RPL5 Antibody

What is RPL5 and why is it important to study?

RPL5 is a component of the ribosome, a large ribonucleoprotein complex responsible for protein synthesis. As part of the 5S RNP/5S ribonucleoprotein particle, it is an essential component of the large subunit (LSU), required for its formation and the maturation of rRNAs . RPL5 is particularly significant in research because:

• It couples ribosome biogenesis to p53/TP53 activation

• Mutations in the RPL5 gene are associated with Diamond-Blackfan anemia (DBA), a form of red blood cell aplasia

• It has been identified as a potential tumor suppressor in multiple cancers including glioblastoma (11%), melanoma (28%), and breast cancer (34%)

• It plays a role in DNA damage repair pathways

What applications can RPL5 antibodies be used for?

RPL5 antibodies have been validated for multiple applications across different research contexts:

It is recommended to optimize antibody dilutions for each specific experimental system .

How do I choose the appropriate RPL5 antibody for my research?

When selecting an RPL5 antibody, consider these key factors:

• Target species compatibility: Confirm reactivity with your species of interest. Many commercial RPL5 antibodies are reactive with human and monkey samples, while some also detect mouse and rat RPL5 .

• Application suitability: Verify the antibody has been validated for your specific application (WB, IHC, IF, etc.) .

• Antibody format: Consider whether you need a polyclonal or monoclonal antibody:

  • Polyclonal antibodies (like 29092-1-AP, 15430-1-AP) offer high sensitivity but may have batch-to-batch variation

  • Monoclonal antibodies (like D5Q5X) provide higher specificity and consistency

• Immunogen region: Check if the antibody targets a relevant epitope of RPL5. For example, some antibodies target regions within amino acids 142-297 or 100 to C-terminus of human RPL5 .

What are the optimal protocols for RPL5 detection by Western blotting?

For optimal RPL5 detection by Western blotting:

• Sample preparation:

  • Extract total protein from cells or tissues using standard lysis buffers containing protease inhibitors

  • For studying ribosomal fractions, consider separating cytoplasmic and nuclear fractions

• Loading and separation:

  • Use 10-12% SDS-PAGE gels for optimal separation

  • Load 20-50 μg of total protein per lane

  • Include positive control samples (e.g., COS-7, MOLT-4, or PC-3 cell lysates)

• Transfer and detection:

  • Transfer to PVDF or nitrocellulose membranes

  • Block with 5% non-fat milk or BSA in TBST

  • Incubate with primary RPL5 antibody (typical dilution: 1:500-1:1000)

  • Expect to detect RPL5 at approximately 34 kDa

• Validation controls:

  • Include RPL5 knockdown samples as negative controls

  • Use multiple RPL5 antibodies targeting different epitopes to confirm specificity

How can I optimize immunofluorescence staining with RPL5 antibodies?

For successful immunofluorescence using RPL5 antibodies:

• Fixation and permeabilization:

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

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

  • For nuclear proteins like RPL5, ensure complete nuclear permeabilization

• Blocking and antibody incubation:

  • Block with 1-5% BSA or normal serum for 1 hour

  • Incubate with RPL5 primary antibody at 1:50-1:500 dilution

  • Optimize incubation time and temperature (typically overnight at 4°C or 1-2 hours at room temperature)

• Subcellular localization:

  • RPL5 shows both cytoplasmic and nucleolar staining patterns

  • Include nuclear counterstains (DAPI/Hoechst) and nucleolar markers for colocalization studies

  • U-251 cells have been validated for RPL5 IF detection

• Control for specificity:

  • Include siRNA knockdown controls

  • Consider peptide competition assays to confirm specificity

What are the common challenges in RPL5 immunohistochemistry and how can they be addressed?

Common challenges and solutions for RPL5 IHC include:

• Antigen retrieval issues:

  • RPL5 detection often requires heat-induced epitope retrieval

  • Use TE buffer pH 9.0 as recommended, or alternatively citrate buffer pH 6.0

  • Optimize retrieval time (typically 15-20 minutes)

• Background staining:

  • Increase blocking time/concentration (3-5% BSA or serum)

  • Optimize antibody dilution (recommended: 1:200-1:800)

  • Include appropriate negative controls (primary antibody omission, isotype controls)

• Tissue-specific considerations:

  • Human prostate cancer tissue has been validated for RPL5 IHC

  • For other tissues, perform titration experiments to determine optimal conditions

  • Include positive control tissues with known RPL5 expression

• Signal amplification:

  • For low-abundance detection, consider using amplification systems (e.g., tyramide signal amplification)

  • Adjust incubation times and temperatures based on sample type

How can RPL5 antibodies be used to study its role in cancer progression?

RPL5 has been implicated as a tumor suppressor in multiple cancers. To investigate its role:

How can I investigate RPL5's role in DNA damage repair using specific antibodies?

Recent research has implicated RPL5 in DNA double-strand break (DSB) repair. To study this function:

• DNA damage induction and recruitment studies:

  • Use laser microirradiation to induce localized DNA damage

  • Perform immunofluorescence with RPL5 antibodies (1:50-1:500 dilution) to visualize recruitment to damage sites

  • Include established DSB markers (γH2AX, 53BP1) for colocalization studies

• DNA repair pathway analysis:

  • Use neutral comet assays following RPL5 knockdown to assess DSB repair efficiency

  • Employ HR, total-EJ, SSA, and alt-EJ reporter assays in U2OS cells to determine which repair pathways are affected

  • Validate knockdown efficiency by Western blot with RPL5 antibodies

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation with RPL5 antibodies to identify repair-related binding partners

  • Include both normal and DNA damage conditions to detect damage-induced interactions

  • For IP applications, use antibodies specifically validated for immunoprecipitation

• Cell cycle dependency:

  • Synchronize cells at different cell cycle phases and analyze RPL5 recruitment to damage sites

  • Use flow cytometry with RPL5 antibodies to assess cell cycle-specific expression and modification

What approaches can be used to study RPL5 in the context of ribosome biogenesis?

To investigate RPL5's role in ribosome biogenesis:

• Polysome profiling:

  • Generate RPL5 knockdown cells and verify by Western blot

  • Perform polysome profiling to analyze ribosome assembly defects

  • Look for specific alterations in 60S subunit and 80S ribosome formation

  • Compare profiles with known ribosome biogenesis defects

• Nucleolar localization studies:

  • Use immunofluorescence with RPL5 antibodies at 1:50-1:500 dilution

  • Co-stain with nucleolar markers (fibrillarin, nucleolin)

  • Monitor changes in localization upon transcription inhibition (actinomycin D treatment)

• 5S RNP complex formation:

  • Perform immunoprecipitation of RPL5 to co-purify 5S rRNA and other components

  • Validate with Western blot and RT-qPCR

  • Study interactions with other ribosomal proteins, particularly RPL11

• Development and differentiation studies:

  • Examine RPL5 expression during embryonic development

  • In Xenopus models, RPL5 expression has been detected throughout development in the anterior neural plate

  • Use immunohistochemistry with RPL5 antibodies to study expression patterns in developing tissues

How can I validate the specificity of my RPL5 antibody?

To ensure RPL5 antibody specificity:

• Genetic validation approaches:

  • Perform siRNA knockdown of RPL5 and confirm reduced signal by Western blot

  • Use CRISPR/Cas9-mediated knockout cells as negative controls

  • Published studies have used these approaches to validate commercial RPL5 antibodies

• Multiple antibody verification:

  • Use multiple antibodies targeting different epitopes of RPL5 (e.g., N-terminal vs. C-terminal)

  • Compare staining patterns across different applications

  • Check if results are consistent with published literature

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide

  • Run parallel assays with blocked and unblocked antibody

  • Specific signal should be significantly reduced with the blocked antibody

• Cross-reactivity assessment:

  • Test the antibody in species with known sequence differences

  • Verify specificity in tissue panels to rule out non-specific binding

What are the common technical issues when working with RPL5 antibodies?

Researchers frequently encounter these challenges with RPL5 antibodies:

• High background in immunostaining:

  • Increase blocking time and concentration

  • Optimize antibody dilution (start with manufacturer's recommendation, then titrate)

  • For IHC, use appropriate quenching of endogenous peroxidases

  • For IF, include autofluorescence reduction steps

• Non-specific bands in Western blot:

  • Increase blocking time and optimize antibody concentration

  • Use freshly prepared samples with protease inhibitors

  • Consider more stringent washing conditions

  • The expected molecular weight of RPL5 is approximately 34 kDa

• Inconsistent immunoprecipitation results:

  • Optimize lysis conditions to preserve protein interactions

  • Test different antibody-to-lysate ratios

  • Consider cross-linking antibodies to beads to prevent heavy chain interference

  • Include appropriate controls (IgG, input)

• Variability between experiments:

  • Use consistent protocols and reagent lots

  • Include internal control samples across experiments

  • Document detailed experimental conditions for reproducibility

How should RPL5 antibodies be stored and handled to maintain their performance?

For optimal antibody performance:

• Storage conditions:

  • Store at -20°C according to manufacturer recommendations

  • RPL5 antibodies are typically stable for one year after shipment

  • Aliquoting is generally unnecessary for -20°C storage

  • Some products (20μl sizes) contain 0.1% BSA for stability

• Buffer composition:

  • Most commercial RPL5 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Avoid repeated freeze-thaw cycles that can degrade antibody performance

  • Do not dilute stock antibody unless preparing working aliquots

• Working dilution preparation:

  • Prepare fresh working dilutions on the day of experiment

  • Use high-quality diluents (filtered, sterile)

  • For long experiments, keep diluted antibody cold during incubation

• Performance monitoring:

  • Include positive control samples in each experiment

  • Monitor signal-to-noise ratio over time

  • If performance decreases, consider purchasing a new lot