RPL35D Antibody

What is RPL35 and why is it important in research?

RPL35 (Ribosomal Protein L35) is a component of the 60S ribosomal subunit that plays a critical role in protein synthesis. It belongs to the L29P family of ribosomal proteins and is located in the cytoplasm. Beyond its canonical role in the ribosome, RPL35 has been implicated in various cellular processes, particularly in cancer biology. Recent research has identified RPL35 as a binding partner for lncNB1 (long non-coding RNA NB1), where this interaction promotes tumorigenesis in neuroblastoma by enhancing E2F1 protein synthesis and N-Myc protein stability . This expanding role of RPL35 beyond ribosomal function makes it an important target for research in molecular biology and cancer studies.

What types of RPL35 antibodies are available for research applications?

Currently, researchers have access to several RPL35 antibodies, primarily polyclonal antibodies derived from rabbit hosts. Notable examples include:

• Rabbit polyclonal anti-RPL35 antibody (catalog #A10561) that reacts with human, mouse, and rat species and is validated for Western blotting applications .

• Rabbit polyclonal anti-RPL35 antibody (ab121244) validated for immunohistochemistry on paraffin-embedded sections (IHC-P), Western blotting (WB), and immunocytochemistry/immunofluorescence (ICC/IF) applications with human samples .

These antibodies are generated against synthetic peptides derived from internal regions of human RPL35, making them suitable for detecting the native protein in various experimental contexts.

How is the specificity of RPL35 antibodies validated?

RPL35 antibodies undergo rigorous validation processes to ensure specificity and minimize cross-reactivity. The validation typically includes:

• Western blot analysis using multiple cell lines (e.g., HeLa, Jurkat, COLO, and 293 cells) to confirm the detection of the correct molecular weight band (~14.5 kDa) .

• Negative controls using non-immune IgG or isotype controls.

• Blocking peptide experiments where the immunizing peptide is used to confirm antibody specificity.

• Cross-species reactivity testing to determine the range of species where the antibody can be effectively utilized.

Researchers should review the validation data provided by manufacturers, including Western blot images showing clear bands at the expected molecular weight, to ensure the antibody's specificity for their intended application.

What are the optimal conditions for using RPL35 antibodies in Western blotting?

For Western blotting applications using RPL35 antibodies, the following protocol recommendations have shown optimal results:

• Sample preparation:

  • Total protein extraction from cells or tissues using standard lysis buffers (RIPA or NP-40 based)

  • Protein concentration determination (BCA or Bradford assay)

  • Loading 20-40 μg of total protein per lane

• Gel electrophoresis and transfer:

  • 12-15% SDS-PAGE gels (due to the small size of RPL35 ~14.5 kDa)

  • Transfer to PVDF or nitrocellulose membranes using standard transfer conditions

• Antibody incubation:

  • Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

  • Primary antibody: Anti-RPL35 at dilutions ranging from 1:500 to 1:3000 in blocking buffer, overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:5000-1:10000 for 1 hour at room temperature

• Detection:

  • Enhanced chemiluminescence (ECL) detection system

  • Expected band at approximately 14.5 kDa

Optimization of antibody dilution is recommended for each specific experimental setup and cell type.

How can RPL35 antibodies be used in immunoprecipitation to study protein interactions?

RNA immunoprecipitation (RIP) assays using RPL35 antibodies have successfully demonstrated interactions between RPL35 protein and RNA molecules, particularly in cancer research. The following methodology has been validated in neuroblastoma studies:

• Cell preparation:

  • Harvest cells at 70-80% confluence

  • Crosslink with 1% formaldehyde for 10 minutes (optional)

  • Lyse cells in RIP buffer containing protease inhibitors and RNase inhibitors

• Immunoprecipitation:

  • Pre-clear cell lysate with protein A/G beads

  • Incubate cleared lysate with anti-RPL35 antibody (5-10 μg) overnight at 4°C

  • Add protein A/G beads and incubate for 2-4 hours

  • Wash extensively with RIP wash buffer

• RNA extraction and analysis:

  • Extract RNA from immunoprecipitated complexes

  • Perform RT-PCR or RNA-seq to identify bound RNA molecules

This approach has successfully demonstrated that RPL35 binds to lncNB1 RNA and E2F1 RNA in neuroblastoma cells, and that transfection with lncNB1 siRNA reduced RPL35 protein binding to E2F1 RNA .

What controls should be included when using RPL35 antibodies in experimental setups?

To ensure the validity and reliability of results when using RPL35 antibodies, the following controls should be incorporated:

• Negative controls:

  • Isotype control (rabbit IgG) for immunoprecipitation and immunohistochemistry

  • No primary antibody control for Western blotting and immunofluorescence

  • siRNA-mediated knockdown of RPL35 as a negative control for antibody specificity

• Positive controls:

  • Cell lines known to express RPL35 (e.g., HeLa, Jurkat, COLO, and 293 cells)

  • Recombinant RPL35 protein (when available)

• Validation controls:

  • Multiple antibodies targeting different epitopes of RPL35

  • Blocking peptide competition assays

  • Mass spectrometry validation of immunoprecipitated proteins

Including these controls enables researchers to confidently interpret their results and address potential criticisms regarding antibody specificity and experimental design.

How has RPL35 antibody research contributed to understanding cancer biology?

RPL35 antibody-based research has revealed critical insights into cancer biology, particularly in neuroblastoma:

• RPL35-lncNB1 interaction mechanism:
  Research using RPL35 antibodies in RNA immunoprecipitation assays has demonstrated that RPL35 specifically binds to lncNB1 RNA in neuroblastoma cells. This interaction is crucial for enhancing E2F1 protein synthesis without affecting E2F1 mRNA levels .

• Regulatory pathway elucidation:
  Through immunoprecipitation and protein analysis with RPL35 antibodies, researchers have uncovered a regulatory pathway where:

  • LncNB1 binds to RPL35

  • This interaction promotes E2F1 protein translation

  • Increased E2F1 activates DEPDC1B transcription

  • DEPDC1B induces ERK phosphorylation

  • Phosphorylated ERK stabilizes N-Myc protein by phosphorylating it at S62

  • Stabilized N-Myc drives oncogenesis in neuroblastoma

• Prognostic significance:
  Studies employing RPL35 antibodies in tissue microarrays have revealed that high expression levels of RPL35 in neuroblastoma tissues correlate with poor patient prognosis, suggesting its potential use as a prognostic biomarker .

These findings position RPL35 and its binding partners as potential therapeutic targets for neuroblastoma and potentially other cancers where similar mechanisms may operate.

What are the challenges in detecting RPL35 in different cellular compartments?

Detecting RPL35 in different cellular compartments presents several challenges that researchers should consider:

• Ribosomal association vs. free form:
  RPL35 primarily associates with ribosomes in the cytoplasm, but it may also exist in free forms or non-ribosomal complexes. Distinguishing between these forms requires careful subcellular fractionation and immunoprecipitation protocols.

• Cross-reactivity with related proteins:
  The L29P family contains structurally similar proteins that may cross-react with RPL35 antibodies. Verification through mass spectrometry or specific knockdown experiments is recommended for definitive identification.

• Epitope accessibility issues:
  When RPL35 is incorporated into the ribosomal structure, certain epitopes may be masked or inaccessible to antibodies. Using antibodies targeting different epitopes can help overcome this limitation.

• Co-localization studies:
  For immunofluorescence applications, co-localization with known ribosomal markers (e.g., RPL7) and non-ribosomal partners (e.g., lncRNAs) requires high-quality antibodies and appropriate imaging techniques to minimize false-positive signals.

Addressing these challenges through carefully designed experiments and appropriate controls is essential for accurate interpretation of RPL35 localization and function studies.

How can RPL35 knockdown be used to validate antibody specificity and study protein function?

RPL35 knockdown experiments serve dual purposes: validating antibody specificity and investigating protein function:

• Antibody validation protocol:

  • Transfect cells with RPL35-specific siRNAs (at least two independent sequences)

  • Include non-targeting control siRNA

  • Perform Western blot analysis 48-72 hours post-transfection

  • A specific antibody should show reduced or absent signal in knockdown samples

  • Quantify knockdown efficiency through densitometry

• Functional studies revealed through knockdown experiments:
  Published research has demonstrated that siRNA-mediated knockdown of RPL35:

  • Significantly down-regulates DEPDC1B, N-Myc, and E2F1 protein expression

  • Reduces DEPDC1B mRNA expression without affecting N-Myc and E2F1 mRNA levels

  • Decreases ERK protein phosphorylation and N-Myc protein phosphorylation at S62

• Rescue experiments:

  • After confirming knockdown phenotypes, introduce siRNA-resistant RPL35 constructs

  • Verify restoration of protein expression using RPL35 antibodies

  • Confirm recovery of downstream effects (e.g., E2F1 and DEPDC1B expression)

These approaches provide robust evidence for both antibody specificity and the functional significance of RPL35 in cellular processes.

What is the cross-species reactivity profile of currently available RPL35 antibodies?

Understanding the cross-species reactivity of RPL35 antibodies is crucial for researchers working with different model organisms. Current data indicates:

When considering using these antibodies in species not explicitly validated by the manufacturer:

• BLAST analysis comparing the immunogen sequence with the target species RPL35 sequence is recommended to predict potential cross-reactivity.

• Pilot validation studies should be performed, particularly when sequence homology is high.

• For novel species applications, multiple detection methods should be employed to confirm specificity (e.g., Western blot plus immunohistochemistry or mass spectrometry).

As noted in customer inquiries, RPL35 antibody A10561 has been successfully used for rat tissue in Western blotting, and there is potential for cross-reactivity with bovine tissues based on sequence homology .

How do you optimize RPL35 antibody protocols for different tissue types?

Optimizing RPL35 antibody protocols for various tissue types requires systematic adjustment of several parameters:

• Tissue-specific protein extraction:

  • Neural tissues: Use specialized buffers containing higher detergent concentrations

  • Muscle tissues: Include additional mechanical homogenization steps

  • Liver and kidney: Consider perfusion with PBS prior to extraction to reduce blood contamination

• Antibody dilution optimization:

  • Start with the manufacturer's recommended range (e.g., 1:500-1:3000 for Western blotting)

  • Perform a dilution series to identify optimal signal-to-noise ratio for each tissue type

  • For highly expressed tissues, higher dilutions may be sufficient

  • For tissues with lower expression, lower dilutions and longer exposure times may be necessary

• Antigen retrieval for immunohistochemistry:

  • Test multiple antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0)

  • Optimize retrieval times (10-30 minutes)

  • For formalin-fixed tissues, longer retrieval times may be necessary

• Signal amplification strategies:

  • Consider biotin-streptavidin systems for low abundance detection

  • Tyramide signal amplification for immunofluorescence applications

  • Enhanced chemiluminescence substrates of varying sensitivity for Western blotting

Tissue-specific optimization should be documented systematically to ensure reproducibility across experiments.

What are the common problems encountered when using RPL35 antibodies and how can they be resolved?

Researchers working with RPL35 antibodies may encounter several challenges:

• High background in Western blots:

  • Problem: Non-specific bands or smears obscuring the target band

  • Solutions:

    • Increase blocking time and concentration (5% BSA or milk)

    • Use more stringent washing conditions (increase TBST concentration)

    • Titrate primary antibody to higher dilutions

    • Pre-adsorb antibody with cell/tissue lysate from a different species

• Weak or absent signal:

  • Problem: Insufficient detection of RPL35 despite proper technique

  • Solutions:

    • Verify protein loading with housekeeping controls

    • Reduce antibody dilution

    • Increase protein loading (40-60 μg)

    • Try enhanced detection systems

    • Consider alternative extraction methods to ensure RPL35 is not lost during processing

• Multiple bands on Western blot:

  • Problem: Detection of bands at unexpected molecular weights

  • Solutions:

    • Verify with RPL35 knockdown to identify specific band

    • Use freshly prepared samples to minimize degradation products

    • Check for post-translational modifications or isoforms

    • Consider subcellular fractionation to separate different pools of RPL35

• Cross-reactivity issues:

  • Problem: Non-specific binding to related proteins

  • Solutions:

    • Use blocking peptides to confirm specificity

    • Try alternative antibodies targeting different epitopes

    • Optimize antibody dilution and incubation conditions

Systematic troubleshooting using these approaches can significantly improve results with RPL35 antibodies.

How should researchers interpret conflicting results between different RPL35 antibodies?

When facing conflicting results between different RPL35 antibodies, a structured approach to interpretation and resolution is necessary:

• Evaluate antibody characteristics:

  • Compare the immunogens used to generate each antibody

  • Assess whether the antibodies target different epitopes

  • Review validation data provided by manufacturers

  • Consider polyclonal vs. monoclonal nature of the antibodies

• Perform validation experiments:

  • siRNA or shRNA knockdown of RPL35 to confirm specificity of each antibody

  • Overexpression studies with tagged RPL35 constructs

  • Peptide competition assays using the specific immunogens

  • Mass spectrometry identification of immunoprecipitated proteins

• Consider biological variables:

  • Different isoforms or post-translational modifications may be detected preferentially by different antibodies

  • Cell type-specific or context-dependent expression patterns

  • Experimental conditions may affect epitope accessibility

• Resolution strategies:

  • Use multiple antibodies in parallel and report all results

  • Focus on results that can be validated by orthogonal methods

  • Correlate antibody results with functional studies (e.g., knockdown phenotypes)

  • Consider developing new validation methods specific to your experimental system

Understanding the basis for conflicting results often leads to new insights about protein behavior and antibody performance.

What complementary techniques should be used alongside RPL35 antibody detection to strengthen research findings?

To strengthen and validate findings obtained with RPL35 antibodies, researchers should incorporate complementary techniques:

• Genetic manipulation approaches:

  • siRNA/shRNA knockdown of RPL35 to confirm antibody specificity and functional effects

  • CRISPR-Cas9 gene editing to generate RPL35 knockout or tagged cell lines

  • Overexpression of wild-type or mutant RPL35 to assess functional consequences

• Transcriptomic analyses:

  • RT-qPCR to measure RPL35 mRNA levels and correlate with protein expression

  • RNA-seq to identify global transcriptional changes associated with RPL35 manipulation

  • Ribosome profiling to assess impacts on translation efficiency

• Protein-protein interaction studies:

  • Co-immunoprecipitation followed by mass spectrometry to identify interaction partners

  • Proximity ligation assays to visualize protein interactions in situ

  • FRET or BiFC assays for real-time analysis of protein interactions in living cells

• Functional assays based on RPL35 biology:

  • Protein synthesis measurements using puromycin incorporation

  • Analysis of downstream signaling pathways (e.g., ERK phosphorylation)

  • Assessment of cellular phenotypes (proliferation, apoptosis, migration)

For example, research on neuroblastoma utilized RPL35 antibodies for immunoprecipitation studies, complemented by siRNA knockdown experiments and functional assays measuring E2F1 protein synthesis, demonstrating how RPL35 binding to lncNB1 promotes tumor growth .