RPL35 Antibody

What is RPL35 and why is it important in biological research?

RPL35 (Ribosomal Protein L35) is a component of the 60S ribosomal subunit with a molecular weight of approximately 15 kDa. It contributes to ribosome formation and stability, which is essential for protein synthesis, cell growth, and proliferation . Recent research has identified RPL35 as potentially important in cancer biology, particularly through its interaction with the long non-coding RNA lncNB1 and its role in N-Myc-driven oncogenesis .

What applications are RPL35 antibodies validated for?

Most commercially available RPL35 antibodies are validated for multiple applications including:

• Western Blotting (WB): Common dilution ranges from 1:200-1:3000

• Immunohistochemistry (IHC): Typically used at 1:50-1:2000 dilutions

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Typically used at 1:50-1:500 dilutions

• ELISA: Usually at higher dilutions of 1:5000-1:40000

The specific dilutions should be optimized for each experimental system to obtain optimal results .

What species reactivity can be expected from RPL35 antibodies?

Most commercially available RPL35 antibodies demonstrate reactivity with human, mouse, and rat samples . Some antibodies may also cross-react with other species including pig, zebrafish, bovine, horse, rabbit, dog, chicken, and xenopus, though this reactivity is often predicted rather than experimentally validated . Cross-reactivity can be estimated by performing BLAST analysis between the target species and the immunogen sequence .

What is the proper storage protocol for RPL35 antibodies?

For long-term storage, RPL35 antibodies should be stored at -20°C, where they typically remain stable for one year after shipment . For short-term storage and frequent use, antibodies can be stored at 4°C for up to one month . Most RPL35 antibodies are formulated in PBS with glycerol (typically 40-50%) and sometimes contain preservatives such as sodium azide (0.02%) . It is crucial to avoid repeated freeze-thaw cycles to maintain antibody activity .

How does RPL35 interact with lncNB1 and what are the implications for cancer research?

Research has demonstrated that RPL35 binds to the long non-coding RNA lncNB1 and to E2F1 RNA . RNA immunoprecipitation assays showed that an anti-RPL35 antibody efficiently pulled down RPL35 protein in BE(2)-C and Kelly cells, demonstrating this interaction . Importantly, knocking down RPL35 with siRNAs significantly reduced DEPDC1B, N-Myc, and E2F1 protein expression, as well as ERK protein phosphorylation and N-Myc protein phosphorylation at S62 . These findings suggest that the lncNB1-RPL35-DEPDC1B axis represents a potential therapeutic target in N-Myc-driven oncogenesis .

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

When using RPL35 antibodies, particularly for Western blot applications, several controls should be included:

• Positive controls: Validated samples include mouse heart tissue, HeLa cells, and mouse skeletal muscle tissue

• Negative controls: Use a control rabbit IgG in place of the primary antibody

• Peptide competition controls: Use the synthesized peptide that was used as the immunogen to confirm specificity

• siRNA knockdown controls: Cells transfected with RPL35 siRNAs can serve as negative controls to validate antibody specificity

For immunohistochemistry applications, positive controls can include rat pancreas tissue, human liver cancer tissue, human pancreas cancer tissue, human stomach cancer tissue, human thyroid cancer tissue, and mouse pancreas tissue .

How can RPL35 protein function be assessed in experimental systems?

RPL35 function can be assessed through various experimental approaches:

• Protein synthesis assays: Puromycin incorporation assays can be used after transfection with control siRNA, lncNB1 siRNAs, or RPL35 siRNAs to measure the impact on protein synthesis

• RNA binding studies: RNA immunoprecipitation assays can be performed using anti-RPL35 antibodies to identify RNA molecules that interact with RPL35

• Protein interaction studies: Co-immunoprecipitation followed by mass spectrometry can identify proteins that interact with RPL35

• Gene expression analysis: qRT-PCR can be used to measure changes in expression of genes potentially regulated by RPL35, such as DEPDC1B

What antigen retrieval methods are recommended for RPL35 immunohistochemistry?

For optimal results in immunohistochemistry applications using RPL35 antibodies, the following antigen retrieval methods are recommended:

• Primary recommendation: TE buffer at pH 9.0

• Alternative method: Citrate buffer at pH 6.0

The choice between these methods may depend on the specific tissue being examined and should be optimized for each experimental system.

How can I troubleshoot weak or absent signals in Western blots using RPL35 antibodies?

Can RPL35 antibodies be used for less common applications like ChIP or flow cytometry?

Most commercial RPL35 antibodies are not explicitly validated for chromatin immunoprecipitation (ChIP) or flow cytometry applications . Since RPL35 is primarily a cytoplasmic protein associated with ribosomes, ChIP applications would not be expected to yield meaningful results unless investigating potential non-canonical functions .

For novel applications, pilot experiments should be conducted with appropriate controls. If pursuing flow cytometry, researchers should:

• Test fixation and permeabilization conditions (since RPL35 is an intracellular protein)

• Optimize antibody concentration starting at higher concentrations than used for WB

• Include isotype controls to assess non-specific binding

• Consider using a known positive control cell line such as HeLa cells

How should I design experiments to study the role of RPL35 in cancer progression?

Based on recent findings linking RPL35 to cancer biology, particularly through the lncNB1-RPL35-DEPDC1B axis in N-Myc-driven oncogenesis , experimental designs might include:

• Expression analysis: Compare RPL35 expression levels across normal tissues and cancer samples using:

  • Western blotting with optimized RPL35 antibodies (1:200-1:1000 dilution)

  • Immunohistochemistry of tissue microarrays (1:500-1:2000 dilution)

  • qRT-PCR for mRNA expression analysis

• Functional studies:

  • siRNA or shRNA knockdown of RPL35 followed by proliferation, migration, and invasion assays

  • Analysis of downstream targets like DEPDC1B, N-Myc, and E2F1 expression

  • Protein synthesis rate measurements via puromycin incorporation assays

• Mechanistic studies:

  • RNA immunoprecipitation to identify RNA binding partners of RPL35

  • Co-immunoprecipitation to identify protein interaction partners

  • Rescue experiments to confirm specificity of phenotypes

• In vivo studies:

  • Xenograft models with RPL35 knockdown or overexpression

  • Analysis of tumor growth, invasion, and metastasis

What criteria should be used when selecting an RPL35 antibody for specific applications?

When selecting an RPL35 antibody for research, consider these key criteria:

• Target epitope: Different antibodies target different regions of RPL35:

  • C-terminal region (AA 86-115)

  • Internal region

  • Full-length protein (AA 1-123)

  Select the appropriate epitope based on research questions (e.g., if studying a specific domain or interaction)

• Validated applications: Ensure the antibody is validated for your specific application:

  • For Western blot: Check for validation images showing the expected 15 kDa band

  • For IHC: Look for validation in tissues similar to your experimental samples

  • For IF/ICC: Confirm subcellular localization patterns in relevant cell types

• Species reactivity: Confirm reactivity with your experimental model species

• Clonality: Most RPL35 antibodies are polyclonal , which may offer broader epitope recognition but potentially more background compared to monoclonal antibodies

• Validation data: Review validation images provided by manufacturers to assess specificity and sensitivity

How can I validate a newly purchased RPL35 antibody in my laboratory?

To validate a new RPL35 antibody in your laboratory setting:

• Western blot validation:

  • Run positive control samples known to express RPL35 (e.g., HeLa cells, Jurkat cells)

  • Confirm the presence of a single band at approximately 15 kDa

  • Perform peptide competition assay with the immunizing peptide

  • Include a knockdown control using RPL35 siRNA

• Immunohistochemistry validation:

  • Use tissues known to express RPL35 (e.g., rat pancreas, human liver)

  • Compare staining pattern with published literature

  • Include negative controls (primary antibody omission and isotype controls)

  • Test different antigen retrieval methods (TE buffer pH 9.0 and citrate buffer pH 6.0)

• Cross-application validation:

  • Compare results across multiple applications (e.g., WB and IHC)

  • Ensure consistent results across different experimental conditions

• Inter-laboratory validation:

  • Compare results with published literature

  • Consider inter-laboratory testing for critical applications

What is known about the role of RPL35 in the lncNB1-mediated oncogenic pathway?

Recent research has revealed that the long non-coding RNA lncNB1 promotes tumorigenesis by interacting with RPL35 . This interaction facilitates translation of E2F1 mRNA, leading to increased DEPDC1B expression . The study identified this pathway as follows:

• LncNB1 binds to RPL35 protein

• The lncNB1-RPL35 complex binds to E2F1 mRNA

• This interaction enhances E2F1 mRNA translation

• Increased E2F1 protein upregulates DEPDC1B transcription

• DEPDC1B promotes ERK protein phosphorylation

• ERK phosphorylation leads to N-Myc protein phosphorylation at S62

• Phosphorylated N-Myc has increased stability and oncogenic activity

This pathway represents a potential therapeutic target in N-Myc-driven oncogenesis, as demonstrated in neuroblastoma models .

How does RPL35 differ from other ribosomal proteins in structure and function?

While the search results don't provide comprehensive information on the unique structural and functional aspects of RPL35 compared to other ribosomal proteins, we can note that:

• RPL35 is a component of the 60S ribosomal subunit with a molecular weight of approximately 15 kDa

• It belongs to the L29P family of ribosomal proteins

• Unlike many ribosomal proteins that function solely in translation, RPL35 has been identified to have additional roles:

  • It binds specifically to lncNB1 and E2F1 mRNA

  • It appears to regulate translation of specific mRNAs rather than just general protein synthesis

  • It influences cell signaling pathways, including ERK phosphorylation

• GO annotations related to RPL35 include poly(A) RNA binding and mRNA binding, suggesting specialized RNA interaction capabilities

These extra-ribosomal functions distinguish RPL35 from many other ribosomal proteins and highlight its potential as a therapeutic target.