)

• Antibody Dilution Range:

  • Primary antibody: 1:500-1:2000 dilution (optimization recommended for each specific lot)

  • Streptavidin-HRP conjugate: 1:5000-1:10000 dilution

• Protocol Modifications:

  • Blocking: 5% non-fat milk or 3% BSA in TBST for 1 hour at room temperature

  • Primary antibody incubation: Overnight at 4°C

  • Washing: 3-5 times with TBST, 5 minutes each

  • Expected band size: 15 kDa

The observed molecular weight of 15 kDa matches the calculated molecular weight based on amino acid sequence, suggesting minimal post-translational modifications affect migration patterns on SDS-PAGE.

How can researchers troubleshoot non-specific binding when using biotin-conjugated RPL23A antibodies?

Non-specific binding is a common challenge when working with biotin-conjugated antibodies. Implement these methodological solutions to improve specificity:

• Endogenous Biotin Blockade:

  • Pre-block samples with avidin followed by free biotin (avidin-biotin blocking kit)

  • This is especially critical for biotin-rich tissues like liver, kidney, and brain

• Optimized Blocking Protocol:

  • Use casein-based blockers instead of BSA (which may contain endogenous biotin)

  • Incorporate 0.1% Tween-20 in blocking buffers to reduce hydrophobic interactions

• Streptavidin-HRP Dilution Series:

  • Test multiple dilutions (1:5000, 1:10000, 1:20000)

  • Select the concentration that provides optimal signal-to-noise ratio

• Sample Pre-absorption:

  • Pre-incubate diluted antibody with non-target tissue/cell lysate

  • Centrifuge and use the supernatant for the primary detection

Cross-reactivity assessments have demonstrated minimal non-specific binding with other ribosomal proteins when using well-validated RPL23A antibodies, ensuring experimental reliability and reproducibility.

What controls should be included when conducting immunoprecipitation experiments with RPL23A antibodies?

Robust immunoprecipitation experiments with RPL23A antibodies require these methodologically essential controls:

• Input Control:

  • 5-10% of the pre-IP lysate should be run alongside IP samples

  • Confirms target protein presence in starting material

• Isotype Control:

  • Use matched isotype antibody (e.g., rabbit IgG) at equivalent concentration

  • Identifies non-specific protein binding to antibody constant regions or beads

• Technical Controls:

  • Beads-only control (no antibody) to assess direct protein binding to matrix

  • Recommended antibody amount: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Validation Approach:

  • Reciprocal IP using antibodies against known interacting partners

  • Verification using multiple antibody clones targeting different epitopes

Successful IP experiments with RPL23A antibodies have been validated in mouse brain tissue, confirming the applicability of these protocols in complex biological samples .

How can RPL23A antibodies be utilized to investigate ribosome biogenesis defects?

RPL23A antibodies provide valuable tools for examining ribosome biogenesis pathways through these methodological approaches:

• Nucleolar Stress Assessment:

  • Immunofluorescence co-localization with nucleolar markers (fibrillarin, nucleolin)

  • Quantification of nucleolar-nucleoplasmic distribution under stress conditions

• Pre-ribosomal Particle Analysis:

  • Sucrose gradient fractionation followed by Western blotting

  • Identification of assembly intermediates using RPL23A as a marker

• Ribosomal Subunit Integration:

  • Pulse-chase experiments with biotin-conjugated antibodies

  • Monitoring temporal incorporation into mature ribosomes

• Biogenesis Defect Characterization:

  • Compare RPL23A levels in nuclear, nucleolar, and cytoplasmic fractions

  • Employ SILAC-based quantitative proteomics with RPL23A immunoprecipitation

This multi-parameter approach enables comprehensive analysis of how ribosomal stress conditions affect various stages of ribosome assembly and maturation.

What is the relationship between RPL23A and the p53 pathway, and how can researchers study this interaction?

The relationship between RPL23A and p53 represents an important extraribosomal function that can be investigated through these methodological strategies:

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation using RPL23A antibodies to pull down MDM2 and p53

  • Proximity ligation assays to visualize in situ interactions

• Functional Analysis:

  • siRNA-mediated RPL23A knockdown followed by p53 stability assessment

  • Evidence suggests RPL23A may promote p53/TP53 degradation through MDM2-mediated polyubiquitination

• Stress Response Dynamics:

  • Monitor RPL23A-p53-MDM2 complex formation under ribosomal stress

  • Track nucleolar-nucleoplasmic translocation following actinomycin D treatment

• Ubiquitination Assays:

  • In vitro ubiquitination assays with recombinant RPL23A, MDM2, and p53

  • Immunoprecipitation with anti-ubiquitin antibodies followed by RPL23A detection

These approaches help elucidate the molecular mechanisms by which RPL23A contributes to p53 regulation, potentially linking ribosomal stress to cell cycle control pathways.

How should researchers interpret differential detection results between RPL23A and RPL23 antibodies?

Distinguishing between RPL23A and RPL23 detection requires careful analysis due to their similar nomenclature but distinct biological identities:

• Protein Characteristics Comparison:

  Feature	RPL23A	RPL23
  UniProt ID	P62750	P62829
  Molecular Weight	17.7 kDa	15 kDa
  Amino Acid Length	156 aa	140 aa
  Common Alternative Names	60S ribosomal protein L23a	60S ribosomal protein L23

• Epitope Mapping Considerations:

  • RPL23A antibodies typically target the C-terminal region (aa 100 to C-terminus)

  • RPL23 antibodies may recognize different epitopes, requiring verification

• Expression Pattern Analysis:

  • Both proteins are ubiquitously expressed but may show tissue-specific regulation

  • Quantitative PCR validation recommended alongside protein detection

• Functional Redundancy Assessment:

  • Despite structural similarities, these proteins may have distinct extraribosomal functions

  • Knockout/knockdown experiments should target genes specifically

When conducting comparative studies, researchers should employ antibodies with validated specificity to avoid cross-reactivity issues that could confound experimental interpretation.

What is the recommended protocol for detecting RPL23A in ELISA applications?

The optimized sandwich ELISA protocol for RPL23A detection includes these methodological steps:

• Assay Preparation:

  • Coat microplate wells with capture antibody (monoclonal anti-RPL23A)

  • Block with 1% BSA in PBS for 1 hour at room temperature

• Sample Processing:

  • Prepare standards ranging from 0.16-10 ng/ml

  • Dilute serum/plasma samples appropriately (validated for EDTA/citrated plasma)

• Detection Procedure:

  • Add samples and incubate

  • Add biotin-labeled antibody followed by Streptavidin-HRP conjugate

  • Wash thoroughly between steps

  • Add TMB substrate and stop solution

• Performance Metrics:

  • Detection sensitivity: 0.059 ng/ml

  • Linear range: 0.16-10 ng/ml

  • Sample linearity validation:

  Sample Dilution	Serum Recovery	EDTA Plasma Recovery
  1:2	89-97%	85-94%
  1:4	93-102%	92-101%
  1:8	81-96%	87-96%
  1:16	93-106%	82-90%

This standardized protocol ensures high reproducibility and accuracy in RPL23A quantification across various biological samples .

What are the optimal conditions for immunohistochemistry applications with RPL23A antibodies?

For successful immunohistochemistry (IHC) applications using RPL23A antibodies, implement these methodological parameters:

• Tissue Preparation:

  • FFPE sections: 4-6 μm thickness

  • Antigen retrieval: TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0 as alternative

• Staining Protocol:

  • Recommended dilution: 1:50-1:500 (antibody-dependent)

  • Incubation time: 1 hour at room temperature or overnight at 4°C

  • Detection system: Biotin-streptavidin-HRP or polymer-based detection

• Signal Development:

  • DAB chromogen development: 5-10 minutes (monitor microscopically)

  • Counterstain: Hematoxylin (30 seconds to 1 minute)

• Validation Controls:

  • Positive control: Human brain tissue (validated)

  • Negative controls: Primary antibody omission and isotype controls

  • Expected localization: Primarily cytoplasmic with nucleolar enrichment

Optimization of antibody concentration should be performed for each new tissue type, particularly when investigating RPL23A expression in pathological specimens with altered protein expression patterns.

How can researchers improve reproducibility when quantifying RPL23A expression levels?

Ensuring reproducible quantification of RPL23A expression requires implementing these methodological strategies:

• Standardized Reference Materials:

  • Include recombinant RPL23A protein standards in each experiment

  • Prepare master aliquots of control cell lysates (e.g., HeLa, Jurkat) to run across experiments

• Normalization Approach:

  • For Western blots: Normalize to multiple housekeeping proteins (β-actin, GAPDH, tubulin)

  • For qPCR: Use geometric mean of multiple reference genes

• Technical Replication:

  • Perform triplicate technical replicates

  • Calculate coefficient of variation (<15% acceptable)

• Image Analysis Standardization:

  • Use identical acquisition parameters across samples

  • Implement automated quantification software with defined intensity thresholds

  • Apply background subtraction algorithms consistently

Following these guidelines helps minimize both technical and biological variability, enabling more reliable detection of true experimental effects when studying RPL23A expression changes.

How can biotin-conjugated RPL23A antibodies be applied in ribosome profiling experiments?

Biotin-conjugated RPL23A antibodies can enhance ribosome profiling through these methodological adaptations:

• Translating Ribosome Affinity Purification (TRAP):

  • Use biotin-conjugated RPL23A antibodies for selective isolation of ribosomes

  • Combine with streptavidin magnetic beads for rapid purification

  • Extract and sequence associated mRNAs to identify actively translated transcripts

• Spatial Ribosome Mapping:

  • Employ proximity ligation assays with biotin-conjugated RPL23A antibodies

  • Map subcellular localization of translation complexes

  • Identify spatially restricted translation events

• Specialized Ribosome Identification:

  • Assess co-localization with other ribosomal proteins to identify heterogeneous ribosomes

  • Investigate potential specialized functions of RPL23A-containing ribosomes

• Integration with CRISPR Screens:

  • Use biotin-conjugated RPL23A antibodies to isolate ribosomes from CRISPR-modified cells

  • Identify genetic dependencies affecting RPL23A incorporation

These applications leverage the high specificity and affinity of the biotin-streptavidin system to enable precise isolation of RPL23A-containing complexes for downstream analysis.

What are the considerations for using RPL23A antibodies in cancer research applications?

When applying RPL23A antibodies in cancer research, researchers should consider these methodological aspects:

• Expression Level Analysis:

  • Compare RPL23A levels across tumor grades and matched normal tissues

  • Correlate with patient survival data and clinical parameters

  • Western blot has been validated across multiple cancer cell lines including HeLa, 293T, PC-3, and Jurkat

• p53 Pathway Interactions:

  • Investigate RPL23A's role in p53 regulation in p53-wild-type versus p53-mutant cancers

  • Examine MDM2-RPL23A interaction as a potential therapeutic target

  • Assess how RPL23A contributes to p53 degradation through MDM2-mediated polyubiquitination

• Ribosomal Stress Response:

  • Evaluate RPL23A as a biomarker for ribotoxic stress in cancer cells

  • Study nucleolar stress responses mediated by RPL23A relocalization

  • Investigate sensitivity to ribosome-targeting therapeutics

• Therapeutic Resistance Mechanisms:

  • Examine RPL23A's potential role in drug resistance pathways

  • Investigate its contribution to stress adaptation in cancer cells

These methodological approaches help characterize the complex roles of RPL23A in cancer biology, potentially identifying novel therapeutic vulnerabilities.