RPL6 Antibody

What applications are RPL6 antibodies typically used for in research?

RPL6 antibodies are utilized across multiple research applications including:

• Western Blotting (WB): Typically used at dilutions of 1:2000-1:10000

• Immunohistochemistry (IHC): Recommended dilutions range from 1:50-1:500

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Used at dilutions of 1:50-1:500 for standard antibodies and 1:300-1:1200 for fluorophore-conjugated variants

• Flow Cytometry (FC): For intracellular detection, approximately 0.20 μg per 10^6 cells in a 100 μl suspension

• ELISA: For protein quantification in complex samples

These applications facilitate the investigation of RPL6 expression, localization, and interactions in various experimental contexts.

What species reactivity can be expected with commercially available RPL6 antibodies?

Most commercially available RPL6 antibodies show cross-reactivity across multiple mammalian species:

This cross-species reactivity is particularly valuable for comparative studies and model organism research, allowing consistent experimental approaches across different biological systems.

How does RPL6 participate in DNA damage response pathways?

RPL6 plays a sophisticated role in DNA damage response that extends beyond its canonical ribosomal functions. When DNA damage occurs, RPL6 is recruited to damage sites in a PARP-dependent manner, where it interacts with histone H2A . This interaction facilitates several critical downstream events:

• Promotes the interaction between MDC1 (mediator of DNA damage checkpoint 1) and γH2AX

• Facilitates the accumulation of MDC1 at DNA damage sites

• Enhances the recruitment of RNF168 (ring finger protein 168)

• Increases H2A Lys-15 ubiquitination (H2AK15ub)

These molecular events subsequently enable the recruitment of downstream repair proteins, including TP53BP1 (tumor protein P53-binding protein 1) and BRCA1 (BRCA1, DNA repair-associated) . When RPL6 is depleted, these processes are impaired, resulting in defects in DNA damage-induced G2-M checkpoint activation, compromised DNA damage repair, and reduced cell survival.

How can RPL6 antibodies be used to investigate its role in non-homologous end joining (NHEJ) and homologous recombination (HR)?

To investigate RPL6's role in NHEJ and HR repair pathways, researchers can employ RPL6 antibodies in several sophisticated experimental approaches:

• Immunofluorescence co-localization studies: Using fluorophore-conjugated RPL6 antibodies (such as CL488-67729) alongside markers for DNA damage (γH2AX) and repair factors (53BP1, BRCA1) to visualize recruitment dynamics at DNA break sites .

• Chromatin immunoprecipitation (ChIP): Utilizing RPL6 antibodies to pull down chromatin regions where RPL6 is recruited following DNA damage induction, followed by sequencing or qPCR analysis of associated DNA.

• Proximity ligation assays (PLA): Combining RPL6 antibodies with antibodies against repair factors to detect and quantify protein-protein interactions specifically at sites of DNA damage.

• siRNA knockdown verification: Confirming successful RPL6 knockdown in experiments measuring repair efficiency through reporter assays for NHEJ and HR pathways .

Research has shown that RPL6 depletion decreases the repair efficiency of both NHEJ and HR pathways, demonstrating its functional importance in these mechanisms .

What are the optimal conditions for using RPL6 antibodies in immunohistochemistry?

For optimal results in immunohistochemistry with RPL6 antibodies, researchers should consider the following methodological parameters:

• Antigen retrieval: Use TE buffer pH 9.0 as the preferred method; alternatively, citrate buffer pH 6.0 may be used but might yield different staining intensity .

• Antibody dilution: The recommended dilution range is 1:50-1:500, but this should be optimized for each specific experimental system and tissue type .

• Tissue positivity: RPL6 antibodies have been validated on human liver tissue, showing reliable detection of the target protein .

• Controls: Include both positive controls (tissues known to express RPL6, such as liver tissue) and negative controls (omitting primary antibody or using isotype control) to validate staining specificity.

• Titration: It is strongly recommended to titrate the antibody for each specific application and tissue type to obtain optimal signal-to-noise ratio .

Researchers should note that cell-type specific expression patterns may vary, and interpretation should consider the subcellular localization patterns expected for RPL6.

What is the recommended protocol for using RPL6 antibodies in western blotting experiments?

For successful western blotting with RPL6 antibodies, the following protocol parameters are recommended:

• Sample preparation:

  • Validated cell lines include HEK-293T, HeLa, HepG2, and Jurkat cells

  • Use standard lysis buffers containing protease inhibitors

  • Determine protein concentration and load 20-50 μg per lane

• Antibody dilution:

  • Primary RPL6 antibody: 1:2000-1:10000 (optimize based on antibody lot and sample type)

  • Secondary antibody: HRP-conjugated anti-rabbit at 1:5000-1:10000

• Expected molecular weight:

  • Calculated molecular weight: 33 kDa

  • Observed molecular weight: 34 kDa

• Blockers and buffers:

  • 5% non-fat milk or BSA in TBST for blocking

  • TBST for washing steps

  • Antibody diluent containing 1-5% blocker

• Incubation conditions:

  • Primary antibody: Overnight at 4°C or 2 hours at room temperature

  • Secondary antibody: 1 hour at room temperature

This protocol has been validated for detecting RPL6 in multiple cell lines and tissue types .

How should flow cytometry experiments with RPL6 antibodies be designed for intracellular detection?

When designing flow cytometry experiments for intracellular detection of RPL6, researchers should follow these methodological guidelines:

• Cell preparation:

  • Harvest cells in exponential growth phase

  • Wash cells in PBS without calcium/magnesium

  • Fix with 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilization:

  • Use 0.1% Triton X-100 or 90% methanol for permeabilization

  • Incubate for 15-30 minutes at room temperature or on ice

• Antibody concentration:

  • Use 0.20 μg of RPL6 antibody per 10^6 cells in a 100 μl suspension

  • Incubate for 30-60 minutes at room temperature

• Controls:

  • Include isotype controls to determine background staining

  • Use positive control cells (HepG2 has been validated)

  • Include a secondary antibody-only control

• Analysis considerations:

  • Set appropriate gates based on forward/side scatter

  • Use compensation controls if multiple fluorophores are employed

  • Consider RPL6's primarily cytoplasmic and nuclear expression

This approach has been validated for the detection of RPL6 in HepG2 cells and should be adaptable to other cell types with appropriate optimization .

How can researchers address non-specific binding issues with RPL6 antibodies?

When encountering non-specific binding with RPL6 antibodies, researchers can implement several strategies:

• Optimize blocking conditions:

  • Increase blocking time (1-2 hours at room temperature)

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

  • Consider adding 0.1-0.3% Triton X-100 to blocking buffer for membrane permeabilization

• Adjust antibody dilution:

  • Increase dilution factor beyond recommended ranges if background is high

  • For WB, test dilutions from 1:2000 up to 1:10000

  • For IF/ICC, test dilutions from 1:50 up to 1:500 or higher

• Modify incubation conditions:

  • Reduce incubation temperature (4°C instead of room temperature)

  • Implement more stringent washing steps (increase number and duration)

  • Consider using different buffer compositions (add 0.05-0.1% Tween-20)

• Validate antibody specificity:

  • Perform peptide competition assays with the immunizing peptide

  • Include RPL6 knockdown or knockout samples as negative controls

  • Compare staining patterns with multiple antibodies targeting different RPL6 epitopes

• Sample preparation considerations:

  • Optimize fixation conditions to preserve epitope accessibility

  • Ensure efficient permeabilization for intracellular targets

  • Use freshly prepared samples and reagents

These approaches collectively minimize background signals while preserving specific RPL6 detection.

How can researchers interpret RPL6 localization data in the context of DNA damage response?

When interpreting RPL6 localization data in DNA damage response contexts, researchers should consider several analytical factors:

• Expected localization patterns:

  • Under normal conditions: Primarily cytoplasmic with some nucleolar staining (consistent with ribosomal function)

  • After DNA damage induction: Increased nuclear localization and co-localization with DNA damage markers (γH2AX foci)

  • PARP inhibition effects: Treatment with PARP inhibitors such as olaparib should suppress RPL6 recruitment to damage sites

• Temporal dynamics:

  • Early response (minutes to hours): Recruitment to DNA damage sites

  • Late response (hours to days): Possible return to baseline distribution

  • Analyze multiple timepoints to capture complete recruitment/resolution kinetics

• Co-localization analysis:

  • Measure co-localization with H2A, MDC1, RNF168, 53BP1, and BRCA1

  • Quantify Pearson's correlation coefficient or Manders' overlap coefficient

  • Compare wild-type versus depleted conditions to establish functional relationships

• Functional correlations:

  • Connect localization patterns with functional outcomes (repair efficiency, checkpoint activation)

  • Assess how RPL6 depletion affects downstream factor recruitment (53BP1, BRCA1)

  • Correlate with cell cycle phase analysis (G2-M checkpoint activation)

• Technical considerations:

  • Use high-resolution imaging (confocal or super-resolution microscopy)

  • Implement deconvolution algorithms for improved spatial resolution

  • Perform quantitative analysis of foci formation and co-localization

This analytical framework allows researchers to distinguish between RPL6's ribosomal and DDR functions, enhancing interpretation of experimental results .

What are the key considerations when selecting an RPL6 antibody for research applications?

When selecting an RPL6 antibody for specific research applications, researchers should evaluate several critical parameters:

• Experimental application compatibility:

  • Different antibodies show optimal performance in specific applications (WB, IHC, IF, FC)

  • Choose antibodies validated for your intended application

  • Consider specialized conjugated versions for applications like flow cytometry or multiplexed IF

• Epitope specificity:

  • N-terminal targeting antibodies may detect different functional aspects than C-terminal targeting ones

  • For DNA damage response studies, consider antibodies targeting regions not involved in H2A interaction

  • The epitope location may affect detection of post-translational modifications

• Species cross-reactivity:

  • Match antibody reactivity to your experimental model organism

  • Consider predicted reactivity percentages (e.g., mouse: 87%, rat: 85%)

  • Validate antibodies when using less common model organisms

• Clonality considerations:

  • Polyclonal antibodies offer broader epitope recognition but possible batch variation

  • All currently available RPL6 antibodies appear to be polyclonal

  • Consider multiple antibody validation for critical experiments

• Technical specifications:

  • Storage conditions (-20°C, with glycerol and sodium azide)

  • Stability (typically one year after shipment)

  • Form (liquid) and purification method (antigen affinity or Protein A)

These considerations help ensure experimental success and data reliability when investigating both canonical and non-canonical functions of RPL6.

How might the extraribosomal functions of RPL6 influence experimental design and interpretation?

The emerging extraribosomal functions of RPL6, particularly in DNA damage response, significantly impact experimental approaches and data interpretation:

• Experimental context awareness:

  • DNA damaging treatments (etoposide, radiation) will alter RPL6 localization and interactions

  • PARP inhibitors (like olaparib) suppress RPL6 recruitment to damage sites

  • Cell cycle phase affects interpretation of RPL6 functions in checkpoint regulation

• Dual function experimental design:

  • Include controls distinguishing between ribosomal and DDR functions

  • Consider combined approaches (protein synthesis inhibitors plus DNA damage induction)

  • Design time-course experiments to separate immediate versus long-term responses

• Protein interaction networks:

  • RPL6-H2A interaction represents a novel nuclear function

  • Consider proximity labeling approaches to identify context-specific interaction partners

  • Investigate potential competition between ribosomal assembly and DNA damage response

• Phenotypic interpretation:

  • RPL6 depletion effects may result from either ribosomal or DNA repair defects

  • G2-M checkpoint defects following RPL6 knockdown reflect its role in DDR signaling

  • Reduced cell survival may have multiple mechanistic explanations

• Therapeutic relevance:

  • Connect findings to potential clinical applications (cancer therapy resistance)

  • Consider implications for combined treatments (PARP inhibitors plus agents affecting RPL6)

  • Explore RPL6 as a biomarker for DNA repair capacity in various cell types