RPL13A Antibody

What is RPL13A and why is it important in research?

RPL13A (ribosomal protein L13a) is a 23 kDa highly basic protein that is a component of the 60S ribosomal subunit. While traditionally associated with ribosomes, RPL13A has significant extra-ribosomal functions . It is a key component of the GAIT (gamma interferon-activated inhibitor of translation) complex, which mediates interferon-gamma-induced transcript-selective translation inhibition during inflammation . Upon interferon-gamma activation and subsequent phosphorylation, RPL13A dissociates from the ribosome and assembles into the GAIT complex, which binds to stem loop-containing GAIT elements in the 3'-UTR of inflammatory mRNAs and suppresses their translation .

Additionally, RPL13A is involved in rRNA methylation processes and is frequently used as a reference gene in qPCR due to its stable expression during differentiation and expansion to higher passages . The gene is co-transcribed with small nucleolar RNA genes U32, U33, U34, and U35, which are located in its second, fourth, fifth, and sixth introns, respectively .

What applications are RPL13A antibodies suitable for?

Most commercially available RPL13A antibodies are validated for multiple applications:

When selecting an antibody, verify that it has been tested in your specific application and experimental system. Many manufacturers provide images of validation data from various applications to help you assess antibody performance .

What is the expected molecular weight of RPL13A in Western blot applications?

When analyzing your Western blot results, be aware that the observed band might not match the predicted molecular weight precisely. If multiple bands appear, validation experiments using positive and negative controls (such as RPL13A knockdown samples) may be necessary to confirm specificity .

How can I validate the specificity of an RPL13A antibody in my experimental system?

Antibody validation is critical for ensuring reliable results. For RPL13A antibodies, consider the following approaches:

• Knockdown/Knockout Controls: Generate RPL13A knockdown samples using siRNA or CRISPR/Cas9 technologies. The specificity of the RPL13A antibody can be confirmed if the signal is substantially reduced or eliminated in these samples compared to controls .

• Known Positive Controls: Include cell lines or tissues known to express RPL13A at detectable levels. According to validation data, A549, Jurkat, HEK-293, HeLa, and RAW264.7 cells have shown positive Western blot results with RPL13A antibodies .

• Immunogen Blocking: Pre-incubate the antibody with excess immunogen peptide before application. A specific antibody will show reduced or no signal when the binding sites are blocked by the immunogen.

• Cross-Species Validation: If working with non-human samples, verify that the antibody has been validated in your species of interest or has high sequence homology. For example, many RPL13A antibodies show reactivity with human samples and predicted reactivity with mouse (95% homology), rat (96%), and other species .

What methodological considerations are important when using RPL13A antibodies in Western blotting?

To optimize Western blot results with RPL13A antibodies:

• Sample Preparation: Since RPL13A can dissociate from ribosomes upon interferon-gamma stimulation, consider how your sample preparation might affect its localization and detection. For studies focusing on the GAIT complex, cytoplasmic fractionation may be necessary.

• Loading Controls: When studying RPL13A's role in specific conditions (e.g., inflammation), traditional housekeeping genes might be affected. Consider using multiple loading controls or total protein normalization.

• Antibody Dilution Optimization: Start with the manufacturer's recommended dilution range (typically 1:500-1:5000 for Western blot) and optimize based on your specific conditions.

• Detection Methods: For studying low-abundance forms of RPL13A (such as phosphorylated RPL13A involved in the GAIT complex), more sensitive detection methods may be required.

• Resolution of Multiple Bands: If you observe multiple bands, consider using phosphatase treatment to determine if bands represent different phosphorylation states, particularly relevant when studying RPL13A's role in the GAIT complex.

How can I effectively use RPL13A antibodies in immunohistochemistry and immunofluorescence?

For optimal IHC and ICC/IF results with RPL13A antibodies:

• Antigen Retrieval: For paraffin-embedded tissues, manufacturers recommend either TE buffer pH 9.0 or citrate buffer pH 6.0 for antigen retrieval .

• Dilution Range: Start with the recommended dilutions (typically 1:100-1:1000 for ICC/IF and 1:50-1:300 for IHC) and optimize for your specific tissue or cell type.

• Verified Positive Controls: Include known positive tissues such as human tonsil or human thyroid cancer tissues, which have been verified for RPL13A expression .

• Subcellular Localization: RPL13A typically shows cytoplasmic localization, but its distribution may change under specific conditions (e.g., interferon-gamma stimulation). When interpreting results, consider whether the observed localization pattern is consistent with the biological context of your experiment.

• Double Staining: Consider double staining with markers for specific subcellular compartments to better understand RPL13A localization, especially when studying its translocation from ribosomes to the GAIT complex.

How can RPL13A antibodies be used to study the GAIT complex and inflammation-related mechanisms?

RPL13A plays a crucial role in the GAIT complex, which mediates interferon-gamma-induced translational repression of inflammatory mRNAs . To study this process:

• Stimulation Experiments: Treat cells with interferon-gamma to induce RPL13A phosphorylation and release from ribosomes. Compare RPL13A localization and protein interactions before and after stimulation using immunoprecipitation followed by Western blotting.

• Phosphorylation-Specific Detection: Consider using phospho-specific antibodies or Phos-tag gels to detect the phosphorylated form of RPL13A that participates in the GAIT complex.

• Co-localization Studies: Perform co-immunofluorescence with antibodies against other GAIT complex components to visualize complex formation and localization.

• RNA-Protein Interaction Analysis: Combine RPL13A antibodies with RNA immunoprecipitation (RIP) assays to identify target mRNAs regulated by the GAIT complex in your specific experimental context.

• Functional Studies: Use RPL13A antibodies in conjunction with translation assays to assess the impact of RPL13A on the translation of specific inflammatory mRNAs containing GAIT elements.

How can I differentiate between ribosome-associated and GAIT complex-associated RPL13A?

Distinguishing between the different functional pools of RPL13A requires specific methodological approaches:

• Subcellular Fractionation: Separate ribosomal and non-ribosomal fractions before Western blot analysis with RPL13A antibodies. The ribosome-free fraction would contain RPL13A involved in the GAIT complex.

• Density Gradient Centrifugation: Use sucrose or glycerol gradients to separate free RPL13A from ribosome-associated RPL13A, followed by Western blot analysis of gradient fractions.

• Immunoprecipitation Studies: Use antibodies against known GAIT complex components to co-immunoprecipitate associated RPL13A, distinguishing it from the ribosomal pool.

• Proximity Ligation Assays: Combine RPL13A antibodies with antibodies against ribosomal or GAIT complex markers to visualize and quantify RPL13A associations in situ.

• Time-Course Experiments: Follow RPL13A localization and associations after interferon-gamma stimulation to track its transition from ribosomes to the GAIT complex.

What considerations are important when using RPL13A as a reference gene in qPCR experiments?

RPL13A is frequently used as a housekeeping gene for normalization in qPCR due to its relatively stable expression , but several factors should be considered:

• Validation in Your Experimental System: Verify that RPL13A expression remains stable under your specific experimental conditions, especially in studies involving inflammation or interferon signaling where RPL13A function may be altered.

• Multiple Reference Genes: Consider using multiple reference genes for more robust normalization, particularly in studies where cellular conditions might affect housekeeping gene expression.

• Primer Design: The validated primers for RPL13A (forward 5'-CAAGCGGATGAACACCAAC-3' and reverse 5'-CGCTTTTTCTTGTCGTAGGGG-3') should amplify a region that doesn't span introns containing snoRNA genes to avoid interference.

• Pseudogene Considerations: RPL13A has multiple processed pseudogenes throughout the genome . Ensure your primers don't amplify these pseudogenes by checking for unique binding sites.

• qPCR Optimization: Optimize annealing temperatures and cycle numbers for your specific PCR system to ensure efficient and specific amplification of RPL13A.

Why might I observe discrepancies in RPL13A molecular weight in Western blots?

Several factors can contribute to variations in observed molecular weight:

• Post-translational Modifications: Phosphorylation of RPL13A occurs during GAIT complex formation, which can alter its migration pattern.

• Splice Variants: Multiple isoforms of RPL13A have been observed due to alternative splicing , potentially resulting in bands of different sizes.

• Protein-Protein Interactions: Incomplete denaturation may result in RPL13A remaining partially bound to other proteins, causing higher molecular weight bands.

• Proteolytic Processing: Sample preparation conditions may affect proteolytic processing of RPL13A.

• Gel Conditions: The percentage of acrylamide, buffer composition, and running conditions can all affect protein migration patterns.

To address these issues, compare your results with positive controls, consider using different sample preparation methods, and potentially perform mass spectrometry analysis to confirm protein identity.

How can I minimize background and non-specific binding with RPL13A antibodies?

To improve signal-to-noise ratio:

• Blocking Optimization: Test different blocking agents (BSA, non-fat milk, commercial blockers) to identify the optimal conditions for your specific antibody.

• Antibody Titration: Perform a dilution series to determine the optimal antibody concentration that maximizes specific signal while minimizing background.

• Washing Conditions: Increase washing steps duration or number to reduce non-specific binding, particularly important in immunohistochemistry applications.

• Pre-adsorption: For tissue samples with high background, consider pre-adsorbing the primary antibody with tissue lysate from a species different from your target.

• Secondary Antibody Selection: Choose a secondary antibody with minimal cross-reactivity to your sample species and consider using highly cross-adsorbed versions for reduced background.

How can RPL13A antibodies be used to study its role in DNA damage pathways?

Recent research has identified extraribosomal functions of ribosomal proteins, including RPL13A, in DNA damage response pathways . To investigate these functions:

• DNA Damage Induction: Treat cells with DNA-damaging agents and assess changes in RPL13A localization, expression, and interactions using RPL13A antibodies.

• Chromatin Association: Perform chromatin immunoprecipitation (ChIP) with RPL13A antibodies to determine if it associates with chromatin in response to DNA damage.

• Protein-Protein Interactions: Use co-immunoprecipitation with RPL13A antibodies to identify interactions with DNA damage response proteins under various conditions.

• RPL13A Knockdown Studies: Combine knockdown approaches with RPL13A antibodies to validate the specificity of observed phenotypes related to DNA damage response.

• Microscopy Approaches: Use immunofluorescence with RPL13A antibodies to track its localization during DNA damage response, particularly in relation to sites of DNA damage or repair complexes.

What methodological approaches are suitable for studying RPL13A's role in translation regulation during inflammation?

To investigate RPL13A's function in translational control during inflammatory responses:

• Polysome Profiling: Combine polysome fractionation with Western blotting using RPL13A antibodies to assess its association with actively translating ribosomes under different inflammatory conditions.

• Ribosome Profiling: Use RPL13A antibodies in conjunction with ribosome profiling to analyze how RPL13A influences the translation of specific mRNAs during inflammation.

• GAIT Element Reporter Assays: Develop reporter constructs containing GAIT elements and use RPL13A antibodies to correlate RPL13A activity with translational repression.

• In vitro Translation Systems: Reconstitute translation systems with and without RPL13A (verified by Western blotting) to directly assess its impact on the translation of target mRNAs.

• Phosphorylation Site Mutants: Generate RPL13A mutants at known phosphorylation sites and use antibodies to track their localization and function in response to inflammatory stimuli.