RPS27 Antibody

What are the optimal applications for RPS27 antibodies in experimental research?

RPS27 antibodies can be successfully employed across multiple experimental techniques with varying recommended dilutions:

When performing immunohistochemistry, optimal results are achieved using TE buffer (pH 9.0) for antigen retrieval, though citrate buffer (pH 6.0) may serve as an alternative . For reproducible results, titration within your specific experimental system is recommended as antibody performance can be sample-dependent .

How can I confirm the specificity of my RPS27 antibody?

Confirming specificity is crucial due to the existence of a highly similar paralog, RPS27L. You should:

• Use positive and negative controls (including RPS27 knockout/knockdown samples if available)

• Validate with recombinant proteins: Previous studies have demonstrated specificity by testing antibodies against both endogenous and HA-tagged RPS27 and RPS27L

• Perform western blot analysis to confirm detection at the expected molecular weight (approximately 9 kDa)

• Consider cross-reactivity: Some commercial antibodies are specifically designed to detect only RPS27 and not RPS27L, as demonstrated in studies where "RPS27L antibody detected both endogenous and HA-tagged RPS27L, but not HA-tagged RPS27, whereas the RPS27 antibody detected both endogenous and S27-HA, but not S27L-HA"

What is the normal tissue expression pattern of RPS27?

RPS27 exhibits a tissue-specific expression pattern that researchers should consider when designing experiments:

• Neural tissue: Strong expression in Purkinje cells of the cerebellum and neurons, while expression in normal astrocytes is minimal or absent

• Gray vs. white matter: Significantly stronger expression in gray matter compared to white matter in normal brain tissue

• Inflammatory conditions: Weak expression in inflammatory astrocytes during multiple sclerosis and encephalitis, while only a minority of macrophages express RPS27

• Neurodegenerative disease: In Alzheimer's disease tissues, RPS27 expression is maintained in Purkinje cells and neurons but remains absent in astrocytes

• Endothelial cells: Normal endothelial cells express RPS27, while endothelial-cell-derived spindle cells in Kaposi's sarcoma (KS) show reduced expression

Understanding this baseline expression is essential for interpreting results in disease models or patient samples.

What storage and handling conditions are recommended for RPS27 antibodies?

For optimal antibody performance and stability:

• Store at -20°C in aliquots to avoid repeated freeze-thaw cycles

• Commercial antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Antibodies remain stable for one year after shipment when properly stored

• Some formulations may contain 0.1% BSA for additional stability

• Handle with appropriate safety precautions due to sodium azide content

How can I distinguish between RPS27 and RPS27L in my experiments?

Distinguishing between RPS27 and its paralog RPS27L is critical for accurate research outcomes:

• Specific antibodies: Use antibodies validated for selective detection of either protein. Studies have developed antibodies that specifically discriminate between these paralogs

• Expression patterns: RPS27 and RPS27L show tissue-specific expression patterns. For example, the spleen (containing abundant B cells) shows high RPS27L expression, while liver tissue (containing hepatocytes) shows high RPS27 expression

• Regulatory differences: RPS27L is induced by p53, while RPS27 is repressed by p53, which can be leveraged in experimental designs

• Western blot confirmation: Both proteins have a molecular weight of approximately 9 kDa but can be differentiated with the right antibodies and gel conditions

• Gene-specific knockdown: Use siRNA targeting unique regions to selectively deplete one paralog while monitoring the other's expression

What role does RPS27 play in cancer biology and what methodologies are recommended for studying this function?

RPS27's role in cancer is complex and context-dependent:

• Expression in gliomas: RPS27 mRNA is significantly overexpressed in gliomas (6.2-fold in WHO grade II/III with IDH mutation, 8.8-fold in WHO grade II/III without IDH mutation, and 4.6-fold in glioblastoma multiforme compared to normal brain)

• Cellular localization: In gliomas, RPS27 is expressed by both astrocytic tumor cells and tumor-associated macrophages, suggesting multiple roles in the tumor microenvironment

• Prognostic significance: While RPS27 expression levels did not affect survival in glioma patients, its expression has been associated with poor prognosis in cutaneous melanoma

• Expression in other cancers: RPS27 is overexpressed in various malignancies including prostate, liver, stomach, colon, and head and neck cancer

• Paradoxical effects: In Kaposi's sarcoma, RPS27 is downregulated, and its inhibition in HUVECs promoted pro-tumor characteristics, suggesting context-dependent functions

Recommended methodologies:

• Immunohistochemical staining of patient samples with quantification of immunoreactive scores

• RNA interference studies combined with cell viability, migration, invasion, and angiogenesis assays

• Analysis of downstream signaling pathways (particularly NF-κB pathway)

• Correlation with clinical parameters and molecular profiles of tumors

What is known about the RNA-binding properties of RPS27 and how can I study its RNA targets?

RPS27 functions as an RNA-binding protein (RBP) with important regulatory roles:

• Structure: RPS27 contains a C4-type zinc finger domain capable of binding to nucleic acids

• RNA targets: Improved RNA immunoprecipitation and sequencing (iRIP-seq) has identified 341 genes associated with RPS27 binding peaks

• Binding preference: Analysis of RPS27 binding sites revealed a G-rich binding signature

• Key RNA interactions:

  • RPS27 globally binds to EEF1A1, an important factor controlling translation elongation

  • It binds to the 5'-untranslated regions of WDR74, involved in rRNA processing

  • It preferentially binds to ribosomal protein transcripts, potentially modulating their processing or translation

Recommended methodology for studying RNA targets:

• iRIP-seq protocol:

  • Crosslink protein-RNA complexes in cells/tissues

  • Immunoprecipitate with RPS27-specific antibody

  • Extract bound RNAs

  • Perform high-throughput sequencing

  • Analyze peaks using bioinformatic tools like Piranha and HOMER for motif analysis

• Validation: Confirm interactions using RIP-qPCR for specific targets

• Functional analysis: Examine the biological relevance of identified interactions through GO analysis and pathway enrichment

• Integration: Combine RNA-binding data with differential expression analysis to identify genes both bound by RPS27 and differentially expressed under experimental conditions

What evidence supports the antiviral function of RPS27 and how can I investigate this property?

RPS27 demonstrates significant antiviral activity through multiple mechanisms:

• Viral challenge response: In shrimp (Marsupenaeus japonicus), RPS27 expression is significantly upregulated following white spot syndrome virus (WSSV) infection

• Effect on viral replication: RPS27 knockdown increases WSSV replication, while overexpression decreases viral replication and increases host survival rate

• Mechanistic pathways:

  • RPS27 activates the NF-κB pathway (through Dorsal and Relish)

  • It regulates antimicrobial peptide (AMP) expression

  • It interacts with viral envelope proteins (VP19, VP24, and VP28), potentially preventing virion assembly

• Human context: RPS27 can activate the NF-κB pathway to regulate antimicrobial peptide expression, potentially impeding viral replication

Recommended methodologies for investigating antiviral properties:

• RNA interference studies:

  • Use siRNA to knock down RPS27 expression

  • Measure viral titers and replication markers before and after viral challenge

• Overexpression experiments:

  • Transfect cells with RPS27 expression constructs

  • Assess viral replication efficiency and host cell survival

• Pathway analysis:

  • Monitor NF-κB activation through nuclear translocation assays

  • Measure expression of antimicrobial peptides

• Protein-protein interaction studies:

  • Co-immunoprecipitation with viral proteins

  • Proximity ligation assays to visualize interactions in situ

How does RPS27 interact with the p53-MDM2 axis and what experimental approaches should be used to study this relationship?

RPS27 exhibits a complex interplay with the p53-MDM2 regulatory network:

• Differential regulation: Unlike RPS27L (which is induced by p53), RPS27 is actually repressed by p53 activation

• Response to DNA damage: Treatment with etoposide (a DNA damaging agent) significantly reduces RPS27 levels in wild-type p53-containing cells (HCT116), but only slightly reduces levels in p53-null cells

• Multi-level interplay: Studies suggest "a multi-level interplay among RPS27L/S27 and p53-MDM2 axis with RPS27L acting as a p53 target, an MDM2 substrate, and a p53 regulator"

Recommended experimental approaches:

• Comparative expression analysis:

  • Treat cells with DNA damaging agents (e.g., etoposide)

  • Compare RPS27 expression in p53-wild-type vs. p53-null cells

  • Monitor expression of p53, MDM2, p21, RPS27, and RPS27L

• Protein stability assays:

  • Cycloheximide chase experiments to determine protein half-life

  • Proteasome inhibition studies to assess degradation mechanisms

• Gain/loss of function experiments:

  • Overexpress or knockdown RPS27 and assess effects on p53 stability and activity

  • Measure p53 target gene expression

• Co-immunoprecipitation:

  • Assess physical interactions between RPS27, p53, and MDM2

  • Map binding domains through mutational analysis

What are the key considerations for using RPS27 antibodies in immunohistochemistry of brain tissues?

When conducting immunohistochemistry of brain tissues using RPS27 antibodies:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded tissues with appropriate antigen retrieval

  • Consider TE buffer (pH 9.0) as the optimal retrieval method

• Quantification methods:

  • Calculate immunoreactive scores based on percentage of stained cells and staining intensity

  • Compare optical density between samples for background assessment

• Regional considerations:

  • Account for significant differences between gray and white matter expression

  • Five of six normal brain white matter specimens typically score negative, with only one sample weakly positive

• Cell type identification:

  • Use co-staining with cell-specific markers (TMEM119 for microglia, GFAP for astrocytes, etc.)

  • RPS27 strongly stains neurons and Purkinje cells but is typically absent in normal astrocytes

• Controls:

  • Include both positive (cerebellar Purkinje cells) and negative (white matter) tissue controls

  • Consider including inflammatory and neurodegenerative disease controls to validate specificity

How can gene replacement strategies be used to study the functional redundancy between RPS27 and RPS27L?

Gene replacement strategies have revealed important insights about the functional relationship between these paralogs:

• Knockin models: Expressing RPS27 from the RPS27L locus completely rescues the early lethality observed upon homozygous truncation of RPS27L, and vice versa

• Compensation mechanisms: Loss of one paralog leads to increased expression of the other:

  • Western blots of tissues from Rps27+/- mice show increased Rps27L protein

  • This effect is most pronounced in the spleen (containing abundant B cells)

  • Similarly, loss of Rps27L leads to increased Rps27 protein, particularly in liver tissue

• Long-term viability: Mice with either gene replacement strategy:

  • Gain weight at similar rates to wild-type mice

  • Are viable to at least 1 year of age

  • Are fertile with normal reproductive capacity

• Phenotypic assessment: Detailed necropsy of homozygous 10-16-week-old males and age-matched wild-type controls revealed no clinically significant differences in:

  • Gross organ weight

  • Gross organ morphology

  • Tissue histology across all major organ systems

Recommended methodological approach:

What protocols are recommended for studying RPS27's interaction with the NF-κB pathway in antiviral responses?

Based on research findings, the following protocols are recommended:

• RPS27 knockdown and overexpression:

  • Use siRNA for transient knockdown or CRISPR/Cas9 for stable knockout

  • Construct expression vectors for overexpression studies

  • Validate expression changes at both mRNA and protein levels

• NF-κB pathway analysis:

  • Monitor mRNA expression levels of NF-κB components (e.g., Dorsal, Relish)

  • Assess nuclear translocation of NF-κB factors using cellular fractionation and Western blotting

  • Measure expression of downstream antimicrobial peptides (AMPs) through qRT-PCR

• Viral challenge experiments:

  • Infect cells/organisms with relevant viruses after RPS27 modulation

  • Quantify viral load using qPCR or plaque assays

  • Assess host survival rates in in vivo models

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation to identify interactions with viral proteins

  • Consider using proximity ligation assays for in situ validation

  • Identify critical binding domains through truncation mutants

• Pathway validation:

  • Use NF-κB inhibitors to confirm the dependence of antiviral effects on this pathway

  • Employ reporter assays to quantify NF-κB activation

  • Perform rescue experiments by modulating both RPS27 and NF-κB components