RPL7A Antibody

What is RPL7A and why is it important in research?

RPL7A (Ribosomal Protein L7a) is a component of the 60S large ribosomal subunit with a molecular weight of approximately 30 kDa. It plays a critical role in protein synthesis as part of the ribosomal machinery. Beyond its structural role in ribosomes, RPL7A has additional functions including:

• Interaction with nuclear hormone receptors, particularly thyroid hormone receptors

• Inhibition of receptor transactivation by preventing DNA binding to response elements

• Potential involvement in ribosome biosynthesis through nucleolar localization and RNA-binding domains

Research on RPL7A is significant for understanding fundamental cellular processes including protein synthesis, hormone signaling, and ribosomal biogenesis.

What types of RPL7A antibodies are available for research?

Several types of RPL7A antibodies are available, each with specific characteristics:

The choice between polyclonal and monoclonal antibodies depends on research needs: polyclonals offer higher sensitivity by recognizing multiple epitopes, while monoclonals provide higher specificity by targeting a single epitope.

Which applications are RPL7A antibodies validated for?

RPL7A antibodies have been validated for multiple applications:

When selecting an antibody, researchers should prioritize products with validation data specific to their application and sample type of interest.

How should I optimize Western blot protocols for RPL7A detection?

Optimizing Western blot protocols for RPL7A requires attention to several parameters:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for efficient extraction

  • Load 20-30 μg total protein for cell lines (based on published protocols)

  • Expected molecular weight: 30-32 kDa

• Gel electrophoresis and transfer:

  • 12% SDS-PAGE shows optimal separation for this molecular weight range

  • Standard semi-dry or wet transfer protocols are suitable

• Antibody incubation:

  • Primary antibody dilution: Start with 1:1000 (polyclonal) or 1:500 (monoclonal)

  • Incubation time: Overnight at 4°C yields better signal-to-noise ratio than 1-2 hours at room temperature

  • Secondary antibody: Anti-rabbit or anti-mouse HRP conjugates at 1:5000-1:10000 dilution

• Detection controls:

  • Positive controls: MCF7, HeLa, 293T cell lysates consistently show strong RPL7A expression

  • Loading control: Consider using other ribosomal proteins or housekeeping proteins

For challenging samples, titration experiments with dilutions ranging from 1:500 to 1:2000 are recommended to determine optimal concentration.

What are the critical factors for successful immunohistochemistry with RPL7A antibodies?

Successful IHC with RPL7A antibodies depends on these critical factors:

• Fixation and antigen retrieval:

  • Formalin-fixed paraffin-embedded (FFPE) tissues are compatible

  • Recommended antigen retrieval: TE buffer pH 9.0; alternatively, citrate buffer pH 6.0

  • Heat-induced epitope retrieval (HIER) for 15-20 minutes

• Antibody selection and dilution:

  • Start with 1:100 dilution for polyclonal antibodies

  • For monoclonal antibodies, 1:500 dilution is typically recommended

  • Optimization ranges: 1:50-1:500

• Detection systems:

  • DAB (3,3'-diaminobenzidine) detection works effectively with RPL7A antibodies

  • Amplification systems may be needed for low-expressing tissues

• Controls and interpretation:

  • Positive tissue controls: Human breast cancer tissue, lung adenocarcinoma

  • Expression pattern: Primarily cytoplasmic and nucleolar localization

  • Pay attention to background staining, particularly with polyclonal antibodies

The typical staining pattern shows predominantly cytoplasmic localization with nucleolar enrichment, consistent with RPL7A's role in ribosome assembly.

How should I optimize immunofluorescence protocols for RPL7A localization studies?

For optimal immunofluorescence with RPL7A antibodies:

• Cell preparation:

  • Fixation options: 4% paraformaldehyde (10 min) or methanol fixation (5 min at -20°C)

  • Methanol fixation has shown good results with A431 cells

  • Permeabilization: 0.1-0.3% Triton X-100 for 5-10 minutes

• Antibody dilution and incubation:

  • Typical starting dilution: 1:100 (range: 1:20-1:200)

  • Primary antibody incubation: Overnight at 4°C recommended

  • Secondary antibody: Anti-rabbit or anti-mouse Alexa Fluor conjugates (1:500-1:1000)

• Counterstaining:

  • DAPI for nuclear visualization

  • Consider co-staining with nucleolar markers (fibrillarin, nucleolin) to confirm nucleolar localization

• Controls and imaging:

  • Positive control cell lines: HepG2, A431

  • Negative controls: Primary antibody omission and non-expressing cells

  • Confocal microscopy recommended for detailed subcellular localization

Expected localization pattern includes strong cytoplasmic signal consistent with RPL7A's role in ribosomes, with additional nucleolar enrichment in many cell types.

How can I design experiments to study RPL7A interactions with hormone receptors?

To investigate RPL7A interactions with hormone receptors:

• Co-immunoprecipitation (Co-IP):

  • Reciprocal Co-IP using both RPL7A antibodies and hormone receptor antibodies

  • Cell lysate preparation: Non-denaturing conditions to preserve protein-protein interactions

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

  • Controls: IgG control, input lysate, and negative control proteins

• Proximity ligation assay (PLA):

  • Provides in situ detection of protein-protein interactions

  • Use RPL7A antibody (1:100) with thyroid hormone receptor antibody

  • Recommended cell lines: Those expressing both proteins at detectable levels

• Functional assays:

  • Reporter gene assays to measure transcriptional effects

  • siRNA knockdown of RPL7A followed by measurement of hormone receptor activity

  • DNA binding assays (EMSA) to verify RPL7A inhibition of receptor-DNA interactions

This experimental design allows for comprehensive characterization of RPL7A's interaction with hormone receptors and its functional consequences, as suggested by previous research showing interactions with thyroid hormone receptors .

What approaches can address the dual function of RPL7A in ribosome assembly and gene regulation?

To investigate RPL7A's dual functionality:

• Subcellular fractionation:

  • Separate cytoplasmic, nucleoplasmic, and nucleolar fractions

  • Analyze RPL7A distribution using Western blot (1:1000 dilution)

  • Compare distribution patterns under different cellular conditions

• RNA immunoprecipitation (RIP):

  • Use RPL7A antibodies to immunoprecipitate RNA-protein complexes

  • Analyze bound RNAs by RT-qPCR or RNA sequencing

  • Controls: IgG control, input RNA, and other ribosomal proteins

• CRISPR-based approaches:

  • Generate domain-specific mutations in RPL7A

  • Target RNA-binding domains versus hormone receptor interaction domains

  • Analyze effects on ribosome assembly versus transcriptional regulation

• Proximity-dependent labeling:

  • RPL7A-BioID or APEX2 fusion proteins to identify proximal proteins

  • Compare interactomes in cytoplasm versus nucleus

  • Validation of novel interactions by Co-IP

This multi-faceted approach can differentiate between RPL7A's canonical role in ribosome assembly and its non-canonical functions in gene regulation, providing insights into how these distinct functions are coordinated.

How can I design RPL7A knockdown experiments to study its function while avoiding global translation effects?

Designing RPL7A knockdown experiments requires careful approaches to distinguish specific functions from global translation defects:

• Partial knockdown strategy:

  • Titrate siRNA/shRNA concentrations to achieve 50-70% reduction

  • Monitor global translation rates using puromycin incorporation assays

  • Use Western blot with validated antibodies (1:1000 dilution) to confirm knockdown levels

• Rescue experiments:

  • Design siRNA-resistant RPL7A constructs

  • Create domain-specific mutants (RNA-binding mutants, hormone receptor interaction mutants)

  • Express at physiological levels to avoid overexpression artifacts

• Acute depletion systems:

  • Auxin-inducible degron (AID) system for rapid, reversible depletion

  • Analyze early timepoints before global translation defects manifest

  • Monitor cell viability and stress responses as controls

• Specific readouts beyond translation:

  • Hormone receptor activity assays

  • RNA subcellular localization

  • Nucleolar stress markers

This experimental design allows researchers to separate RPL7A's specific roles from its general contribution to translation, providing clearer insights into its non-canonical functions.

What are common issues with RPL7A Western blots and how can they be resolved?

Common issues and solutions for RPL7A Western blots:

When addressing these issues, remember that RPL7A's observed molecular weight is typically 30-32 kDa, which may vary slightly between species and cell types .

How can I validate the specificity of my RPL7A antibody?

To validate RPL7A antibody specificity:

• Genetic approaches:

  • CRISPR knockout or knockdown (siRNA/shRNA) of RPL7A

  • Western blot comparison between wild-type and depleted samples

  • Expected: Substantial reduction or elimination of the specific band at 30-32 kDa

• Immunizing peptide competition:

  • Pre-incubate antibody with excess immunizing peptide

  • Run parallel Western blots with competed and non-competed antibody

  • Specific bands should disappear in the competed sample

• Multiple antibody validation:

  • Test multiple antibodies targeting different RPL7A epitopes

  • Compare staining patterns and band recognition

  • Consistent results across antibodies suggest specificity

• Mass spectrometry confirmation:

  • Immunoprecipitate with RPL7A antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm presence of RPL7A peptides in the immunoprecipitated material

These validation approaches ensure that observed signals truly represent RPL7A, which is particularly important for functional studies where antibody specificity directly impacts data interpretation.

What controls should be included when using RPL7A antibodies in various applications?

Essential controls for RPL7A antibody applications:

For quantitative applications, standard curves using recombinant protein or calibrated cell lysates should be included. Additionally, validation across multiple experimental conditions helps ensure reproducibility of results.

How can I investigate the role of RPL7A in nucleolar stress responses?

To investigate RPL7A in nucleolar stress responses:

• Stress induction methods:

  • Actinomycin D (low dose: 5-10 nM) to inhibit RNA polymerase I

  • 5-Fluorouracil treatment to disrupt rRNA processing

  • Serum starvation or nutrient deprivation

• Analytical approaches:

  • Time-course immunofluorescence (1:100 dilution) to track RPL7A relocalization

  • Co-staining with nucleolar markers (nucleolin, fibrillarin)

  • Live-cell imaging with fluorescently tagged RPL7A

• Molecular readouts:

  • p53 stabilization and activation

  • Changes in pre-rRNA processing

  • Alterations in RPL7A-associated protein complexes (analyzed by IP-mass spectrometry)

• Functional assessments:

  • Effect of RPL7A depletion on stress resistance

  • Impact on nucleolar recovery after stress

  • Consequences for cell cycle progression and survival

This experimental approach can reveal RPL7A's potential role in sensing or mediating nucleolar stress responses, which may represent an important function beyond its structural role in ribosomes.

How does RPL7A contribute to specialized ribosomes and selective translation?

To investigate RPL7A's role in specialized ribosomes:

• Polysome profiling with RPL7A immunoblotting:

  • Fractionate polysomes on sucrose gradients

  • Analyze RPL7A distribution across fractions by Western blot (1:1000 dilution)

  • Compare normal conditions versus cellular stress

• Ribosome footprinting approaches:

  • RPL7A immunoprecipitation followed by ribosome profiling

  • Identify mRNAs preferentially associated with RPL7A-containing ribosomes

  • Bioinformatic analysis of mRNA features (5'UTR structures, sequence motifs)

• Tissue-specific analyses:

  • Compare RPL7A expression and incorporation into ribosomes across tissues

  • Use immunohistochemistry (1:100 dilution) for tissue distribution patterns

  • Correlate with tissue-specific translation programs

• RPL7A variant studies:

  • Identify natural variants or post-translational modifications

  • Generate mutants mimicking or blocking these modifications

  • Assess impact on ribosome composition and mRNA selectivity

This experimental approach can determine whether RPL7A contributes to ribosome heterogeneity and specialized translation, which represents an emerging area in ribosome biology with implications for development and disease.

What techniques can resolve contradictory findings regarding RPL7A subcellular localization?

To resolve contradictions in RPL7A localization:

• Comprehensive fixation method comparison:

  • Paraformaldehyde (4%) versus methanol fixation

  • Glutaraldehyde (0.1-0.5%) for enhanced structural preservation

  • Live-cell imaging with tagged RPL7A to avoid fixation artifacts

• Subcellular fractionation with biochemical validation:

  • Isolate cytoplasmic, nucleoplasmic, and nucleolar fractions

  • Western blot analysis with multiple RPL7A antibodies (1:1000 dilution)

  • Validation with compartment-specific markers

• Super-resolution microscopy:

  • STED or STORM imaging for nanoscale resolution

  • Co-localization with defined subcellular markers

  • Quantitative image analysis for distribution patterns

• Cell cycle synchronization:

  • Analyze RPL7A localization across cell cycle phases

  • Use immunofluorescence (1:100 dilution) with cell cycle markers

  • Correlate with ribosome biogenesis activity

• Antibody epitope mapping:

  • Use antibodies targeting different RPL7A regions

  • Compare staining patterns to identify epitope-specific effects

  • Validate with tagged RPL7A constructs

This multi-faceted approach can resolve contradictory findings by accounting for technical variables (fixation methods, antibody epitopes) and biological variables (cell cycle stage, cell type differences) that may influence RPL7A's apparent localization.

What emerging applications of RPL7A antibodies should researchers consider?

Emerging applications for RPL7A antibodies include:

• Single-cell analyses:

  • Imaging mass cytometry with metal-conjugated RPL7A antibodies

  • Single-cell Western blotting for heterogeneity assessment

  • Multiplexed immunofluorescence with other ribosomal and regulatory proteins

• Extracellular and non-canonical localization studies:

  • Investigation of RPL7A in extracellular vesicles

  • Membrane association under specific cellular conditions

  • Potential mitochondrial localization and function

• Cancer biomarker applications:

  • Quantitative IHC in tumor progression studies

  • Correlation with treatment response

  • Multiplexed analysis with other ribosomal proteins and translation factors

• Therapeutic target validation:

  • Antibody-based inhibition of specific RPL7A interactions

  • Targeted protein degradation approaches

  • Validation of RPL7A-directed therapeutics

These emerging applications highlight the evolving understanding of RPL7A beyond its canonical role in ribosomes, opening new research directions with potential diagnostic and therapeutic implications.

How can researchers integrate RPL7A studies with emerging ribosome heterogeneity concepts?

To integrate RPL7A studies with ribosome heterogeneity research:

• Specialized ribosome immunoprecipitation:

  • Use RPL7A antibodies (0.5-4.0 μg for IP) to isolate specific ribosome subpopulations

  • Analyze associated proteins and RNAs

  • Compare composition across developmental stages or disease states

• Cryo-EM structural studies:

  • Immunoaffinity purification of RPL7A-containing ribosomes

  • Structural analysis of RPL7A positioning and interactions

  • Comparison with ribosomes lacking or with modified RPL7A

• Translational specificity analysis:

  • Ribosome profiling of RPL7A-depleted or -modified cells

  • Identification of mRNAs with altered translation efficiency

  • Motif analysis for potential regulatory elements

• Systems biology integration:

  • Network analysis of RPL7A interactors under various conditions

  • Integration with transcriptomics and proteomics data

  • Mathematical modeling of RPL7A contributions to translation dynamics

This integrated approach positions RPL7A research within the broader context of ribosome heterogeneity, potentially revealing specialized functions in development, stress responses, and disease states.