RPS10 Antibody

What is RPS10 and why is it important in cellular research?

RPS10 is a 165 amino acid component of the small ribosomal 40S subunit with a molecular weight of approximately 19 kDa. This protein plays a crucial role in ribosome assembly and protein synthesis, serving as an essential component in the cellular translation machinery .

RPS10 has gained significant research interest due to:

• Its role in ribosome biogenesis and translational regulation

• The discovery that it undergoes important post-translational modifications, particularly methylation at Arg158 and Arg160 residues by protein-arginine methyltransferase 5 (PRMT5)

• Its unique subcellular localization pattern, being found in both cytoplasm and nucleoli (specifically in the granular component region where ribosome assembly occurs)

• Its genetic link to Diamond-Blackfan anemia type 9 (DBA9), a congenital hypoplastic anemia characterized by macrocytic anemia, erythroblastopenia, and increased malignancy risk

Understanding RPS10 function and regulation provides valuable insights into fundamental cellular processes and disease mechanisms, making RPS10 antibodies essential tools for researchers in molecular biology, cell biology, and biomedical research.

What types of RPS10 antibodies are available for research applications?

The research landscape offers a diverse array of RPS10 antibodies optimized for different experimental applications:

Host Species and Clonality:

Application Compatibility:

Species Reactivity:

Most commercially available RPS10 antibodies demonstrate reactivity against human, mouse, and rat samples , facilitating cross-species research applications.

Conjugated Options:

For specialized applications, conjugated antibodies are available including:

• Alexa Fluor® 647 conjugated variants

• Alexa Fluor® 488 conjugated variants

• Biotin, FITC, and HRP conjugates

This diversity enables researchers to select the most appropriate RPS10 antibody based on their specific experimental design, target species, and detection method requirements.

How should I optimize Western blot protocols for specific detection of RPS10?

Western blot optimization for RPS10 detection requires careful consideration of several parameters to ensure specific and sensitive results:

Sample Preparation and Separation:

• Use appropriate lysis buffers containing protease inhibitors to prevent degradation of RPS10

• RPS10 has a calculated molecular weight of 19 kDa , requiring higher percentage gels (12-15% SDS-PAGE) for optimal separation

• Load adequate protein amounts (typically 20-40 μg of total cell lysate) for detection

Transfer Parameters:

• For smaller proteins like RPS10, use PVDF membranes with 0.2 μm pore size to prevent protein pass-through

• Optimize transfer conditions (80-100V for 1-2 hours or 30V overnight) for efficient transfer of low-molecular-weight proteins

Antibody Selection and Incubation:

• Follow specific dilution recommendations: 1:500-1:1000 for polyclonal antibody 14894-1-AP or 1:1000-1:2000 for monoclonal antibody 67609-1-Ig

• Primary antibody incubation is typically performed overnight at 4°C to enhance sensitivity while maintaining specificity

• Use 5% non-fat dry milk or BSA in TBST for blocking and antibody dilution

Controls for Validation:

• Include positive control lysates: Raji cells , HepG2 cells, Jurkat cells, K-562 cells

• Implement loading controls (β-tubulin, GAPDH) for normalization

• Consider RPS10 knockdown/knockout controls to confirm band specificity

Troubleshooting Multiple Bands:

If multiple bands appear, consider:

• Post-translational modifications: PRMT5-mediated methylation may cause band shifts

• Proteolytic degradation: Ensure complete protease inhibition during sample preparation

• Cross-reactivity: Verify specificity through knockdown experiments

• Non-specific binding: Further optimize blocking and antibody dilutions

A carefully optimized Western blot protocol enables reliable detection of RPS10, facilitating accurate analysis of its expression levels across different experimental conditions.

What are the optimal conditions for immunohistochemical detection of RPS10?

Successful immunohistochemical detection of RPS10 requires attention to several critical parameters:

Tissue Preparation and Fixation:

• Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used for RPS10 immunohistochemistry

• Optimal section thickness is typically 4-6 μm to balance signal strength and resolution

Antigen Retrieval Methods:

• Heat-mediated antigen retrieval is recommended with either:

  • TE buffer at pH 9.0 (primary recommendation)

  • Citrate buffer at pH 6.0 (alternative approach)

• Heating duration of 10-20 minutes in a pressure cooker or microwave provides optimal epitope exposure

Antibody Selection and Dilution:

• For polyclonal antibodies (e.g., 14894-1-AP), use dilutions between 1:20-1:200

• For monoclonal antibodies (e.g., EPR8545), follow manufacturer-specific recommendations

• Incubation times typically range from 1 hour at room temperature to overnight at 4°C

Detection Systems:

• Polymer-based detection systems often provide superior sensitivity and reduced background compared to traditional avidin-biotin methods

• DAB (3,3'-diaminobenzidine) is the most common chromogen for RPS10 visualization

• Hematoxylin counterstaining provides cellular context

Validation Controls:

• Positive tissue controls: Human lymphoma tissue has demonstrated positive RPS10 staining

• Negative controls: Include serial sections with primary antibody omitted and replaced with isotype-matched IgG or PBS

• Absorption controls: Pre-incubation of the antibody with immunizing peptide should eliminate specific staining

Evaluation Criteria:

• RPS10 typically shows both cytoplasmic and nucleolar staining patterns

• Assess staining intensity, distribution, and subcellular localization

• Compare staining patterns with literature reports on RPS10 distribution

Optimizing these parameters enables specific detection of RPS10 in tissue sections, supporting investigations into its expression patterns in normal and pathological conditions.

How can I use RPS10 antibodies to study subcellular localization through immunofluorescence?

RPS10 exhibits a distinctive subcellular localization pattern that can be effectively visualized using immunofluorescence techniques:

Cell Preparation and Fixation:

• Culture cells on glass coverslips or chamber slides to 60-80% confluence

• For optimal preservation of RPS10's nucleolar localization, fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Methanol fixation (-20°C for 10 minutes) may provide enhanced nuclear permeabilization for nucleolar proteins

Permeabilization and Blocking:

• Permeabilize with 0.2-0.5% Triton X-100 in PBS for 10 minutes to facilitate antibody access to nuclear and nucleolar compartments

• Block with 5% normal serum (matching secondary antibody host) or 3% BSA in PBS for 30-60 minutes to reduce background

Antibody Incubation:

• Use RPS10 antibodies validated for immunofluorescence applications at recommended dilutions:

  • For EPR8545 antibody: Follow manufacturer guidelines for optimal dilution

  • For fluorophore-conjugated antibodies: Consider direct detection to eliminate secondary antibody cross-reactivity

• Incubate primary antibody overnight at 4°C for optimal signal-to-noise ratio

Co-localization Studies:

• To confirm nucleolar localization, co-stain with established nucleolar markers:

  • Nucleophosmin/B23: A known RPS10 interaction partner concentrated in the granular component region

  • Fibrillarin: Marks the dense fibrillar component of nucleoli

  • UBF: Localizes to the fibrillar centers

• Include DAPI or Hoechst staining to visualize nuclei

Advanced Microscopy:

• Confocal microscopy provides superior resolution for nucleolar substructures compared to widefield fluorescence

• Super-resolution techniques (STED, STORM, SIM) can further resolve the precise localization within nucleolar subcompartments

• Z-stack imaging enables three-dimensional analysis of RPS10 distribution

Experimental Controls and Comparisons:

• Compare wild-type RPS10 localization with the R158K/R160K methylation mutant, which fails to concentrate in the granular component region

• Include PRMT5 knockdown experiments to evaluate how reduced methylation affects RPS10 localization

• Use GFP-tagged RPS10 constructs as complementary approaches to validate antibody staining patterns

This methodological approach enables detailed characterization of RPS10's dynamic subcellular localization and provides insights into how post-translational modifications and protein interactions influence its distribution and function.

How does post-translational methylation affect RPS10 function and research applications?

Post-translational methylation of RPS10 by protein-arginine methyltransferase 5 (PRMT5) represents a critical regulatory mechanism with profound implications for both RPS10 function and experimental considerations:

Methylation Sites and Functional Impact:

• PRMT5 catalyzes the symmetric dimethylation of RPS10 specifically at arginine residues Arg158 and Arg160

• This methylation is essential for:

  • Proper assembly of ribosomes

  • Optimal protein synthesis

  • Normal cell proliferation

  • RPS10 protein stability

Structural and Molecular Consequences:

• Methylation-deficient mutants (RPS10-R158K/R160K) demonstrate:

  • Inefficient incorporation into ribosomes

  • Increased susceptibility to proteasomal degradation

  • Weak interaction with nucleophosmin/B23

  • Failure to concentrate in the granular component region of nucleoli

Experimental Approaches to Study RPS10 Methylation:

Considerations for Antibody-Based Detection:

• Standard RPS10 antibodies may not distinguish between methylated and unmethylated forms

• Western blot may reveal subtle mobility shifts between methylated and unmethylated RPS10

• Immunoprecipitation with methylation-specific antibodies followed by RPS10 detection can assess methylation status

Disease Relevance and Therapeutic Implications:

• PRMT5 is characterized as an oncoprotein with roles in tumorigenesis

• RPS10 methylation represents a potential mechanism through which PRMT5 promotes cancer progression

• Targeting the PRMT5-RPS10 axis may offer therapeutic opportunities in cancer and ribosomopathies

Understanding RPS10 methylation provides critical insights into ribosome assembly regulation and protein synthesis control, highlighting the importance of considering post-translational modifications when interpreting RPS10 antibody-based experimental results.

What is the connection between RPS10 mutations and Diamond-Blackfan anemia?

RPS10 mutations have been directly implicated in Diamond-Blackfan anemia type 9 (DBA9), establishing a critical link between ribosomal protein dysfunction and hematological disease:

Genetic Basis:

• At least five distinct RPS10 gene mutations have been identified in individuals with Diamond-Blackfan anemia

• These mutations typically result in abnormally short, nonfunctional RPS10 protein that impairs ribosome assembly

Clinical Presentation of DBA9:

• Congenital non-regenerative hypoplastic anemia presenting in early infancy

• Moderate to severe macrocytic anemia and erythroblastopenia

• Increased risk of malignancy development

• Variable physical abnormalities affecting multiple body systems

Molecular Pathophysiology:

• Defective RPS10 leads to impaired 40S ribosomal subunit assembly

• Shortage of functioning ribosomes increases apoptosis of blood-forming cells in bone marrow

• Erythroid progenitors appear particularly sensitive to ribosomal stress

• Abnormal regulation of cell division or inappropriate apoptosis triggering contributes to associated physical abnormalities

Research Approaches for Studying RPS10-DBA Connection:

RPS10 Antibody Applications in DBA Research:

• Assessing RPS10 protein levels in patient cells

• Studying ribosome assembly defects through subcellular fractionation and Western blotting

• Investigating compensatory mechanisms in response to RPS10 deficiency

• Evaluating therapeutic approaches aimed at rescuing RPS10 function or bypassing ribosomal defects

Therapeutic Implications:

• Current treatments primarily supportive: corticosteroids, blood transfusions, hematopoietic stem cell transplantation

• Emerging research on gene therapy approaches to correct RPS10 mutations

• Potential for therapeutics targeting downstream pathways activated by ribosomal stress

The established connection between RPS10 mutations and Diamond-Blackfan anemia highlights the crucial role of ribosomal proteins in normal development and hematopoiesis, providing valuable insights into both fundamental biology and potential therapeutic strategies.

How can RPS10 antibodies be used to study ribosome assembly and protein synthesis?

RPS10 antibodies serve as powerful tools for investigating ribosome assembly and protein synthesis through multiple experimental approaches:

Ribosomal Fractionation Analysis:

• Separate ribosomal components on sucrose gradients

• Use RPS10 antibodies to track the distribution of RPS10 across fractions containing:

  • Free 40S subunits

  • 80S monosomes

  • Actively translating polysomes

• Compare distributions between normal and perturbed conditions to assess assembly defects

Immunoprecipitation-Based Approaches:

• Use RPS10 antibodies to immunoprecipitate RPS10-containing complexes

• Identify co-precipitating proteins through mass spectrometry or Western blotting

• Study interactions with known partners like nucleophosmin/B23, which is essential for RPS10 localization to nucleolar granular components

• Analyze associated RNAs to understand RPS10's role in ribosome assembly

Subcellular Localization Studies:

• Perform immunofluorescence to visualize RPS10 distribution between cytoplasm and nucleoli

• Track changes in localization during cell cycle progression or stress responses

• Investigate how mutations or post-translational modifications (particularly methylation) affect localization patterns

• Co-localize with markers of ribosome biogenesis to identify specific roles in assembly

Pulse-Chase Analysis:

• Label newly synthesized proteins with radioactive amino acids or non-radioactive analogs

• Immunoprecipitate RPS10 at various chase timepoints

• Monitor incorporation of RPS10 into ribosomal complexes over time

• Compare wild-type with methylation-deficient mutants (R158K/R160K) to assess assembly kinetics

Translational Activity Assessment:

• Combine RPS10 antibodies with techniques like polysome profiling or ribosome footprinting

• Compare global translation patterns between normal cells and those with RPS10 deficiency

• Identify specific mRNAs whose translation is particularly sensitive to RPS10 dysfunction

Advanced Microscopy Techniques:

• Use RPS10 antibodies in proximity ligation assays (PLA) to visualize interactions with assembly factors

• Apply super-resolution microscopy to precisely localize RPS10 within nucleolar subcompartments

• Perform live-cell imaging with fluorescent-tagged RPS10 to complement antibody-based approaches

Disease Model Applications:

• Compare RPS10 expression, localization, and incorporation into ribosomes between normal and Diamond-Blackfan anemia cells

• Investigate how therapeutic interventions affect RPS10 function in disease models

• Study compensatory mechanisms activated in response to RPS10 deficiency

These methodological approaches demonstrate how RPS10 antibodies can be strategically employed to gain insights into fundamental aspects of ribosome assembly and protein synthesis, as well as their dysregulation in disease states.

How should I validate the specificity of an RPS10 antibody for my research?

Rigorous validation of RPS10 antibody specificity is essential for generating reliable and reproducible research data. A comprehensive validation approach should incorporate multiple complementary strategies:

Genetic Knockdown/Knockout Approaches:

• Implement RPS10 siRNA knockdown using validated sequences:

  • 5′-GAGCTGGCAGACAAGAAT-3′

  • 5′-GAGTCATGGTGGCCAAGAA-3′

• Generate CRISPR/Cas9 knockout cell lines (when complete knockout is viable)

• Confirm specific signal reduction/loss in Western blot, immunofluorescence, or other applications

• Non-specific signals will remain unchanged in knockdown/knockout samples

Overexpression Validation:

• Express tagged RPS10 constructs (FLAG-tagged, Myc-tagged, or GFP-fusion proteins)

• Verify antibody detection of both endogenous and overexpressed protein

• The tagged protein should appear at a slightly higher molecular weight due to the tag

• Consider using the methylation-deficient mutant (R158K/R160K) as a biological control

Peptide Competition/Absorption Testing:

• Pre-incubate the RPS10 antibody with excess immunizing peptide or recombinant RPS10

• Apply the neutralized antibody in parallel with non-neutralized antibody

• Specific signals should be significantly reduced or eliminated in the neutralized sample

• This approach is particularly valuable for polyclonal antibodies like 14894-1-AP

Multi-antibody Concordance:

• Compare results using antibodies targeting different RPS10 epitopes:

  • Polyclonal antibody 14894-1-AP (targets full RPS10 fusion protein)

  • Monoclonal antibody 67609-1-Ig (targets different RPS10 fusion protein)

  • Monoclonal antibody EPR8545 (specific epitope)

• Consistent results across multiple antibodies strongly support specificity

Mass Spectrometry Verification:

• Immunoprecipitate RPS10 using the antibody of interest

• Identify precipitated proteins through mass spectrometry analysis

• Confirmation of RPS10 as a predominant hit provides strong evidence for specificity

Recombinant Protein Controls:

• Test antibody against purified recombinant RPS10 in Western blot

• Verify recognition at the correct molecular weight (19 kDa)

• Compare with other recombinant ribosomal proteins to assess cross-reactivity

Application-Specific Validation:

• For immunofluorescence: Confirm expected subcellular localization pattern (cytoplasmic and nucleolar distribution)

• For IHC: Verify staining patterns in tissues with known RPS10 expression (e.g., human lymphoma tissue)

• For Western blot: Confirm single band at ~19 kDa in positive control cells (Raji, HepG2, Jurkat)

Method-Specific Controls:

• Include isotype controls (especially for monoclonal antibodies)

• Omit primary antibody to assess secondary antibody specificity

• Include unrelated primary antibodies of the same isotype to control for non-specific binding

Thorough validation using multiple complementary approaches ensures that experimental observations genuinely reflect RPS10 biology rather than antibody artifacts, enhancing research reliability and reproducibility.

What are the most effective troubleshooting strategies for common issues with RPS10 antibodies?

When working with RPS10 antibodies, researchers may encounter several common challenges. Here are methodological approaches to address these issues:

Issue 1: Multiple Bands in Western Blot

Methodological solution: Perform band verification by either:

• RPS10 knockdown experiment to identify which band specifically decreases

• Immunoprecipitation with the RPS10 antibody followed by mass spectrometry analysis

• Pre-incubation with immunizing peptide to identify which bands disappear

Issue 2: Weak or No Signal in Western Blot

Methodological solution: Implement a systematic optimization protocol:

• Prepare a positive control lysate (Raji cells, HepG2)

• Test multiple membrane types (PVDF with 0.2 μm pore size recommended)

• Perform antibody titration to determine optimal concentration

• Extend primary antibody incubation time (overnight at 4°C)

Issue 3: High Background in Immunohistochemistry/Immunofluorescence

Methodological solution: Implement a systematic background reduction protocol:

• Test multiple blocking agents (5% BSA, normal serum, commercial blockers)

• Perform serial dilutions of primary antibody to determine optimal concentration

• Include additional washing steps with 0.1% Tween-20 in PBS

• For IHC, consider quenching endogenous peroxidase activity more thoroughly

Issue 4: Inconsistent Immunohistochemistry Results

Methodological solution: Implement a systematic optimization matrix:

• Test multiple antigen retrieval methods with varying durations

• Compare different detection systems with the same tissue sections

• Include internal positive control tissue in each experiment

• Consider automated staining systems for improved consistency

Issue 5: Difficulty Detecting Nucleolar RPS10

Methodological solution: Implement nucleolar visualization enhancement:

• Use methanol fixation (-20°C for 10 minutes) to enhance nuclear permeabilization

• Co-stain with nucleolar marker nucleophosmin/B23

• Employ confocal microscopy for improved resolution of nucleolar structures

• Consider pre-extraction protocols to reduce cytoplasmic signal

These methodological troubleshooting approaches provide systematic strategies for addressing common challenges with RPS10 antibodies across different applications, enabling researchers to generate more reliable and reproducible results.