RPL18A Antibody

Basic Research Questions

• What is RPL18A and what is its functional significance in cellular biology?

  RPL18A (Ribosomal Protein L18a) is a component of the 60S ribosomal subunit and belongs to the L18AE family of ribosomal proteins. It has a molecular weight of approximately 21-23 kDa and plays a crucial role in protein synthesis as part of the translational machinery. The protein is encoded by a gene located on chromosome 19p13.11 in humans. Beyond its structural role in ribosomes, RPL18A has been implicated in viral replication mechanisms, particularly through its interaction with the hepatitis C virus internal ribosome entry site (IRES) . This interaction suggests RPL18A may serve functions beyond basic protein synthesis. The protein is widely expressed across numerous tissue types, making it an important target for studying ribosomal biology and translational regulation.

• What applications are RPL18A antibodies commonly used for in research settings?

  RPL18A antibodies have been validated for multiple experimental applications with varying recommended dilutions:

  Application	Common Dilution Ranges	Notes
  Western Blot (WB)	1:500-1:50000	Depending on antibody sensitivity
  Immunohistochemistry (IHC)	1:20-1:4000	Often requires antigen retrieval with TE buffer pH 9.0
  ELISA	1:5000-1:20000	High sensitivity allows greater dilution
  Immunoprecipitation (IP)	0.5-4.0 μg per mg lysate	Effective for protein interaction studies
  Flow Cytometry (FACS)	1:200-1:400	For detecting cellular expression levels
  Immunocytochemistry (ICC)	1:200-1:1000	For subcellular localization studies

  When designing experiments, researchers should perform antibody titration to determine optimal working concentrations for their specific experimental conditions and sample types.

• What are the recommended storage conditions for RPL18A antibodies to maintain functionality?

  Proper storage is critical for maintaining antibody activity and specificity. Most RPL18A antibodies should be stored at -20°C for long-term preservation, where they typically remain stable for one year after shipment . For short-term storage and frequent use, 4°C is suitable for up to one month. Many commercial preparations contain 50% glycerol and 0.02% sodium azide as preservatives to prevent microbial growth and maintain stability . Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and diminished antibody performance. For antibodies received in liquid form, aliquoting into smaller volumes prior to freezing is recommended to minimize freeze-thaw cycles. Always refer to the manufacturer's specific storage recommendations, as formulations may vary between suppliers.

• What species reactivity do commercially available RPL18A antibodies typically exhibit?

  Commercial RPL18A antibodies demonstrate cross-reactivity with multiple species due to the high conservation of ribosomal proteins across evolution. Most commonly available antibodies react with:

  • Human samples (universal across most antibodies)

  • Mouse samples (common in polyclonal antibodies)

  • Rat samples (verified for many antibodies)

  Some antibodies also show predicted reactivity with additional species based on sequence homology:

  • Zebrafish (for specific antibodies with verified sequence identity)

  • Bovine (based on sequence conservation)

  When selecting an antibody for cross-species applications, researchers should verify that the immunogen sequence used for antibody generation shares high homology with the target species, or that the antibody has been experimentally validated in that species.

Advanced Research Questions

• What strategies can be employed to validate the specificity of RPL18A antibodies?

  Comprehensive validation of RPL18A antibodies is essential to ensure experimental reliability. Recommended validation strategies include:

  1. Genetic strategies: Utilize RPL18A knockdown/knockout cells as negative controls to verify antibody specificity. This approach is particularly powerful as it directly tests whether the detected signal depends on the presence of the target protein.

  2. Independent antibody verification: Compare results using multiple antibodies targeting different RPL18A epitopes. Concordant results with antibodies recognizing distinct regions (e.g., N-terminal vs. C-terminal) provide strong evidence of specificity .

  3. Orthogonal validation: Correlate antibody-based protein detection with RNA-seq data for RPL18A expression across tissues or experimental conditions. Prestige Antibodies® from Sigma-Aldrich utilize this approach for validation .

  4. Immunoprecipitation followed by mass spectrometry: Confirm that immunoprecipitated material contains RPL18A by mass spectrometry analysis, which can determine if the antibody pulls down the intended target .

  5. Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to block specific binding. This should eliminate or significantly reduce specific signal in applications like Western blot or immunohistochemistry.

• How can researchers differentiate between RPL18A and other closely related ribosomal proteins in experimental settings?

  Distinguishing RPL18A from other ribosomal proteins requires careful experimental design due to structural similarities and sequence homology within the ribosomal protein family:

  1. Epitope selection: Choose antibodies raised against unique regions of RPL18A that have minimal homology with other ribosomal proteins. Antibodies targeting amino acids 100-180 or 140-166 of human RPL18A have shown good specificity .

  2. Molecular weight verification: RPL18A has a distinct molecular weight of 21-23 kDa, which differs from other ribosomal proteins. In Western blot applications, careful calibration with molecular weight markers can help distinguish RPL18A from similar proteins .

  3. Two-dimensional electrophoresis: Separate proteins by both isoelectric point and molecular weight to further distinguish RPL18A from other ribosomal proteins with similar molecular weights.

  4. Mass spectrometry validation: For definitive identification, tryptic digestion followed by mass spectrometry analysis can provide peptide fingerprints unique to RPL18A .

  5. Parallel detection with RPL18-specific antibodies: Using antibodies specific to the related but distinct RPL18 protein (as opposed to RPL18A) can help confirm specificity through differential detection patterns .

• What are the optimal conditions for immunoprecipitation of RPL18A and its interacting partners?

  Successful immunoprecipitation of RPL18A requires careful optimization of experimental conditions:

  1. Antibody selection: Use antibodies specifically validated for immunoprecipitation. The Proteintech antibody (15751-1-AP) has been verified for this application with Jurkat cells .

  2. Lysate preparation: Prepare cell lysates in NETN buffer or other compatible buffers that maintain protein-protein interactions. For ribosomal proteins, inclusion of RNase inhibitors may be necessary if studying RNA-dependent interactions.

  3. Antibody concentration: Use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate for optimal results .

  4. Cross-linking considerations: For transient or weak interactions, consider using cross-linking reagents like formaldehyde or DSP (dithiobis[succinimidyl propionate]) before cell lysis.

  5. Washing conditions: Use stringent washing conditions to reduce non-specific binding, but be cautious not to disrupt specific interactions of interest. A series of washes with decreasing salt concentrations can help balance specificity and sensitivity.

  6. Elution strategy: For mass spectrometry applications, elution with SDS sample buffer is common. For functional studies of interacting partners, milder elution conditions using competing peptides may better preserve protein activity.

• How do monoclonal and polyclonal RPL18A antibodies compare in performance across different applications?

  The choice between monoclonal and polyclonal RPL18A antibodies significantly impacts experimental outcomes:

  Attribute	Monoclonal RPL18A Antibodies	Polyclonal RPL18A Antibodies
  Epitope Recognition	Single epitope (e.g., clone 6G6G10 targets AA 50-176)	Multiple epitopes across the protein
  Batch-to-Batch Variability	Minimal variation, highly reproducible	May show greater variation between lots
  Signal Strength	May have lower sensitivity in some applications	Often provide stronger signals due to multiple binding sites
  Background	Often cleaner background in IHC and IF	May show higher background in some applications
  Applications	Excellent for highly specific detection needs	Superior for applications requiring robust detection
  Recommended Uses	Flow cytometry, assays requiring high specificity	Western blotting, IHC of low-abundance targets

  For critical research applications, parallel validation using both monoclonal and polyclonal antibodies may provide complementary data and increase confidence in results, particularly when studying novel aspects of RPL18A biology.

• What are the key considerations when using RPL18A antibodies in virus-infected cell systems?

  When studying RPL18A in the context of viral infections, particularly in relationship to its role in viral replication mechanisms:

  1. Baseline expression analysis: Establish baseline RPL18A expression in your cell system before viral infection using validated antibodies by Western blot or immunofluorescence. RPL18A expression has been shown to change during viral infections .

  2. Time-course experiments: Monitor RPL18A expression at multiple time points post-infection to capture dynamic changes. In some viral systems, RPL18A levels may initially increase and then decrease as infection progresses.

  3. Subcellular localization: Use immunofluorescence to track potential redistribution of RPL18A during infection, as viral hijacking of ribosomal machinery may involve relocalization of ribosomal proteins.

  4. Co-immunoprecipitation studies: Employ RPL18A antibodies for co-IP studies to identify viral proteins that directly interact with RPL18A, particularly in hepatitis C virus systems where RPL18A interacts with the IRES .

  5. Controls for specificity: Include appropriate controls such as uninfected cells and cells infected with mutant viruses lacking specific binding domains to differentiate between direct and indirect effects on RPL18A.

  6. RNA-protein interaction studies: Consider RNA-immunoprecipitation (RIP) approaches using RPL18A antibodies to capture viral RNA-protein complexes and understand the functional significance of these interactions.

• What troubleshooting approaches should be employed when facing inconsistent results with RPL18A antibodies?

  When encountering variability or unexpected results with RPL18A antibodies:

  1. Antibody validation: Verify antibody specificity using positive control lysates known to express RPL18A. HeLa, Jurkat, and NIH/3T3 cells have been confirmed to express detectable levels of RPL18A .

  2. Sample preparation optimization:

     • For Western blot: Ensure complete denaturation of samples; ribosomal proteins may require stronger denaturation conditions

     • For IHC: Test multiple antigen retrieval methods—both TE buffer pH 9.0 and citrate buffer pH 6.0 have been successful for RPL18A antibodies

  3. Dilution optimization: Perform careful titration experiments. RPL18A antibodies show wide effective dilution ranges (1:500-1:50000 for WB), and optimal concentration may vary by sample type .

  4. Cross-reactivity assessment: Test for potential cross-reactivity with other ribosomal proteins by comparing observed band patterns with predicted molecular weights of similar ribosomal proteins.

  5. Fixation effects: For immunofluorescence or IHC applications, compare multiple fixation methods (paraformaldehyde, methanol, acetone) as these can dramatically affect epitope accessibility for ribosomal proteins.

  6. Buffer composition: For immunoprecipitation of RPL18A, test different lysis buffers as ribosomal proteins exist in large complexes that may require specific solubilization conditions to maintain native conformation while allowing antibody access.

• How can RPL18A antibodies be integrated into single-cell analysis approaches?

  Incorporating RPL18A antibodies into single-cell analysis requires special considerations:

  1. Flow cytometry optimization: For intracellular staining, use a fixation and permeabilization protocol optimized for nuclear/ribosomal proteins. RPL18A antibodies have been validated for flow cytometry at dilutions of 1:200-1:400 .

  2. Single-cell immunofluorescence: When studying heterogeneity of RPL18A expression across cell populations, optimize immunofluorescence protocols for quantitative imaging. This may include:

     • Standardized fixation and permeabilization

     • Careful titration of primary and secondary antibodies

     • Inclusion of reference markers for normalization

  3. Imaging mass cytometry: For multiplexed protein detection at single-cell resolution, metal-conjugated RPL18A antibodies can be integrated into imaging mass cytometry panels. This requires careful panel design to avoid signal spillover.

  4. Single-cell Western blot: For quantitative protein analysis in individual cells, RPL18A antibodies have been successfully used in microfluidic single-cell Western blot systems. This approach allows correlation of RPL18A levels with other protein markers at the single-cell level.

  5. Single B-cell technology: For developing new RPL18A-targeting antibodies, single B-cell isolation and cloning approaches can yield native antibody pairs with potentially improved specificity. This technology allows for the isolation of antibodies with the natural pairing of VH and VL chains .

• What are the current methodological advances in RPL18A antibody production and bioengineering?

  Recent technological developments have enhanced RPL18A antibody production:

  1. Recombinant antibody technology: Newer recombinant RPL18A antibodies, such as Proteintech's 83928-1-RR, offer improved batch-to-batch consistency compared to traditional polyclonal antibodies . These are generated through cloning and expression of antibody genes in controlled expression systems.

  2. Phage display libraries: This technology enables selection of high-affinity antibody fragments against specific RPL18A epitopes. Libraries of VH and VL genes expressed on phage surfaces can be screened for optimal binding properties, followed by cloning into expression vectors .

  3. Single B-cell technology: This approach uses B-cells from immunized donors to create monoclonal antibodies with naturally paired heavy and light chains, potentially improving specificity. Methods include fluorescent-activated cell sorting (FACS), micro-engraving, and fluorescent-activated droplet sorting (FADS) .

  4. Antibody fragment engineering: For applications requiring better tissue penetration or reduced immunogenicity, RPL18A antibody fragments (Fabs, scFvs) can be produced in systems with reduced post-translational modification capacity and high expression yield, such as E. coli, plant, or insect cell-based systems .

  5. Enhanced validation technology: Modern validation approaches align with International Working Group for Antibody Validation (IWGAV) standards, using genetic strategies, independent antibody verification, orthogonal validation with RNA-seq, and functional assay validation to ensure antibody specificity .