RPL11 Antibody Pair

What is RPL11 and why is it important in research?

RPL11 (ribosomal protein L11) is a component of the 60S ribosomal subunit and belongs to the L5P family of ribosomal proteins. It plays crucial roles in ribosome biogenesis and protein synthesis. RPL11 is significant in research because it functions as part of the 5S ribonucleoprotein particle (5S RNP) essential for large subunit formation and rRNA maturation . Additionally, RPL11 serves as a key mediator in the ribosomal stress response pathway by binding to MDM2 and activating p53, making it relevant for cancer research .

What applications are RPL11 antibody pairs typically used for?

RPL11 antibody pairs are primarily used in sandwich ELISA applications for the quantitative detection of RPL11 across multiple species. The validated pairs have been tested in humans, rats, mice, guinea pigs, bovine, horse, pig, dog, chicken, and goat samples . Beyond sandwich ELISA, individual RPL11 antibodies have applications in Western blotting, immunohistochemistry (IHC), immunofluorescence (IF), immunocytochemistry (ICC), and immunoprecipitation (IP) .

What sample types can be analyzed using RPL11 antibody pairs?

RPL11 antibody pairs have been validated for use with multiple sample types including:

• Cell lysates (from various cell lines like BxPC-3)

• Tissue homogenates (liver, lung, pancreas from human, mouse, and rat)

• Serum and plasma samples

• Recombinant protein standards

The antibody pairs must be optimized specifically for each sample type as matrix effects can influence detection sensitivity .

What are the recommended dilutions for RPL11 antibody applications?

The recommended dilutions vary by application and specific antibody:

Optimal dilutions should be determined empirically for specific sample types and experimental conditions .

How should I validate the specificity of RPL11 antibody pairs?

To validate RPL11 antibody pair specificity:

• Perform cross-reactivity testing with related ribosomal proteins

• Include knockdown/knockout controls (siRNA or CRISPR-mediated)

• Use recombinant RPL11 protein as a positive control

• Conduct epitope mapping to ensure the antibody pairs recognize distinct, non-overlapping epitopes

• Verify results using alternative detection methods (Western blot or IF)

• Confirm protein size matches the expected molecular weight (~20 kDa)

Multiple publications have validated RPL11 antibodies in knockout/knockdown systems, providing confidence in antibody specificity .

What factors affect the sensitivity of RPL11 detection in sandwich ELISA?

Several factors influence RPL11 detection sensitivity:

• Antibody pair compatibility - The capture and detection antibodies must recognize non-overlapping epitopes

• Blocking reagents - Optimize to minimize background while maintaining specific signal

• Sample preparation - Proper cell/tissue lysis ensures complete protein extraction

• Incubation conditions - Temperature and time significantly impact antigen-antibody binding kinetics

• Detection system - Enzyme-substrate combinations vary in sensitivity

• Washing steps - Insufficient washing causes high background; excessive washing can reduce signal

The reported sensitivity for optimal RPL11 antibody pairs can detect protein levels in the picogram to nanogram range .

How can I optimize a sandwich ELISA protocol for RPL11 detection?

For optimal RPL11 sandwich ELISA performance:

• Plate coating: Use the capture antibody at 0.5 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C

• Blocking: 1-2% BSA or milk protein in PBS or TBS for 1-2 hours at room temperature

• Sample dilution: Prepare a dilution series to determine the linear range

• Detection antibody: Apply at 0.34 μg/ml in blocking buffer

• Signal development: Optimize substrate incubation time to maximize signal-to-noise ratio

• Standard curve: Use recombinant RPL11 protein to establish quantitative measurements

• Controls: Include positive controls (known RPL11-positive samples) and negative controls

Always validate new lots of antibodies as performance may vary between batches .

How can RPL11 antibodies be used to study the MDM2-p53 pathway?

RPL11 antibodies are valuable tools for investigating the MDM2-p53 pathway:

• Co-immunoprecipitation: Use RPL11 antibodies to pull down and analyze MDM2-RPL11 complexes

• Immunofluorescence: Track RPL11 relocalization from nucleolus to nucleoplasm during ribosomal stress

• ChIP assays: Investigate RPL11's potential role in transcriptional regulation through p53

• Proximity ligation assays: Visualize RPL11-MDM2 interactions in situ

• Western blot: Monitor changes in RPL11, MDM2, and p53 levels during stress response

Research has revealed that RPL11 binds to MDM2 through an extensive interface involving an acidic domain and two zinc fingers in MDM2, leading to conformational changes in both proteins that are crucial for p53 activation .

What methods can detect changes in RPL11 localization during ribosomal stress?

To monitor RPL11 subcellular redistribution:

• Immunofluorescence microscopy: Use RPL11 antibodies with nucleolar markers (fibrillarin, nucleolin) for co-localization studies

• Subcellular fractionation: Separate nuclear, nucleolar, and cytoplasmic fractions followed by Western blot analysis

• Live-cell imaging: Create RPL11-fluorescent protein fusions to track dynamic changes

• Super-resolution microscopy: Achieve nanoscale resolution of RPL11 localization changes

• Chromatin immunoprecipitation: Determine if RPL11 associates with specific genomic regions

Studies have shown that during ribosomal stress, RPL11 accumulates in the nucleoplasm where it can bind MDM2, inhibiting its activity and stabilizing p53 .

How can antibody pairs be used to study the RPL11-5S rRNA-RPL5 complex?

To investigate the RPL11-5S rRNA-RPL5 complex:

• Sequential immunoprecipitation: First pull down with RPL11 antibody, then with RPL5 antibody

• RNA immunoprecipitation: Use RPL11 antibodies to precipitate the complex followed by RT-PCR for 5S rRNA

• Size exclusion chromatography: Fractionate complexes and detect components using respective antibodies

• Mass spectrometry: Identify additional components of RPL11-containing complexes

• Glycerol gradient centrifugation: Separate complexes based on size and detect RPL11 and RPL5 by Western blot

Research has shown that the RPL11-5S rRNA-RPL5 complex is critical for p53 activation during ribosomal stress and may be stabilized in the nucleoplasm upon disruption of ribosome biogenesis .

What are common pitfalls when using RPL11 antibody pairs in sandwich ELISA?

Common challenges include:

• High background signal: Often caused by insufficient blocking or washing, or cross-reactivity

  • Solution: Optimize blocking buffer composition and washing steps; test different antibody pairs

• Poor sensitivity: May result from suboptimal antibody concentrations or sample preparation

  • Solution: Titrate antibodies; ensure RPL11 is properly extracted from samples

• Non-linear standard curve: Often indicates detection range issues

  • Solution: Use narrower dilution ranges; ensure recombinant standards are properly folded

• Matrix effects: Sample components may interfere with detection

  • Solution: Develop matrix-matched standards; consider sample clean-up procedures

• Hook effect: Very high concentrations of RPL11 can cause falsely low readings

  • Solution: Test multiple sample dilutions to identify potential hook effects

How can I distinguish between free RPL11 and RPL11 in ribosomal complexes?

To differentiate free RPL11 from complex-bound forms:

• Sucrose gradient fractionation: Separate free proteins from ribosomal subunits and intact ribosomes

• Size exclusion chromatography: Differentiate based on molecular weight

• Differential centrifugation: Use increasing centrifugal forces to pellet progressively smaller complexes

• Immunoprecipitation with conformation-specific antibodies: Some antibodies may preferentially recognize free vs. bound forms

• Native PAGE: Preserve protein complexes for size-based separation prior to Western blotting

This distinction is particularly important when studying ribosomal stress, where free RPL11 accumulates and can bind MDM2 .

How should I validate RPL11 antibodies for cross-reactivity with other species?

To validate cross-species reactivity:

• Sequence alignment: Compare RPL11 sequences across target species to identify conserved epitopes

• Western blot: Test antibodies against samples from each species of interest

• Peptide competition: Confirm specificity using blocking peptides from different species

• Immunoprecipitation: Verify pull-down efficacy with mass spectrometry validation

• Positive and negative controls: Include samples known to express or lack RPL11

The high conservation of RPL11 across species (human, mouse, rat, and others) enables many antibodies to work across species, but validation is essential .

How can RPL11 antibody pairs contribute to Diamond-Blackfan anemia research?

RPL11 antibody pairs offer valuable tools for Diamond-Blackfan anemia (DBA) research:

• Diagnostic screening: Quantify RPL11 levels in patient samples to identify potential RPL11 haploinsufficiency (DBA7)

• Genotype-phenotype correlation: Compare RPL11 protein levels with clinical manifestations

• Drug screening: Monitor RPL11 levels and localization in response to potential therapeutics

• Pathway analysis: Study the relationship between RPL11 deficiency and p53 activation

• Patient stratification: Group patients based on molecular signatures for personalized treatment approaches

Research has shown that mutations in RPL11 (associated with DBA7) affect ribosome biogenesis and trigger p53-dependent cell cycle arrest, potentially explaining the erythroid-specific pathology of DBA .

What role might RPL11 antibodies play in cancer research?

RPL11 antibodies contribute significantly to cancer research by:

• Biomarker development: Assess RPL11 levels in tumors with disrupted ribosome biogenesis

• Nucleolar stress response: Study how cancer cells respond to ribosomal/nucleolar stress

• MDM2 inhibitor development: Screen compounds that mimic RPL11-MDM2 interaction

• Therapy response monitoring: Evaluate ribosomal stress pathway activation during treatment

• Structure-function studies: Investigate how RPL11 structural changes affect cancer-related pathways

The RPL11-MDM2-p53 pathway represents a potential therapeutic target, and antibodies that can detect specific conformations or modifications of RPL11 may provide insights into novel cancer treatments .

How can multidimensional analysis incorporate RPL11 antibody data with other molecular techniques?

Integrative approaches combining RPL11 antibody data with other techniques include:

• Proteogenomic analysis: Correlate RPL11 protein levels (antibody-based) with gene expression data

• Single-cell multi-omics: Combine immunofluorescence with RNA-seq at single-cell resolution

• Proximity-dependent biotinylation (BioID): Identify novel RPL11 interactors in different cellular compartments

• CRISPR screening: Correlate genetic dependencies with RPL11 protein levels and localization

• Computational modeling: Incorporate antibody-derived quantitative data into pathway models

By combining antibody-based detection with genomic, transcriptomic, and other proteomic analyses, researchers can develop a more comprehensive understanding of RPL11's roles in normal and pathological conditions.

What are the best fixation and permeabilization methods for RPL11 immunofluorescence?

Optimal fixation and permeabilization for RPL11 immunofluorescence:

• Fixation options:

  • 4% paraformaldehyde (10-15 minutes at room temperature) preserves structure while maintaining epitope accessibility

  • Methanol fixation (-20°C for 10 minutes) may better preserve nuclear antigens

• Permeabilization options:

  • 0.1-0.5% Triton X-100 (5-10 minutes) for thorough permeabilization

  • 0.1-0.5% Saponin for milder permeabilization that preserves membrane structures

• Combined protocols:

  • Fix with 4% PFA for 10 minutes, then permeabilize with 0.2% Triton X-100 for 5 minutes

  • For nucleolar proteins like RPL11, methanol fixation often provides superior results

• Antigen retrieval:

  • For tissues, use TE buffer at pH 9.0 or citrate buffer at pH 6.0 for optimal epitope exposure

Published protocols recommend optimization for each cell type and specific antibody .

What strategies exist for multiplexing RPL11 detection with other proteins?

For multiplexing RPL11 with other protein markers:

• Multi-color immunofluorescence:

  • Use antibodies from different host species

  • Apply directly conjugated antibodies with compatible fluorophores

  • Employ sequential staining protocols for same-species antibodies

• Immunohistochemical multiplexing:

  • Sequential chromogenic IHC with stripping or blocking between rounds

  • Tyramide signal amplification for spectral unmixing

• Mass cytometry (CyTOF):

  • Label antibodies with distinct metal isotopes for highly multiplexed detection

• Co-immunoprecipitation coupled with Western blot:

  • Pull down with RPL11 antibody then probe for interacting partners

• Recommended protein combinations:

  • RPL11 + MDM2 + p53 for stress response pathway

  • RPL11 + RPL5 + fibrillarin for nucleolar studies

  • RPL11 + RPS14/RPS19 for DBA research

Validated RPL11 antibodies have been successfully used in multiplexed applications with both nucleolar and stress response markers .

How should I design controls for experiments using RPL11 antibody pairs?

Essential controls for RPL11 antibody experiments:

• Positive controls:

  • Cell lines with confirmed RPL11 expression (BxPC-3, human liver tissue)

  • Recombinant RPL11 protein standards

• Negative controls:

  • RPL11 knockdown or knockout samples

  • Primary antibody omission controls

  • Isotype controls with irrelevant antibodies of the same class

• Specificity controls:

  • Peptide competition assays using the immunogen

  • Western blot verification showing a single band at ~20 kDa

• Technical controls:

  • Standard curves with recombinant protein

  • Serial dilutions to confirm linearity

  • Inter- and intra-assay calibrators for quantitative applications

• Biological context controls:

  • Ribosomal stress inducers (actinomycin D, 5-FU) to verify expected RPL11 relocalization

  • Comparisons across multiple species when using cross-reactive antibodies

Proper controls ensure reliable and reproducible results across different experimental conditions.