RPL37 Antibody

What is RPL37 and why is it important for ribosomal research?

RPL37 (Ribosomal Protein L37) is a component of the large 60S ribosomal subunit. In humans, the canonical protein has 97 amino acid residues with a molecular mass of 11.1 kDa and is primarily localized in the cytoplasm . As a member of the Eukaryotic ribosomal protein eL37 family, RPL37 contains a C2C2-type zinc finger-like motif and surrounds the polypeptide exit tunnel, playing a critical role in ribosome structure and function.

RPL37 is particularly significant because:

• It is essential for 60S ribosomal subunit formation

• It plays a crucial role in pre-rRNA processing, specifically in the removal of ITS2 spacer from 27SB pre-rRNA

• It functions in the recruitment of factors required for 27SB pre-rRNA processing (Nsa2 and Nog2)

• It has extraribosomal functions, including regulation of the Mdm2-p53-MdmX network

• Orthologs have been identified across multiple species, including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken

What applications are RPL37 antibodies commonly used for?

RPL37 antibodies can be employed in multiple experimental approaches:

Based on commercial antibody information, Western Blot appears to be the most widely validated application, with many antibodies also tested for ELISA, ICC/IF, and IHC applications .

How do I select the most appropriate RPL37 antibody for my research?

Selection should be based on several key factors:

• Species reactivity: Most commercial antibodies recognize human RPL37, with many cross-reacting with mouse and rat orthologs. For other species like Chlamydomonas, specialized antibodies are available .

• Epitope location: Consider whether the antibody targets a region that might be masked when RPL37 is incorporated into ribosomes or complexed with other proteins. Antibodies against different epitopes include:

  • N-terminal region

  • C-terminal region (common in commercial offerings)

  • Middle region antibodies (available from some suppliers)

• Validated applications: Ensure the antibody has been tested for your specific application with supporting data:

  • Western blot validation should show a distinct band at ~11 kDa

  • ICC/IF validation should demonstrate appropriate subcellular localization

  • IHC validation should show expected tissue distribution patterns

• Clonality: Consider polyclonal for higher sensitivity or monoclonal for higher specificity. Most available RPL37 antibodies are rabbit polyclonals .

• Conjugation options: If needed for specific applications, antibodies conjugated with biotin, FITC, HRP, or fluorescent dyes are available from suppliers like G Biosciences .

What optimization steps are necessary for Western blot detection of RPL37?

Given RPL37's small size (11.1 kDa) and potential for cross-reactivity with similar ribosomal proteins, several optimization steps are critical:

• Gel percentage: Use 15% SDS-PAGE gels for optimal resolution of small proteins

• Protein loading: Load 30 μg of total protein from whole cell lysates for optimal detection

• Transfer conditions:

  • Use nitrocellulose membranes for better binding of small proteins

  • Consider wet transfer methods rather than semi-dry for more efficient transfer of small proteins

• Blocking conditions:

  • 5% non-fat dry milk in TBS-T for 1 hour at room temperature

  • Some antibodies may perform better with BSA-based blocking buffers

• Antibody dilution: Typically 1:1000-1:10,000, but optimize based on signal-to-noise ratio

• Positive controls:

  • HEK-293T and HeLa cell lysates are commonly used as positive controls for human RPL37

  • For mouse studies, consider mouse tissue lysates with high ribosome content (liver, kidney)

• Detection system:

  • ECL-based detection systems work well for most applications

  • Fluorescent secondary antibodies (IRDye systems) offer quantitative advantages

How can RPL37 antibodies be used to study the MDM2-p53-MdmX regulatory network?

RPL37 has been identified as one of several ribosomal proteins that can bind to MDM2 and inhibit its E3 ligase activity, leading to p53 stabilization and cell cycle arrest . This represents an important ribosome biogenesis surveillance pathway.

For studying this regulatory network, consider these methodological approaches:

• Co-immunoprecipitation assays:

  • Immunoprecipitate RPL37 and probe for MDM2, p53, and MdmX in the precipitate

  • Use cell lysates prepared with buffers containing 25 mM Tris-HCl pH 7.5, 137 mM NaCl, 2.7 mM KCl, and 0.5% Igepal CA-630 with protease inhibitors

  • Include RNase treatment controls to determine if interactions are RNA-dependent

• Ubiquitination assays:

  • Transfect cells with HA-Ubiquitin, p53, Flag-MDM2, and Myc-RPL37

  • Treat cells with proteasome inhibitor MG132 (25 μM for 6 hours)

  • Immunoprecipitate p53 or MDM2 and detect ubiquitinated species using anti-HA antibody

• Protein stability analysis:

  • Perform cycloheximide chase assays (100 μg/mL cycloheximide) in cells overexpressing RPL37

  • Harvest cells at various time points and quantify p53 levels by Western blot

  • Calculate protein half-life using exponential decay modeling

• Transcriptional activity assessment:

  • Analyze p53 target gene expression via qRT-PCR after RPL37 overexpression or depletion

  • Design primers for p53 target genes and normalize expression to housekeeping genes like GAPDH

  • Use the ΔΔCt method to determine relative gene expression changes

• Cell cycle analysis:

  • Transfect cells with RPL37 expression constructs

  • After 24 hours, fix cells and stain with propidium iodide

  • Analyze cell cycle distribution by flow cytometry

Interestingly, while RPL37, RPS15, and RPS20 can all regulate the MDM2-p53-MdmX network, research has shown they employ different mechanisms to do so, resulting in distinct patterns of p53 target gene regulation .

What are the key considerations when studying RPL37's role in ribosome biogenesis?

RPL37 plays a critical role in ribosome biogenesis, particularly in pre-rRNA processing. When investigating this function:

• Pre-ribosomal particle analysis:

  • Use GFP-tagged RPL37 for immunoprecipitation of pre-ribosomal complexes

  • Extract RNA from purified pre-ribosomes and analyze pre-rRNA species by northern blotting

  • Design oligonucleotide probes complementary to specific regions of pre-rRNAs

• Depletion studies:

  • In yeast, both RPL37A (YLR185W) and RPL37B (YDR500C) genes must be considered

  • Use conditional depletion systems (e.g., GAL1 promoter control) rather than direct deletion if the gene is essential

  • Monitor pre-rRNA processing by northern blotting and primer extension analysis

• Localization studies:

  • Track RPL37 localization during normal growth and under ribosomal stress

  • Compare localization patterns with other assembly factors and ribosomal proteins

  • Use nucleolar markers (e.g., nucleolin, fibrillarin) as references

• Functional complementation:

  • Test if human RPL37 can complement yeast rpl37 mutants

  • Create point mutations in zinc finger motifs to assess structural requirements

  • Express truncated versions to map functional domains

• Interaction partner identification:

  • Identify RPL37-interacting proteins specific to ribosome assembly

  • Focus on interactions with pre-rRNA processing factors like Nsa2 and Nog2

  • Distinguish between stable and transient interactions using different crosslinking approaches

Research has shown that depletion of RPL37 in yeast leads to defects in ITS2 processing from 27SB pre-rRNA, indicating its essential role in 60S ribosomal subunit biogenesis .

How can RPL37 antibodies be used in multiplexed imaging applications?

Multiplexed imaging with RPL37 antibodies requires careful optimization to achieve specific detection alongside other markers:

• Antibody selection considerations:

  • Choose antibodies raised in different host species to enable simultaneous detection

  • Verify minimal cross-reactivity between secondary antibodies

  • Test antibodies individually before combining to establish optimal working dilutions

• Recommended protocol for multiplexed immunofluorescence:

  • Fixation: 4% paraformaldehyde for 15 minutes at room temperature

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

  • Blocking: 1% BSA + 10% normal serum from secondary antibody host species

  • Primary antibody incubation: Anti-RPL37 (1:500) with other primary antibodies overnight at 4°C

  • Secondary antibody selection: Use fluorophores with minimal spectral overlap

  • Sequential detection may be necessary if antibodies are from the same host

• Marker combinations for studying ribosome biology:

  • RPL37 + nucleolar markers (nucleolin, fibrillarin): Assess RPL37 incorporation into pre-ribosomes

  • RPL37 + MDM2 + p53: Investigate stress response pathways

  • RPL37 + translation initiation factors: Study translation regulation

• Advanced multiplexing techniques:

  • Cyclic immunofluorescence for detecting >5 markers in the same sample

  • Mass cytometry for highly multiplexed tissue imaging

  • Proximity ligation assays to visualize RPL37 interactions with specific partners

• Imaging platforms:

  • Confocal microscopy for subcellular localization

  • Super-resolution microscopy (STED, STORM) for detailed structural analysis

  • Automated high-content imaging for quantitative analysis of large sample sets

Sample preparation and quality are critical for successful multiplexed imaging. HeLa cells have been successfully used for immunofluorescence detection of RPL37, showing predominantly cytoplasmic localization with nuclear/nucleolar enrichment .

How do you distinguish between RPL37 and its paralog RPL37A in experimental settings?

RPL37 and RPL37A are distinct ribosomal proteins with separate functions, despite similar nomenclature. Discriminating between them requires specific strategies:

Methodological approaches to ensure specificity:

• Antibody validation:

  • Test antibodies against recombinant RPL37 and RPL37A proteins

  • Perform peptide competition assays with specific peptides from each protein

  • Verify antibody specificity in cells with siRNA knockdown of either RPL37 or RPL37A

• PCR-based discrimination:

  • Design primers specific to unique regions of each transcript

  • Use qRT-PCR to quantify expression levels of each paralog

  • Sequence amplicons to confirm identity

• Protein analysis:

  • Use high-resolution gel systems (15-20% acrylamide) to separate based on size differences

  • Consider 2D gel electrophoresis to separate based on both size and charge

  • Perform mass spectrometry on immunoprecipitated samples to identify unique peptides

• Functional distinction:

  • RPL37 has been specifically implicated in MDM2-p53 regulation

  • RPL37A functions may be more restricted to ribosome structure

  • Design experiments to test each protein's specific role

While both are components of the large ribosomal subunit, they occupy distinct positions and likely have non-overlapping functions in ribosome assembly and function .

What methodological approaches are required for studying post-translational modifications of RPL37?

Investigating post-translational modifications (PTMs) of RPL37 requires specialized techniques:

• Modification-specific antibody development:

  • Generate antibodies against specific modified peptides (phosphorylated, ubiquitinated, etc.)

  • Validate specificity using in vitro modified recombinant RPL37

  • Test against cell lysates treated with modification-inducing or -inhibiting agents

• Enrichment strategies for PTM detection:

  • Immunoprecipitate RPL37 from large-scale cultures

  • Enrich for specific modifications:

    • Phosphopeptides: TiO₂ or IMAC chromatography

    • Ubiquitinated peptides: Anti-diGly antibodies

    • Acetylated peptides: Anti-acetyllysine antibodies

• Mass spectrometry analysis:

  • Perform tryptic digestion of purified RPL37

  • Use LC-MS/MS with high resolution and mass accuracy

  • Search for both known and unexpected modifications

  • Quantify modification stoichiometry using labeled standards

• Functional studies of modifications:

  • Generate modification-mimicking mutants (e.g., S→D for phosphorylation)

  • Create modification-resistant mutants (e.g., K→R for ubiquitination/acetylation)

  • Assess impact on RPL37's role in ribosome assembly and p53 regulation

  • Determine effect on RPL37-MDM2 interaction

• Dynamic regulation of modifications:

  • Study changes in modification patterns during cell cycle

  • Investigate stress-induced modification changes

  • Examine modification differences between free and ribosome-incorporated RPL37

When analyzing PTMs of ribosomal proteins like RPL37, it's essential to ensure complete coverage of the protein sequence and to distinguish between modifications that occur before versus after ribosome incorporation.

What are common challenges in RPL37 antibody-based experiments and how can they be addressed?

Researchers often encounter several challenges when working with RPL37 antibodies:

• Cross-reactivity with other ribosomal proteins:

  • Solution: Validate antibody specificity using RPL37 knockdown controls

  • Pre-absorb antibodies with recombinant proteins of potential cross-reactants

  • Use antibodies targeting unique epitopes of RPL37

• High background in immunostaining:

  • Solution: Increase blocking time/concentration (5% BSA for 2 hours)

  • Optimize antibody concentration using titration experiments

  • Include additional washing steps with higher salt concentration

  • For tissues, use specialized blocking reagents to reduce endogenous biotin/peroxidase activity

• Multiple bands in Western blot:

  • Solution: Optimize sample preparation to minimize protein degradation

  • Adjust gel percentage (15% SDS-PAGE recommended)

  • Verify antibody specificity using recombinant RPL37

  • Test different extraction methods to ensure complete denaturation of complexes

• Weak signal in immunoprecipitation:

  • Solution: Use gentler lysis buffers to preserve native protein conformation

  • Cross-link antibodies to beads to prevent heavy chain contamination

  • Increase antibody and lysate amounts

  • Try different antibody clones that recognize exposed epitopes

• Inconsistent results across different cell types:

  • Solution: Optimize fixation and permeabilization conditions for each cell type

  • Adjust antibody concentration based on RPL37 expression levels

  • Consider cell type-specific RPL37 interaction partners that might mask epitopes

  • Validate antibody performance in each new cell line

• Detection in nucleolus:

  • Solution: Optimize nuclear permeabilization (0.5% Triton X-100 for 15 minutes)

  • Use specialized fixatives that better preserve nucleolar structure

  • Consider pre-extraction methods to remove cytoplasmic ribosomes

  • Use higher antibody concentrations for detecting nucleolar RPL37

Each antibody may require specific optimization based on the application, cell type, and experimental conditions.

What controls are essential when working with RPL37 antibodies?

Proper controls are crucial for ensuring reliable results in RPL37 antibody-based experiments:

• Positive controls:

  • Cell lines with confirmed RPL37 expression (HEK-293T, HeLa)

  • Recombinant RPL37 protein for Western blots

  • Tissues with high ribosome content (liver, pancreas)

• Negative controls:

  • siRNA/shRNA knockdown of RPL37

  • Secondary antibody-only controls for immunostaining

  • Isotype control antibodies of the same species/class

  • Peptide competition assays using the immunizing peptide

• Specificity controls:

  • RPL37A-specific antibodies to confirm differential detection

  • Cells expressing tagged RPL37 (verify co-localization)

  • Comparison of multiple antibodies targeting different epitopes

• Application-specific controls:

  • For Western blot: Loading controls (β-actin, GAPDH)

  • For IF/ICC: Counterstains for cellular compartments (DAPI for nucleus, phalloidin for cytoskeleton)

  • For IP: IgG control immunoprecipitations

  • For qPCR: No-RT controls, reference gene controls

• Experimental manipulation controls:

  • Ribosomal stress induction (low dose actinomycin D)

  • Proteasome inhibition (MG132) for ubiquitination studies

  • Protein synthesis inhibition (cycloheximide) for half-life studies

• Cross-species controls:

  • Test antibodies on samples from multiple species if cross-reactivity is claimed

  • Include species-specific positive controls when testing new applications

Documenting and reporting these controls increases the reliability and reproducibility of research findings involving RPL37.

How are RPL37 antibodies being used in cancer research?

RPL37's involvement in the MDM2-p53 pathway makes it particularly relevant to cancer research:

• Expression analysis in tumors:

  • IHC studies comparing RPL37 levels between normal and tumor tissues

  • Correlation of expression with clinical outcomes and p53 status

  • Analysis of RPL37 alterations across cancer types using tissue microarrays

• Therapeutic targeting potential:

  • Investigating small molecules that might modulate RPL37-MDM2 interaction

  • Exploring synthetic lethal interactions in p53-deficient cancers

  • Studying RPL37's role in response to ribosome-targeting cancer therapies

• Biomarker development:

  • Assessing RPL37 as a prognostic or predictive biomarker

  • Developing immunoassays for detecting RPL37 in patient samples

  • Examining RPL37 modifications as cancer-specific markers

• Mechanistic studies:

  • Investigating cancer-specific alterations in RPL37-dependent p53 regulation

  • Studying RPL37's role in cancer cell response to nucleolar stress

  • Examining changes in ribosome composition and function in cancer cells

• Translational regulation:

  • Analyzing how altered RPL37 affects translation of specific mRNAs in cancer

  • Investigating specialized ribosomes in different cancer types

  • Exploring potential alterations in the translation of cancer-relevant mRNAs

Recent advances in antibody-based detection methods, including multiplexed immunofluorescence and spatial proteomics, are enhancing our understanding of RPL37's role in cancer biology.

What developments are occurring in antibody technology for ribosomal protein research?

Several technological advances are improving ribosomal protein antibody research:

• Single-domain antibodies and nanobodies:

  • Smaller size enables access to cryptic epitopes in assembled ribosomes

  • Greater stability for in vivo applications

  • Potential for intracellular expression to track ribosome dynamics

• Active learning approaches for antibody development:

  • Machine learning models predicting antibody-antigen binding

  • Library-on-library screening approaches for identifying optimal antibody-antigen pairs

  • Out-of-distribution prediction methods to reduce experimental testing requirements

• Site-specific conjugation strategies:

  • Precisely positioned fluorophores or affinity tags

  • Orientation-controlled immobilization for better sensitivity

  • Reduced impact on antibody binding properties

• Proximity labeling applications:

  • Antibody-mediated targeting of enzymes (BioID, APEX) to specific ribosome populations

  • Mapping the local interactome of ribosomal proteins in different cellular contexts

  • Identifying transient interactions during ribosome assembly or stress response

• Multiplex detection systems:

  • Simultaneous detection of multiple ribosomal proteins

  • Combined protein-RNA detection methods

  • Spatial transcriptomics integration with antibody-based protein detection

• Ribosome-specific recombinant antibodies:

  • In vitro selection of antibodies against native ribosomal complexes

  • Conformation-specific antibodies recognizing assembly intermediates

  • Antibodies distinguishing between different functional states of ribosomes

Recent research has demonstrated that active learning strategies can reduce the number of experiments needed to develop effective antibodies by up to 35%, potentially accelerating ribosomal protein research .

What are emerging research questions about RPL37 that antibody-based approaches could help address?

Several frontier research areas could benefit from advanced antibody-based approaches:

• Extraribosomal functions:

  • Is RPL37 involved in other cellular processes beyond the MDM2-p53 pathway?

  • Does RPL37 have chromatin-associated functions like some other ribosomal proteins?

  • Are there tissue-specific roles for RPL37 outside of ribosome biogenesis?

• Stress response mechanisms:

  • How does RPL37 localization and modification change under different cellular stresses?

  • Is RPL37 involved in stress granule formation or regulation?

  • Does RPL37 participate in non-canonical translation during stress?

• Specialized ribosomes:

  • Is RPL37 incorporation regulated to create specialized ribosomes for specific mRNAs?

  • Does RPL37 content vary across different tissues or developmental stages?

  • How might RPL37 variants affect ribosome function?

• Post-translational regulation:

  • What is the complete map of RPL37 modifications?

  • How do these modifications change during development or disease?

  • Which enzymes regulate RPL37 modifications?

• Evolutionary considerations:

  • How has RPL37 function evolved across species?

  • Are there species-specific interactions or regulations?

  • What can we learn from comparing RPL37 to bacterial homologs?

Antibodies specifically designed to recognize different forms, modifications, and interaction states of RPL37 will be essential tools for addressing these questions.

How might single-cell approaches using RPL37 antibodies advance ribosome biology?

Single-cell technologies combined with RPL37 antibodies could revolutionize our understanding of ribosome heterogeneity:

• Single-cell proteomics:

  • Quantify RPL37 levels across individual cells in tissues

  • Correlate with other ribosomal proteins to identify specialized ribosomes

  • Link ribosome composition to cell state or function

• Spatial transcriptomics integration:

  • Combine RPL37 antibody staining with RNA sequencing

  • Map ribosome distribution and mRNA localization simultaneously

  • Identify cell type-specific patterns of ribosome organization

• Live-cell imaging applications:

  • Track ribosome dynamics in single cells using fluorescent antibody fragments

  • Monitor stress responses at the single-cell level

  • Observe heterogeneity in ribosome biogenesis across a population

• CyTOF and imaging mass cytometry:

  • Multi-parameter analysis of ribosomal proteins and regulatory factors

  • Tissue-level mapping of ribosome composition

  • Correlation with cell cycle and differentiation markers

• Microfluidic applications:

  • High-throughput screening of RPL37 interactions

  • Single-cell western blotting for quantification

  • Droplet-based assays for RPL37 function

• CRISPR screening combined with antibody detection:

  • Identify genes affecting RPL37 localization or modification

  • Screen for factors involved in specialized ribosome formation

  • Discover new regulatory pathways controlling ribosome composition