RPL23A Antibody, HRP conjugated

What is RPL23A and what are its biological functions?

RPL23A (Ribosomal Protein L23a) is a component of the large 60S ribosomal subunit. This 18 kDa protein plays a critical role in protein synthesis as part of the ribosome, which is the large ribonucleoprotein complex responsible for cellular protein production. RPL23A specifically binds to a defined region on the 26S rRNA, forming part of the structural framework of the ribosome .

Beyond its canonical role in protein synthesis, RPL23A has been identified as having extraribosomal functions. Notably, it may promote p53/TP53 degradation through stimulation of MDM2-mediated TP53 polyubiquitination, suggesting involvement in cellular stress responses and potentially cancer progression .

The amino acid sequence of RPL23A is 100% conserved between mice and humans, indicating its evolutionary importance . RPL23A mRNA is ubiquitously expressed across a wide range of healthy human tissues, reflecting its fundamental role in cellular processes .

RPL23A Antibody Applications and Methodologies

Optimal dilutions for RPL23A antibody applications:

For HRP-conjugated antibodies specifically, it's recommended to:

• Perform initial titration experiments to determine optimal concentration

• Include appropriate blocking reagents to minimize background signal

• Use enhanced chemiluminescence (ECL) substrate for Western blot detection

• Include controls to verify specificity

For Western blotting, RPL23A antibodies have been successfully tested with:

• HeLa cells

• HepG2 cells

• Human liver tissue

• Human heart tissue

• Mouse liver tissue

• Rat liver tissue

How do I optimize ELISA protocols using RPL23A antibodies?

For ELISA-based detection of RPL23A:

• Assay range: The typical assay range is 0.16-10 ng/ml

• Sensitivity: Detection limit approximately 0.059 ng/ml

• Sample types: Validated for human serum, plasma, and cell culture supernatants (especially validated for citrated/EDTA plasma)

Procedural recommendations:

• Ensure proper washing of plates to remove excess un-reacted reagents

• Be aware of potential High Dose Hook Effect in samples with very high analyte concentrations

• Avoid samples containing sodium azide (NaN3) as it can destroy HRP activity

• Maintain consistent timing between wells

• Run samples in duplicate or triplicate for reliability

How can I detect T-cell responses to RPL23A in autoimmunity research?

RPL23A has been identified as a potentially important self-antigen in autoimmune diseases, particularly rheumatoid arthritis. For researchers investigating T-cell responses:

• Antibody selection: Use anti-RPL23A antibodies for co-staining with T-cell markers

• Tissue analysis: Serial sections can be stained with anti-RPL23A and other markers (e.g., anti-CD55) to identify co-expressing cells

• Serum analysis: Patient serum can be tested for RPL23A autoantibodies as a clinical marker

• T-cell assays: RPL23A protein can be used to stimulate T cells and measure cytokine production

Research has shown that recombinant RPL23A protein can induce dose-dependent interleukin-2 production by specific TCR-expressing T cell hybridomas and stimulate inflammatory cytokine production by CD4+ T cells from the draining lymph nodes of arthritic joints .

What is the role of RPL23A in ubiquitination pathways and cancer progression?

Recent research has uncovered that:

• HERC3 (a ubiquitin ligase) directly targets RPL23A for ubiquitination-dependent degradation

• The HERC3-RPL23A axis plays a significant role in colorectal cancer (CRC) proliferation and cell cycle regulation

• RPL23A can independently regulate cell cycle and cell proliferation in CRC

• HERC3 modulates the ubiquitination of p21 and regulates the expression of c-Myc and p21

• RPL23A can rescue the effects of HERC3 on these pathways

Methodology for studying ubiquitination:

• Western blot analysis following treatment with proteasome inhibitors

• In vivo ubiquitylation assays

• Cycloheximide analysis for protein stability

• Mass spectrometry analysis

• Gene Set Enrichment Analysis (GSEA)

How do I troubleshoot non-specific binding issues with RPL23A antibodies?

When experiencing high background or non-specific binding:

• Antibody validation: Confirm antibody specificity via knockout/knockdown controls

• Blocking optimization: Test different blocking reagents (BSA, non-fat milk, commercial blockers)

• Dilution series: Perform careful titration of primary and secondary antibodies

• Cross-reactivity testing: Test against recombinant protein controls

• Buffer optimization: Adjust salt concentration and detergent levels in wash buffers

For HRP-conjugated antibodies specifically, potential issues include:

• Enzymatic activity loss during storage (maintain at -20°C with appropriate stabilizers)

• Interference from endogenous peroxidases (use appropriate quenching steps)

• Cross-reactivity with endogenous biotin (if using biotin-streptavidin systems)

What recent discoveries link RPL23A to disease mechanisms beyond its ribosomal function?

Several emerging research areas have identified RPL23A's roles beyond protein synthesis:

• Autoimmunity: RPL23A has been identified as a self-antigen in rheumatoid arthritis, with a significantly greater proportion of patients having IgG antibodies specific for RPL23A compared to healthy controls

• Cancer biology: RPL23A is involved in:

  • Regulation of cell proliferation and cell cycle progression

  • Interactions with the p53 pathway through MDM2-mediated mechanisms

  • Modulation of c-Myc expression in colorectal cancer

• Ubiquitination pathways: RPL23A is targeted for degradation by HERC3 via the ubiquitination proteasome-dependent pathway, suggesting a role in protein quality control mechanisms

How can I develop a multiplex detection system for ribosomal proteins including RPL23A?

For developing multiplex detection systems:

• Antibody selection: Choose antibodies with different host species or isotypes to prevent cross-reactivity

• Conjugation strategy: Select different reporter molecules (fluorophores, enzymes) for each target

• Validation approach:

  • Test each antibody individually before multiplexing

  • Use specific blocking peptides to confirm specificity

  • Include appropriate controls for each detection channel

For RPL23A specifically, consider:

• Combining with antibodies against other ribosomal components (e.g., RPL proteins)

• Using co-immunoprecipitation to identify interaction partners

• Implementing proximity ligation assays to detect in situ protein interactions

What are the comparative sensitivities of different detection methods for RPL23A?