RPL23A Antibody

What is RPL23A and what is its primary biological function?

RPL23A is a component of the 60S subunit in the ribosome responsible for protein synthesis. It belongs to the L23P family of ribosomal proteins and is located primarily in the cytoplasm. This protein binds to a specific region on the 26S rRNA and plays a crucial role in the assembly and function of the large ribosomal subunit . Beyond its canonical role in translation, RPL23A may also promote p53/TP53 degradation through the stimulation of MDM2-mediated TP53 polyubiquitination . The RPL23A gene is co-transcribed with several small nucleolar RNA genes (U42A, U42B, U101A, and U101B) located within its introns .

Which experimental applications are most suitable for RPL23A antibodies?

RPL23A antibodies have been successfully validated for multiple applications:

The optimal working dilution varies depending on the specific antibody clone and application, so it's advisable to perform preliminary titration experiments to determine optimal conditions for your specific experimental system .

How should I validate the specificity of an RPL23A antibody for my research application?

Validation of RPL23A antibody specificity requires a multi-faceted approach:

• Knockout/knockdown controls: Generate RPL23A-depleted samples using siRNA or CRISPR-Cas9 systems as negative controls. For example, using stable shRNA expression to target RPL23A in HLE and MHCC97H cells has been successful in previous studies .

• Western blot validation: Confirm a single band at the expected molecular weight (18 kDa) across multiple cell lines or tissues. Published studies have demonstrated successful detection in HeLa, HepG2, Jurkat, 293T cell lysates, and human/mouse/rat liver tissues .

• Peptide competition assay: Pre-incubate the antibody with recombinant RPL23A protein or the immunizing peptide to confirm signal abolishment.

• Cross-reactivity assessment: Test the antibody against closely related ribosomal proteins to ensure specificity, particularly important given the sequence conservation of ribosomal proteins.

• Multiple antibody approach: Compare staining patterns using antibodies raised against different epitopes of RPL23A. For instance, antibodies targeting the N-terminal (aa 1-100) versus C-terminal (aa 100 to C-terminus) regions should show concordant results .

What controls are essential when using RPL23A antibodies in disease-related research?

When investigating RPL23A in disease contexts, particularly cancer and autoimmune conditions, the following controls are critical:

• Matched normal-disease tissue pairs: When studying hepatocellular carcinoma or melanoma, always include paired adjacent non-tumor tissues as controls. Studies have shown significant expression differences between tumor and adjacent normal tissues .

• Multiple cell line validation: Confirm findings across several disease-relevant cell lines. For HCC studies, HLE and MHCC97H cells have been successfully used .

• Isotype controls: Include appropriate isotype control antibodies to distinguish non-specific binding, especially in immunohistochemistry applications.

• Disease-specific negative controls: For autoimmune studies like rheumatoid arthritis research, include serum samples from other autoimmune conditions (e.g., systemic lupus erythematosus, osteoarthritis) as controls to verify specificity of RPL23A antibody responses .

• Recombinant protein controls: Use purified recombinant RPL23A protein as a positive control, particularly in immunoprecipitation experiments .

How is RPL23A involved in cancer progression, particularly hepatocellular carcinoma?

RPL23 (distinct from but related to RPL23A) has been identified as a driver of metastasis in hepatocellular carcinoma (HCC) . Research findings demonstrate:

• Upregulation in HCC tissues: RPL23 is significantly upregulated in human HCC tissues compared to adjacent non-tumoral tissues, with increased expression observed in 87% of HCC samples .

• Association with poor prognosis: Higher RPL23 expression correlates with poor survival outcomes in HCC patients .

• Mechanistic role in metastasis: RPL23 promotes HCC metastasis by stabilizing MMP9 mRNA. Specifically, RPL23 binds to the 3'UTR of MMP9 and enhances its mRNA stability, thereby increasing MMP9 expression, which is a known promoter of cancer cell invasion .

• Effects on cellular phenotype: Knockdown of RPL23 in HCC cell lines (HLE and MHCC97H) significantly decreases cell proliferation, migration, and invasion capacities .

• In vivo validation: RPL23 knockdown suppresses tumor growth and lung metastasis in orthotopic xenograft models .

These findings suggest that targeting the RPL23/MMP9 pathway could represent a potential therapeutic strategy for HCC treatment.

What is the connection between RPL23A and autoimmune diseases like rheumatoid arthritis?

RPL23A has been identified as a target self-antigen in autoimmune arthritis :

• T-cell recognition: Arthritogenic T cells from SKG mice (which spontaneously develop rheumatoid arthritis-like disease) recognize RPL23A. Specifically, a T-cell receptor designated 7-39 TCR showed high reactivity to RPL23A .

• Autoantibody production: Both mice with arthritis and human rheumatoid arthritis patients demonstrate elevated levels of anti-RPL23A antibodies compared to healthy controls .

• Inflammatory response: Recombinant RPL23A protein induces dose-dependent interleukin-2 production by T cells bearing the arthritogenic TCR, and stimulates inflammatory cytokine production by CD4+ T cells from arthritic joints .

• Human disease relevance: RPL23A is ubiquitously expressed in human tissues, and patients with rheumatoid arthritis show significantly higher levels of IgG antibodies specific for RPL23A compared to healthy controls or patients with other autoimmune conditions .

• Cellular localization: Immunohistochemical studies have shown co-localization of RPL23A with CD55 (a marker for fibroblast-like synoviocytes) in rheumatoid arthritis synovial tissues .

These findings suggest that RPL23A could be a clinically relevant autoantigen in rheumatoid arthritis and potentially a diagnostic marker or therapeutic target.

What are the optimal protocols for detecting RPL23A in Western blot applications?

Based on validated protocols for RPL23A antibodies:

• Sample preparation:

  • For cell lines: Lyse cells in RIPA buffer containing protease inhibitors

  • For tissues: Homogenize in lysis buffer, then centrifuge to remove debris

  • Use 15-50 μg of total protein per lane

• Gel electrophoresis:

  • Use 12-15% SDS-PAGE gels for optimal resolution of the 18 kDa RPL23A protein

  • Include molecular weight markers that clearly demarcate the 15-25 kDa range

• Transfer conditions:

  • Semi-dry or wet transfer at 100V for 60-90 minutes

  • PVDF membranes are generally preferred for detecting lower molecular weight proteins

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Primary antibody dilutions range from 1:500 to 1:12000 depending on the specific antibody

  • Incubate primary antibody overnight at 4°C

  • Secondary antibody at 1:5000 to 1:50000 for 1 hour at room temperature

• Detection:

  • Enhanced chemiluminescence (ECL) provides sufficient sensitivity

  • Exposure times typically range from 10 seconds to 2 minutes

Example protocol: Western blot analysis with anti-RPL23A antibody (ab223089) at 1/500 dilution detected a clear 18 kDa band in rat brain lysate .

What are the recommended procedures for immunohistochemical detection of RPL23A?

For optimal immunohistochemical staining of RPL23A:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissue sections (4-6 μm thickness)

  • For fresh tissues, fix in 4% paraformaldehyde before processing

• Antigen retrieval:

  • Heat-induced epitope retrieval is recommended

  • Use TE buffer at pH 9.0 for optimal results

  • Alternative: citrate buffer pH 6.0 can also be effective

• Antibody dilution and incubation:

  • Recommended dilutions range from 1:50 to 1:2400 depending on the antibody

  • Incubate primary antibody overnight at 4°C or 1-2 hours at room temperature

  • Use appropriate HRP-conjugated secondary antibody

• Detection system:

  • DAB (3,3'-diaminobenzidine) is commonly used for visualization

  • Counterstain with hematoxylin for nuclear definition

• Controls:

  • Positive control: human testis tissue has been validated for RPL23A staining

  • Negative control: omit primary antibody or use isotype control

The antibody has been successfully used to detect RPL23A in human normal colon tissue .

What are common causes of non-specific or weak signals when using RPL23A antibodies?

Several factors can contribute to suboptimal results with RPL23A antibodies:

• Non-specific binding issues:

  • Cause: Insufficient blocking or excessive antibody concentration

  • Solution: Increase blocking time/concentration (try 5% BSA instead of milk), optimize antibody dilution, add 0.1% Tween-20 to wash buffers

• Weak or absent signal:

  • Cause: Insufficient antigen, degraded protein, ineffective antigen retrieval

  • Solution: Increase protein loading (30-50 μg), ensure proper sample preparation with protease inhibitors, optimize antigen retrieval conditions (test both pH 6.0 citrate and pH 9.0 TE buffers)

• Multiple bands in Western blot:

  • Cause: Cross-reactivity, protein degradation, post-translational modifications

  • Solution: Use freshly prepared samples, add additional protease inhibitors, try antibodies targeting different epitopes, consider performing peptide competition assays

• Irregular staining patterns in IHC/ICC:

  • Cause: Uneven fixation, over-fixation, inappropriate antigen retrieval

  • Solution: Standardize fixation protocols, titrate fixation time, optimize antigen retrieval methods

• Background in immunofluorescence:

  • Cause: Autofluorescence, non-specific binding

  • Solution: Include an autofluorescence quenching step, use appropriate blocking serum (e.g., normal serum from secondary antibody host species), increase washing steps

How can I distinguish between RPL23A and its closely related pseudogene RPL23AP53 in my research?

Differentiating between RPL23A and its pseudogene RPL23AP53 requires specific approaches:

• PCR-based discrimination:

  • Design primers that target unique regions not conserved between RPL23A and RPL23AP53

  • For RPL23AP53, validated primers include: forward 5'-GAA GAT CCG CAT GTC ACT CA-3′ and reverse 5′-TGG TCA GCG GAA ACT TGA TA-3′

  • Confirm primer specificity using NCBI BLAST before use

• Expression analysis:

  • Normalize against housekeeping genes such as GAPDH (primers: forward 5′-CCA CTC CTC CAC CTT TGA CG-3′ and reverse 5′-CCA CCA CCC TGT TGC TGT AG-3′)

  • Calculate expression using the 2−ΔCT method

• Antibody selection:

  • Choose antibodies specifically validated against the protein of interest

  • Confirm epitope regions to ensure they don't cross-react with pseudogene products

• Bioinformatic approaches:

  • For RNA-seq data, apply specific alignment parameters that account for pseudogenes

  • Use tools designed to distinguish between highly similar sequences

• Functional validation:

  • When studying cellular effects, perform rescue experiments with constructs expressing either RPL23A or RPL23AP53 to confirm specificity

Research has shown that RPL23AP53 expression differs significantly between primary and metastatic melanoma samples and correlates with changes in immune cell infiltration, distinguishing it functionally from RPL23A .

How can RPL23A antibodies be utilized to investigate its role in p53/TP53 regulation?

RPL23A has been implicated in p53/TP53 regulation through stimulation of MDM2-mediated TP53 polyubiquitination . To investigate this relationship:

• Co-immunoprecipitation (Co-IP) studies:

  • Use RPL23A antibodies to pull down protein complexes

  • Probe for MDM2 and p53 in the immunoprecipitated material to assess protein-protein interactions

  • Perform reciprocal Co-IPs with MDM2 or p53 antibodies and probe for RPL23A

• Proximity ligation assay (PLA):

  • Utilize antibodies against RPL23A and p53/MDM2 to visualize and quantify their interactions in situ

  • This technique allows detection of protein interactions with high sensitivity

• Cellular localization studies:

  • Perform immunofluorescence co-staining of RPL23A with p53 and MDM2

  • Analyze subcellular distribution under normal conditions and after cellular stress

• Functional assays:

  • Combine RPL23A knockdown or overexpression with analysis of p53 stability and activity

  • Measure p53 half-life in the presence or absence of RPL23A

  • Assess p53 target gene expression following manipulation of RPL23A levels

• Ubiquitination assays:

  • Following RPL23A manipulation, immunoprecipitate p53 and probe for ubiquitin to assess polyubiquitination levels

  • Use proteasome inhibitors to accumulate ubiquitinated proteins for easier detection

What novel applications of RPL23A antibodies are emerging in cancer research?

RPL23A antibodies are finding new applications in cancer research beyond traditional protein detection:

• Prognostic marker development:

  • RPL23 expression correlates with poor prognosis in hepatocellular carcinoma

  • Immunohistochemical staining with RPL23A antibodies could be developed into a prognostic panel

• Therapeutic target validation:

  • RPL23 promotes HCC metastasis through MMP9 mRNA stabilization

  • Antibodies can help validate this pathway as a therapeutic target through in vitro and in vivo studies

• Biomarker identification:

  • In melanoma, RPL23AP53 expression correlates with immune cell infiltration profiles

  • RPL23A/RPL23AP53 antibodies could help develop biomarkers for immunotherapy response prediction

• RNA-protein interaction studies:

  • RNA immunoprecipitation (RIP) assays using RPL23A antibodies can identify RNA targets like MMP9 mRNA

  • This application helps elucidate post-transcriptional regulatory mechanisms in cancer

• Therapeutic antibody development:

  • Research into RPL23A as an antigen in autoimmune conditions could potentially be leveraged for developing therapeutic antibodies that block pathogenic interactions

These emerging applications highlight the continuing evolution of RPL23A antibody use in cancer research beyond basic protein detection.