RPL10A Antibody

What is RPL10A and what is its biological function?

RPL10A (Ribosomal Protein L10a) is a component of the large (60S) ribosomal subunit. It functions primarily as part of the massive ribonucleoprotein complex responsible for protein synthesis in cells . RPL10A is also known by several alternative names including NEDD6 (Neural precursor cell expressed developmentally down-regulated protein 6), CSA-19, and large ribosomal subunit protein uL1 .

Beyond its canonical role in translation, research suggests RPL10A may have additional functions. In plants, RPL10A has been implicated in early development processes and abscisic acid (ABA) signaling pathways . The protein exhibits high expression during germination and early developmental stages, suggesting it plays a critical role during these processes .

RPL10A is localized in multiple cellular compartments including the nucleoli, cytosol, and endoplasmic reticulum, indicating diverse cellular functions beyond protein synthesis . The calculated molecular weight of human RPL10A is approximately 25 kDa, which is consistently observed in Western blot experiments .

What types of RPL10A antibodies are commercially available for research?

Several types of RPL10A antibodies are available for research applications, primarily rabbit polyclonal antibodies with different immunogens and validated applications:

These antibodies are generated using different immunogens (synthetic peptides or recombinant fragments), which can affect their epitope recognition and performance in specific applications . When selecting an antibody, researchers should consider which applications have been validated for their experimental system.

What are the most common applications for RPL10A antibodies in research?

RPL10A antibodies are utilized in multiple research applications with varying dilution recommendations:

Western blotting is among the most frequently used applications, with RPL10A antibodies detecting the protein in various human cell lines including HeLa, HEK-293T, Jurkat, and HepG2, as well as in tissue samples from brain and liver . Immunofluorescence studies have revealed the subcellular localization of RPL10A in nucleoli, cytosol, and endoplasmic reticulum .

How should I choose the appropriate RPL10A antibody for my specific application?

Selecting the optimal RPL10A antibody requires careful consideration of multiple factors:

• Experimental application: Different antibodies are validated for specific applications. For example, ab187998 is validated for IHC-P and ICC/IF, while ab226381 is validated for IP and WB .

• Species reactivity: Ensure the antibody recognizes RPL10A in your experimental organism. Most commercially available RPL10A antibodies react with human and mouse samples, but cross-reactivity with other species varies .

• Immunogen region: Antibodies generated against different regions of RPL10A may perform differently. For instance, ab187998 targets aa 100-200, while ab226381 recognizes aa 50-100 .

• Published validation: Review publications that have used the antibody for your specific application. For example, antibody 16681-1-AP has been cited in multiple publications for WB, IF, FC, and CoIP applications .

• Subcellular localization studies: If studying RPL10A localization, choose antibodies validated for IF/ICC with demonstrated subcellular detection capabilities. ab187998 has been shown to detect RPL10A in nucleoli, cytosol, and endoplasmic reticulum .

When possible, perform pilot experiments with small quantities of different antibodies to determine which performs best in your experimental system before committing to larger studies.

What are the recommended protocols for using RPL10A antibodies in Western blot experiments?

For successful Western blot detection of RPL10A, follow these methodological guidelines:

• Sample preparation:

  • Use NETN lysis buffer for efficient protein extraction

  • Load approximately 50 μg of total protein lysate per lane

  • Include positive control samples (HeLa, HEK-293T, or Jurkat cell lysates)

• Gel electrophoresis and transfer:

  • Use standard SDS-PAGE conditions for a protein of ~25 kDa

  • Ensure complete transfer to membrane (PVDF or nitrocellulose)

• Antibody incubation:

  • For ab226381: Use at 0.1 μg/mL concentration

  • For 16681-1-AP: Dilute 1:1000-1:4000 in blocking buffer

  • Incubate with primary antibody overnight at 4°C for optimal results

• Expected results:

  • RPL10A appears as a single band at approximately 25 kDa

  • Signal intensity may vary across different cell types and tissues

• Controls and validation:

  • Include positive control samples (HeLa cells show consistent RPL10A expression)

  • Consider including RPL10A knockdown/knockout samples as negative controls if available

This protocol consistently detects RPL10A in human cell lines (HeLa, HEK-293T, Jurkat, HepG2) and tissue samples (brain, liver) .

What are the optimal conditions for immunofluorescence experiments with RPL10A antibodies?

For optimal immunofluorescence detection of RPL10A, follow these protocol recommendations:

• Cell preparation:

  • Culture cells on coverslips or chamber slides

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

• Antibody incubation:

  • For ab187998: Use at 2 μg/ml concentration

  • For 16681-1-AP: Dilute 1:10-1:100 in blocking buffer

  • Incubate with primary antibody for 1-2 hours at room temperature or overnight at 4°C

• Expected localization patterns:

  • Nuclear/nucleolar localization (prominent)

  • Cytosolic distribution

  • Endoplasmic reticulum association

• Model cell lines:

  • U-2 OS cells show strong nucleolar and cytosolic RPL10A staining

  • HepG2 cells are also validated for RPL10A immunofluorescence

• Advanced applications:

  • Co-staining with markers for nucleoli (e.g., fibrillarin), endoplasmic reticulum (e.g., calnexin), or other ribosomal proteins can provide contextual information

  • In plant cells, RPL10A has been localized in guard cells, providing insights into its role in ABA responses

These protocols have been successfully used to demonstrate the subcellular distribution of RPL10A in various cell types, revealing its localization pattern consistent with its multiple cellular functions .

How can I perform successful immunoprecipitation using RPL10A antibodies?

For effective immunoprecipitation of RPL10A and associated proteins, follow these methodological guidelines:

• Sample preparation:

  • Prepare cell lysates using NETN buffer or other non-denaturing lysis buffers

  • Clear lysates by centrifugation at 12,000g for 10 minutes at 4°C

  • Use fresh lysates for optimal results

• Antibody amounts:

  • For 16681-1-AP: Use 0.5-4.0 μg antibody per 1-3 mg of total protein lysate

  • For ab226381: Follow manufacturer's recommended amounts for IP applications

• IP procedure:

  • Pre-clear lysate with protein A/G beads if background is a concern

  • Incubate cleared lysate with antibody for 2-4 hours or overnight at 4°C

  • Add protein A/G beads and incubate for additional 1-2 hours

  • Wash beads 3-5 times with cold lysis buffer

  • Elute with SDS sample buffer for Western blot analysis

• Known interacting partners:

  • RPL10A has been shown to interact with TGG1 and TGG2 myrosinases in co-immunoprecipitation studies

  • It also interacts with the glycine-rich protein GRP7

  • Co-IP can be used to validate these and other potential interactions

• Controls:

  • Include an isotype control antibody IP to identify non-specific binding

  • Consider using RPL10A-depleted cells as negative controls if available

This approach has been successfully used to identify RPL10A-interacting proteins in both human cell lines like HeLa and in plant systems, providing insights into its diverse cellular functions beyond ribosome assembly .

How is RPL10A involved in ABA signaling pathways in plants?

Research has revealed that RPL10A plays a significant role in abscisic acid (ABA) signaling pathways in plants:

• ABA-induced expression: RPL10A expression is induced approximately 1.6-fold by 1 μM ABA treatment, with induction observed as early as 4 hours after exposure to ABA . This induction appears to be specific to RPL10A, as other RPL10 family members (RPL10B and RPL10C) do not show altered expression upon ABA treatment .

• Transcriptional regulation: The induction of RPL10A by ABA depends on ABI3 and ABI5 transcription factors. In abi3 and abi5 mutant plants, RPL10A transcript levels do not increase in response to ABA treatment as they do in wild-type plants .

• Promoter analysis: In silico analysis of the RPL10A promoter reveals the presence of several cis-regulatory elements associated with hormone regulation, including:

  • G-box elements involved in ABA responses

  • Binding sites for bZIP transcription factors like ABI5

  • RY elements for B3-type transcription factors like ABI3

• Guard cell function: RPL10A is localized in guard cells and affects stomatal aperture:

  • RPL10A-overexpressing plants show increased stomatal closure and reduced water loss

  • rpl10A/+ mutants exhibit decreased ABA sensitivity in guard cells and increased water loss after ABA treatment

• Protein interactions: RPL10A interacts with proteins involved in ABA signaling:

  • TGG1 and TGG2 myrosinases, which are involved in ABA signaling in guard cells

  • Predicted interactions with ATH-BTB domain proteins (BPMs) and ATHB6, a negative regulator of ABA response

These findings suggest that RPL10A could regulate ABA responses at multiple levels, potentially through transcriptional regulation, post-transcriptional processes, or by influencing the translation of key components in ABA signaling pathways .

What roles does RPL10A play in early plant development?

RPL10A has been implicated in various aspects of early plant development:

• Expression pattern: RPL10A exhibits highest expression during germination and early developmental stages, suggesting it is the main contributor to these processes among the RPL10 family members .

• Developmental significance: Homozygous rpl10a mutants are not viable, indicating that RPL10A is essential for normal development. This is in contrast to RPL10B and RPL10C, where homozygous mutants are viable .

• Heterozygous mutant phenotypes: rpl10A/+ heterozygous mutant plants show:

  • Decreased sensitivity to ABA during germination and early development

  • Altered water loss regulation

  • Changes in stomatal aperture response to ABA

• Overexpression effects: Plants overexpressing RPL10A demonstrate:

  • Enhanced sensitivity to ABA

  • Increased stomatal closure under light conditions

  • Reduced water loss from leaves and rosettes

• Genetic interactions: The expression of other RPL10 family members (RPL10B and RPL10C) is not altered in rpl10A/+ mutants, suggesting there is no functional compensation within this gene family during development .

These findings highlight the critical and non-redundant role of RPL10A in plant development, particularly during early developmental stages and in mediating ABA responses that affect growth and water conservation .

What protein-protein interactions have been identified for RPL10A?

Several protein-protein interactions involving RPL10A have been identified, providing insights into its functions beyond ribosome assembly:

• Experimentally validated interactions:

  • TGG1 and TGG2 myrosinases: Co-immunoprecipitation studies have shown that RPL10A interacts with these proteins, which are involved in ABA signaling in guard cells

  • Glycine-rich protein GRP7: RPL10A co-immunoprecipitates with this protein involved in mRNA stability

• Predicted interactions from protein-protein interaction networks:

  • ATH-BTB domain proteins (BPMs): These proteins regulate stomatal closure

  • ATHB6: A class I homeobox-leucine zipper transcription factor that functions as a negative regulator of ABA response

  • RNA-binding proteins: Involved in exon junction and RNA processing

• Ribosomal and translational interactions:

  • As a component of the large ribosomal subunit, RPL10A interacts with other ribosomal proteins and ribosomal RNA

  • It may have specific roles in selective translation of certain mRNAs during stress responses

• Methodology for studying interactions:

  • Co-immunoprecipitation with antibodies like 16681-1-AP has been used successfully to identify RPL10A-interacting proteins

  • Proximity-dependent biotin identification (BioID) and yeast two-hybrid systems can provide additional insights into RPL10A interaction networks

These interactions suggest that RPL10A may function through multiple mechanisms, including direct protein-protein interactions that affect signaling pathways and interactions with RNA-binding proteins that influence post-transcriptional regulation .

How does RPL10A expression change under different experimental conditions?

RPL10A expression exhibits dynamic changes in response to various experimental conditions:

• ABA treatment responses:

  • In wild-type plants, 1 μM ABA induces RPL10A expression approximately 1.6-fold

  • Induction is observed as early as 4 hours after exposure to 5, 10, or 20 μM ABA

  • No significant differences in induction levels are observed between 10 and 20 μM ABA treatments

• Time-dependent expression changes:

  • RPL10A induction is detectable at 4 hours post-ABA treatment

  • The induction persists at 24 hours post-treatment

• Genetic background effects:

  • In abi3-1 mutants, ABA induction of RPL10A is reduced compared to wild-type plants

  • In abi5-1 mutants, RPL10A is not induced by ABA treatment

  • In rpl10A/+ heterozygous mutants, basal RPL10A expression is lower than in wild-type plants, but the fold-change induction by ABA is similar (1.7-fold in mutants vs. 1.6-fold in wild-type)

• Developmental stage variations:

  • RPL10A shows highest expression during germination and early development stages

  • Expression patterns differ from other RPL10 family members (RPL10B and RPL10C)

• Cell-type specific expression:

  • In plants, RPL10A is expressed in guard cells, consistent with its role in stomatal responses to ABA

  • In human cells, RPL10A is detected in various cell types including HeLa, HEK-293T, Jurkat, and HepG2 cells

This dynamic regulation of RPL10A expression under different conditions suggests that its levels are tightly controlled to respond to specific cellular needs, particularly during stress responses and developmental transitions .

How should I interpret variations in RPL10A expression across different cell types?

When analyzing RPL10A expression across different cell types, consider these important interpretive guidelines:

• Baseline expression levels:

  • RPL10A is ubiquitously expressed as a ribosomal protein, but expression levels can vary significantly between cell types

  • In human samples, RPL10A is detected in various cell lines (HeLa, HEK-293T, Jurkat, HepG2) and tissues (brain, liver, pancreas)

  • In pancreatic tissue, RPL10A is observed in the cytoplasm of exocrine glandular cells

• Subcellular localization differences:

  • The predominant localization pattern (nucleolar, cytosolic, or ER-associated) may vary between cell types

  • In U-2 OS cells, RPL10A shows strong nucleolar localization with additional cytosolic and ER staining

  • These differences may reflect cell-type specific functions of RPL10A beyond ribosome assembly

• Quantitative analysis considerations:

  • When comparing expression levels between cell types, normalize to appropriate housekeeping genes or total protein content

  • For plant studies, ACTIN2 has been used successfully as a reference gene

  • Consider using multiple normalization controls to ensure robust quantification

• Functional implications:

  • Higher RPL10A expression may correlate with increased protein synthesis demands

  • Cell-type specific expression patterns may reflect specialized functions

  • In plants, guard cells express RPL10A, corresponding to its role in stomatal regulation

• Experimental validation:

  • Confirm expression variations using multiple techniques (e.g., WB, IF, qPCR)

  • Consider the use of knockout/knockdown approaches to assess functional significance of expression differences

These interpretive frameworks help researchers contextualize RPL10A expression patterns and develop hypotheses about its cell-type specific functions .

What are important controls to include in RPL10A antibody experiments?

Robust RPL10A antibody experiments require appropriate controls to ensure result validity:

• Positive controls:

  • Cell/tissue types with known RPL10A expression:

    • HeLa, HEK-293T, Jurkat, or HepG2 cells for human studies

    • Brain, liver, or pancreatic tissue for human tissue experiments

  • Recombinant RPL10A protein or overexpression systems can serve as strong positive controls

• Negative controls:

  • Primary antibody omission to assess secondary antibody specificity

  • Isotype control antibodies to identify non-specific binding

  • RPL10A knockdown/knockout samples where available (noting that complete knockout may be lethal in some systems)

  • Pre-adsorption with immunizing peptide to demonstrate specificity

• Loading and technical controls:

  • For Western blotting: Housekeeping proteins (β-actin, GAPDH, α-tubulin)

  • For immunofluorescence: DNA staining (DAPI, Hoechst) for nuclear localization reference

  • For qPCR analysis of RPL10A expression: ACTIN2 has been validated as a reference gene in plant studies

• Application-specific controls:

  • For immunoprecipitation: IgG control to identify non-specific binding

  • For immunohistochemistry: Normal (non-diseased) tissue sections as baseline references

  • For co-localization studies: Known markers for subcellular compartments (nucleolin for nucleoli, calnexin for ER)

• Experimental validation controls:

  • Multiple antibodies targeting different epitopes of RPL10A

  • Multiple detection methods (e.g., fluorescence, chromogenic)

  • Correlation with mRNA expression data where possible

Including these controls ensures experimental robustness and facilitates accurate interpretation of RPL10A antibody data across different experimental systems .

How can I troubleshoot common issues with RPL10A antibody experiments?

When troubleshooting RPL10A antibody experiments, consider these methodological solutions for common issues:

• Western blot problems:

  • No signal detected:

    • Increase antibody concentration (try 1:1000 for 16681-1-AP or 0.2-0.5 μg/mL for ab226381)

    • Extend primary antibody incubation to overnight at 4°C

    • Use NETN lysis buffer, which has been validated for RPL10A extraction

    • Increase protein loading to 50-75 μg total protein

  • Multiple bands:

    • Use fresher antibody aliquots to avoid degradation

    • Increase blocking time/concentration

    • Consider using different antibody (ab226381 shows clean single band at 25 kDa)

• Immunofluorescence issues:

  • Weak signal:

    • Increase antibody concentration (try 5 μg/mL for ab187998)

    • Optimize fixation method (4% PFA for 15-20 minutes works well)

    • Use signal amplification systems if needed

  • High background:

    • Increase blocking time/stringency

    • Dilute antibody in fresh blocking buffer

    • Increase wash steps after antibody incubation

• Immunoprecipitation challenges:

  • Poor pull-down efficiency:

    • Increase antibody amount to 2-4 μg per sample

    • Extend incubation time to overnight at 4°C

    • Use crosslinking approaches for transient interactions

  • Non-specific binding:

    • Pre-clear lysates with protein A/G beads

    • Use more stringent wash buffers

    • Compare results with isotype control antibodies

• Tissue-specific considerations:

  • Plant tissues:

    • For ABA-treated samples, collect tissue 4-24 hours post-treatment for optimal RPL10A induction

    • Use ACTIN2 as reference gene for expression studies

  • Human tissues:

    • For pancreatic tissue, focus on exocrine glandular cells which show clear RPL10A expression

• Species cross-reactivity issues:

  • For non-human/mouse studies, perform sequence alignment to predict antibody recognition

  • Consider using antibodies raised against conserved epitopes

  • Validate with recombinant protein from the species of interest

These troubleshooting approaches address common technical challenges when working with RPL10A antibodies across different experimental systems .

How do I reconcile contradictory findings about RPL10A in my research?

When faced with contradictory findings about RPL10A, employ these analytical strategies to resolve discrepancies:

• Methodological differences assessment:

  • Antibody considerations:

    • Different antibodies target distinct epitopes (ab187998 targets aa 100-200, while ab226381 targets aa 50-100)

    • Polyclonal antibodies may recognize different isoforms or post-translationally modified variants

    • Compare antibody validation data and choose the most appropriate for your specific application

  • Experimental conditions:

    • For ABA studies, timing is critical—RPL10A induction occurs by 4 hours and persists at 24 hours

    • ABA concentration affects results (1-20 μM range has been validated)

    • Developmental stage impacts RPL10A expression and function

• Biological context evaluation:

  • Cell/tissue type differences:

    • RPL10A functions may vary between cell types (e.g., guard cells vs. other plant cells)

    • Expression patterns differ between tissues (cytoplasmic in pancreatic exocrine cells, nucleolar and cytosolic in U-2 OS cells)

  • Genetic background effects:

    • RPL10A induction by ABA depends on ABI3 and ABI5 transcription factors

    • rpl10A/+ heterozygous mutants show distinct phenotypes from wild-type plants

• Data integration approaches:

  • Multi-omics integration:

    • Combine protein expression data with transcriptomics and interactome studies

    • Consider post-translational modifications affecting RPL10A function

  • Functional validation:

    • Use gain- and loss-of-function approaches (RPL10A overexpression and knockdown)

    • Evaluate phenotypic outcomes to determine biological significance

• Technical reconciliation strategies:

  • Reproducibility assessment:

    • Replicate experiments under identical conditions

    • Standardize protocols across laboratories

  • Independent validation:

    • Use orthogonal methods to confirm findings

    • Employ multiple antibodies targeting different epitopes

• Literature contextualization:

  • Evolutionary perspective:

    • Consider functional divergence between plant and mammalian RPL10A

    • Evaluate conservation of interaction networks and regulatory mechanisms

  • Emerging research integration:

    • RNA-seq analysis of WT, mutant, and transgenic plants can help characterize regulatory networks

    • Consider newly identified RPL10A interacting partners

These analytical frameworks help reconcile apparently contradictory findings by considering methodological, biological, and technical factors that influence RPL10A research outcomes .