RPL13 Antibody

What is RPL13 and why is it important in research?

RPL13 (Ribosomal Protein L13) is a component of the 60S ribosomal subunit with a molecular weight of approximately 24 kDa . Beyond its canonical role in protein synthesis, RPL13 plays crucial roles in bone development as evidenced by its high expression in chondrocytes and osteoblasts in mouse growth plates . RPL13 is also implicated in immune responses, particularly in antiviral defense mechanisms . These diverse functions make RPL13 antibodies valuable tools for investigating ribosomal biology, skeletal development, and immune regulation.

Most commercial RPL13 antibodies demonstrate reactivity with human, mouse, and rat samples . When working with other species, verify cross-reactivity in the product documentation. Some antibodies show broader reactivity across species, including dog, cow, guinea pig, zebrafish, and xenopus . For highly conserved regions of RPL13, cross-reactivity is more likely, but validation in your specific species is recommended before proceeding with extensive experiments.

What are the optimal conditions for Western blot analysis using RPL13 antibodies?

For Western blot applications using RPL13 antibodies:

• Sample preparation: Total protein extraction from cells (e.g., HeLa, SGC-7901) or tissues (e.g., human lung) using RIPA buffer containing protease inhibitors

• Protein loading: 10-20 μg of total protein per lane

• Recommended dilutions: 1:500-1:1000 for polyclonal antibodies ; check specific product documentation

• Expected band size: 24 kDa (observed molecular weight)

• Positive controls: HeLa cells, SGC-7901 cells, and human lung tissue have been validated

• Detection system: HRP-conjugated secondary antibodies (e.g., anti-rabbit IgG at 1:20,000 dilution)

Acetylated-tubulin can serve as an effective loading control (1:10,000 dilution) .

How should I optimize immunohistochemistry protocols for RPL13 antibodies?

For optimal IHC results with RPL13 antibodies:

• Antigen retrieval: Use TE buffer pH 9.0 or citrate buffer pH 6.0

• Blocking: 5% normal serum in PBS for 1 hour at room temperature

• Primary antibody incubation: Use dilution range of 1:20-1:200 ; optimize for your specific tissue

• Human colon cancer tissue has been validated as a positive control

• For RPL13 detection in bone tissue, special decalcification procedures may be required to preserve epitope integrity while removing calcium

• Signal amplification systems may be necessary when studying tissues with lower RPL13 expression levels

What considerations are important for immunoprecipitation experiments with RPL13 antibodies?

For successful immunoprecipitation of RPL13:

• Antibody amount: Use 0.5-4.0 μg of RPL13 antibody for 1.0-3.0 mg of total protein lysate

• Validated cell lines: HeLa cells have been confirmed for IP applications

• Pre-clearing step: Incorporate to reduce non-specific binding

• Buffer selection: Use buffers that maintain native protein conformation if studying RPL13 interactions

• Controls: Include IgG control and input samples to validate specificity

• For RNA-protein interaction studies, consider cross-linking before IP to capture transient interactions between RPL13 and RNA molecules

How can I use RPL13 antibodies to study its role in ribosome biogenesis and function?

Advanced research on RPL13's role in ribosome biology can employ:

• Polysome profiling: Isolate ribosome fractions on sucrose gradients and analyze RPL13 distribution to assess incorporation into functional ribosomes

• Ribosome subunit analysis: Compare RPL13 variant distribution in 40S, 60S, 80S, and polysome fractions

• Translation dynamics: Measure changes in polysome profiles following RPL13 knockdown or overexpression

• Puromycin incorporation assays: Quantify global protein synthesis rates when RPL13 expression is altered

• Pull-down assays using RPL13 antibodies to identify bound cofactors that confer specificity in ribosome activity

• RPL13-bound mRNA identification: Immunoprecipitate RPL13-containing ribosomes and perform RNA-seq to identify specifically translated mRNAs

What approaches can I use to investigate RPL13's role in immune responses?

To study RPL13's immunological functions:

• Overexpression and knockdown experiments combined with cytokine measurements (IFN-β, IL-6) following immune stimulation

• Promoter activity assays for NF-κB and IFN-β genes in response to RPL13 modulation

• Co-immunoprecipitation studies to identify RPL13 interactions with immune signaling components or pathogen proteins

• Time-course experiments tracking RPL13 expression and localization during immune challenges

• ELISA quantification of RPL13 levels in serum or plasma during different immune states (detection range: 0.78-50ng/mL; sensitivity: 0.39ng/mL)

How can I study RPL13 variants implicated in skeletal dysplasia?

For research on RPL13 variants associated with spondyloepimetaphyseal dysplasia:

• Molecular modeling to predict how variants (e.g., 18-amino acid insertion or p.Arg183Pro) affect protein structure and rRNA interactions

• Pre-rRNA processing analysis via northern blot to assess impact on ribosome biogenesis

• Sucrose gradient analysis to examine the incorporation of variant RPL13 into ribosomes

• Translation efficiency measurements using reporter assays

• Chondrocyte and osteoblast culture models to analyze cellular consequences of RPL13 variants

• Immunofluorescence to determine subcellular localization changes caused by RPL13 variants

How can I verify the specificity of my RPL13 antibody?

To confirm antibody specificity:

• Western blot validation using RPL13 knockdown or knockout samples as negative controls

• Peptide competition assays by pre-incubating the antibody with the immunizing peptide

• Testing multiple antibodies targeting different epitopes of RPL13

• Confirming the observed molecular weight matches the expected 24 kDa

• Using positive control samples with known RPL13 expression (HeLa cells, SGC-7901 cells, human lung tissue)

• For immunofluorescence, confirming the expected nucleolar/cytoplasmic localization pattern

What are common pitfalls when working with RPL13 antibodies and how can I address them?

Common challenges and solutions:

• Cross-reactivity with other ribosomal proteins:

  • Use highly purified antibodies

  • Validate with specific knockout controls

  • Consider recombinant monoclonal antibodies for higher specificity

• Variable expression levels across tissues:

  • Adjust antibody concentrations based on target tissue expression

  • Use signal amplification for tissues with low expression

  • Include positive control tissues (e.g., human colon cancer tissue for IHC)

• Background signals in immunostaining:

  • Optimize blocking conditions (BSA percentage, serum type)

  • Increase washing steps and duration

  • Titrate antibody concentration carefully (1:20-1:200 for IHC)

• Storage-related antibody degradation:

  • Store at -20°C with 50% glycerol

  • Avoid repeated freeze-thaw cycles

  • For long-term storage, aliquot the antibody solution

How should I approach conflicting results between different RPL13 antibodies?

When faced with discrepant results:

• Compare the epitopes recognized by each antibody – different epitopes may be accessible in different contexts

• Evaluate the antibody formats (monoclonal vs. polyclonal) – each has distinct advantages

• Validate with orthogonal methods (e.g., mass spectrometry, RNA expression)

• Consider post-translational modifications or protein interactions that might mask epitopes

• Test antibodies in multiple applications to identify context-dependent performance

• Perform side-by-side validation in known positive and negative control samples

• Consider the possibility that different antibodies may detect distinct RPL13 variants or isoforms

How can RPL13 antibodies be used to study specialized ribosomes and selective translation?

For investigating ribosome heterogeneity:

• Polysome profiling with RPL13 antibodies to identify specific mRNA populations translated by RPL13-containing ribosomes

• Single-cell translatomic profiling combined with transcriptomics to correlate RPL13 expression with translation patterns

• Proximity labeling approaches (BioID, APEX) using RPL13 as bait to identify proteins near the ribosome exit tunnel

• Super-resolution microscopy to visualize RPL13-containing ribosomes in specific subcellular locations

• Targeted ribosome profiling to identify transcripts preferentially translated by RPL13-containing ribosomes versus ribosomes with RPL13 variants

What is known about RPL13's extraribosomal functions, and how can they be studied?

To investigate non-canonical roles of RPL13:

• Subcellular fractionation combined with immunoblotting to detect non-ribosomal RPL13 pools

• Co-immunoprecipitation followed by mass spectrometry to identify novel interaction partners

• ChIP-seq approaches to investigate potential chromatin association

• Imaging techniques to track RPL13 under stress conditions or during specific cellular processes

• Proximity labeling in different cellular compartments to identify context-specific interactors

• Functional assays focused on immune signaling pathways, where RPL13 has been shown to modulate NF-κB and interferon responses

How do disease-associated RPL13 variants affect protein function and cellular phenotypes?

For functional characterization of pathogenic variants:

• CRISPR-Cas9 genome editing to introduce specific variants (e.g., splice variants leading to 18-amino acid insertions)

• RNA-seq to identify transcriptome-wide changes caused by RPL13 variants

• Ribosome profiling to detect alterations in translation efficiency and start site selection

• Cryo-EM structural analysis to visualize how variants affect ribosome structure

• Patient-derived cell models (fibroblasts, iPSCs) to study cellular consequences in disease-relevant contexts

• In vitro translation assays to measure kinetic parameters of protein synthesis with variant RPL13