RPS23 Antibody

What is RPS23 and what cellular functions does it perform?

RPS23, also known as small ribosomal subunit protein uS12, is a subunit of the 40S ribosome and serves as the first precursor of the small eukaryotic ribosomal subunit . It plays a crucial role in maintaining translational fidelity by monitoring the complementarity between mRNA codons being translated and the anti-codons of aminoacyl-tRNAs . This protein is strategically positioned in the decoding center of the ribosome, making it essential for accurate protein synthesis.

The protein has a calculated molecular weight of 16 kDa, though its observed molecular weight typically ranges from 16-18 kDa in experimental conditions . RPS23 is primarily localized in the cytoplasm and has also been detected in melanosomes .

What applications are RPS23 antibodies most commonly used for?

RPS23 antibodies are utilized in multiple research applications, with the most common being:

Positive Western blot detection has been confirmed in A431 cells, U2OS cells, human placenta tissue, and mouse ovary tissue . For immunofluorescence, MCF-7 cells have shown reliable positive detection .

What are the key differences between polyclonal and monoclonal RPS23 antibodies?

Polyclonal RPS23 antibodies (such as ABIN6257642) recognize multiple epitopes on the protein, typically producing stronger signals but potentially lower specificity . These antibodies are often raised against synthesized peptides derived from specific regions of human RPS23, such as the N-terminal amino acids .

Monoclonal RPS23 antibodies (such as NBP3-23644, clone YWHAE/8309R) recognize a single epitope, offering higher specificity but sometimes lower sensitivity . They are frequently generated using recombinant human full-length RPS23 protein as the immunogen .

When selecting between these options, researchers should consider:

• Polyclonal antibodies are often preferred for detection of low-abundance proteins

• Monoclonal antibodies are superior for applications requiring high reproducibility

• Recombinant monoclonal antibodies (like NBP3-23644) offer the specificity of monoclonals with improved batch-to-batch consistency

What species reactivity can researchers expect from commercial RPS23 antibodies?

RPS23 antibodies show varied species reactivity profiles depending on the specific product:

• Most tested RPS23 antibodies show confirmed reactivity with human and mouse samples

• Some antibodies additionally react with rat samples

• Predicted reactivity (though requiring validation) has been reported for pig, zebrafish, bovine, horse, sheep, rabbit, dog, and Xenopus samples

Species cross-reactivity is likely due to the high conservation of RPS23 across species. When working with non-validated species, researchers should perform preliminary testing at multiple antibody concentrations to confirm specificity.

How does OGFOD1-mediated prolyl hydroxylation affect RPS23 function and antibody recognition?

OGFOD1 (2-oxoglutarate and iron-dependent oxygenase domain containing 1) catalyzes prolyl hydroxylation of RPS23 at the Pro-62 position . This post-translational modification is nearly complete in most cell types, with >95% of ribosomal RPS23 being hydroxylated in normal tissue and across various cell lines .

The OGFOD1-RPS23 interaction forms a high-affinity complex that is stable even under SDS-PAGE conditions, resulting in the detection of both the standard 16 kDa RPS23 band and a 100 kDa OGFOD1-RPS23 complex when analyzed by immunoblotting .

For researchers studying RPS23 biology, this has several important implications:

• Antibodies raised against regions containing or adjacent to Pro-62 may show differential recognition of hydroxylated versus non-hydroxylated forms

• When analyzing OGFOD1 knockout models, researchers should be aware that RPS23 will be predominantly non-hydroxylated

• The presence of the 100 kDa band in immunoblots may be mistakenly interpreted as non-specific binding, when it actually represents the OGFOD1-RPS23 complex

How can researchers distinguish between RPS23 and its retroposed gene products?

The Rps23rg gene family originated through retroposition of the ribosomal protein S23 (Rps23) mRNA . Two functionally expressed genes in mice, Rps23rg1 and Rps23rg2, are reversely transcribed relative to Rps23 .

When designing experiments to differentiate between RPS23 and its retroposed gene products:

• Select antibodies specifically validated against the target of interest

• Use genetic models (knockout or siRNA) to confirm specificity

• Be aware that standard Western blotting may not differentiate between these proteins if they have similar molecular weights

• Consider using unique peptide sequences for targeted mass spectrometry approaches

RPS23RG family members interact with adenylate cyclases to upregulate PKA activity and downregulate GSK-3 activity, potentially reducing AD-like pathologies such as Aβ levels and tau phosphorylation . Therefore, when studying neurodegenerative disease models, researchers should carefully validate which protein family member they are detecting.

What controls should be included when validating RPS23 antibody specificity?

To ensure experimental rigor when working with RPS23 antibodies, researchers should implement the following controls:

• Positive controls: Use cell lines with confirmed RPS23 expression such as A431, U2OS, or MCF-7 cells

• Negative controls:

  • Primary antibody omission

  • Isotype controls (Rabbit IgG for polyclonal antibodies )

  • siRNA/shRNA knockdown of RPS23 (note that complete knockout may be lethal due to essential ribosomal function)

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to confirm specificity

• Molecular weight verification: Confirm detection at the expected 16-18 kDa range

• OGFOD1 knockout comparison: To distinguish hydroxylated vs. non-hydroxylated forms

For advanced applications, consider using OGFOD1 knockout/knockdown cells as these will contain non-hydroxylated RPS23, providing a control for hydroxylation-sensitive antibodies .

What are the optimal protocols for using RPS23 antibodies in Western blotting?

For successful Western blot detection of RPS23, follow these methodological guidelines:

• Sample preparation:

  • Lyse cells in RIPA buffer containing protease inhibitors

  • Use fresh samples when possible or store at -80°C

  • Load 20-50 μg of total protein per lane

• Gel selection:

  • 12-15% polyacrylamide gels are recommended due to RPS23's low molecular weight (16-18 kDa)

  • Consider gradient gels (4-20%) if analyzing both free RPS23 and the OGFOD1-RPS23 complex (~100 kDa)

• Transfer conditions:

  • Semi-dry transfer: 15V for 30 minutes

  • Wet transfer: 100V for 60 minutes

  • Use 0.2 μm PVDF membrane (preferred over nitrocellulose for low molecular weight proteins)

• Antibody incubation:

  • Primary antibody dilution: Start with 1:1000 and adjust based on signal strength

  • Incubate overnight at 4°C for optimal results

  • Secondary antibody: Anti-rabbit HRP conjugate at 1:5000 dilution for 1 hour at room temperature

• Detection:

  • Enhanced chemiluminescence (ECL) is suitable for most applications

  • For low abundance detection, consider using more sensitive ECL substrates

Be aware that RPS23 may appear as both the 16-18 kDa monomer and as part of the 100 kDa OGFOD1-RPS23 complex in some samples .

How should tissue samples be prepared for immunohistochemistry with RPS23 antibodies?

For optimal immunohistochemical detection of RPS23 in paraffin-embedded tissues, follow this protocol:

• Tissue fixation and processing:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard protocols

  • Section at 4-5 μm thickness

• Antigen retrieval (critical step):

  • Heat tissue sections in 10mM Tris with 1mM EDTA, pH 9.0, for 45 min at 95°C

  • Allow to cool at room temperature for 20 minutes

  • This step is essential for exposing the epitope recognized by the antibody

• Antibody incubation:

  • Use antibody at 1-2 μg/ml concentration

  • Incubate for 30 minutes at room temperature

  • For polyclonal antibodies, consider longer incubation times (1 hour)

• Detection system:

  • Use a polymer-based detection system for improved sensitivity

  • Include appropriate positive and negative controls

Cytoplasmic staining should be observed, consistent with RPS23's known localization . Be aware that melanosomes may also show positive staining.

What troubleshooting approaches are recommended for non-specific binding of RPS23 antibodies?

When encountering non-specific binding with RPS23 antibodies, implement these troubleshooting strategies:

• High background issues:

  • Increase blocking time (use 5% BSA in TBST for 2 hours)

  • Increase washing steps (5x5 minutes with TBST)

  • Reduce antibody concentration

  • Filter antibody solution before use to remove aggregates

• Multiple bands on Western blot:

  • Verify if the ~100 kDa band represents the OGFOD1-RPS23 complex

  • Use fresher lysates to minimize degradation products

  • Add additional protease inhibitors to extraction buffer

  • Perform peptide competition assay to identify true versus non-specific bands

• No signal detected:

  • Verify protein loading with total protein stain

  • Increase antibody concentration or incubation time

  • Try alternative antigen retrieval methods

  • Check antibody storage conditions and expiration date

• Cross-reactivity concerns:

  • Test antibody on known negative control samples

  • Consider antibodies targeting different epitopes of RPS23

  • Use antibodies purified by antigen affinity chromatography

If persistent issues occur with commercial antibodies, antigen affinity purification of the antibody may improve specificity .

How can RPS23 antibodies be utilized in neurodegeneration research?

Recent research has identified connections between RPS23-related proteins and neurodegenerative conditions, particularly Alzheimer's disease. The Rps23rg gene family has been shown to regulate β-amyloid levels and tau phosphorylation, two major pathological features of Alzheimer's disease .

When applying RPS23 antibodies to neurodegeneration research:

• Experimental design considerations:

  • Carefully validate antibody specificity between RPS23 and RPS23RG proteins

  • Include relevant controls for each disease model

  • Consider both cellular and animal models for comprehensive analysis

• Functional analysis approach:

  • Investigate interactions between RPS23RG proteins and adenylate cyclases

  • Monitor effects on cAMP levels, PKA activity, and GSK-3 activity

  • Assess downstream effects on tau phosphorylation and Aβ generation

• Potential therapeutic implications:

  • Screen for compounds that might modulate RPS23/RPS23RG function

  • Evaluate effects on disease progression in animal models

  • Develop targeted approaches based on protein-protein interactions

These applications require careful antibody selection and experimental design to distinguish between RPS23 and its retroposed gene products.

What considerations should researchers have when investigating RPS23 prolyl hydroxylation?

The high degree of RPS23 hydroxylation (>95%) in normal tissues presents interesting research challenges and opportunities . When studying this post-translational modification:

• Experimental models:

  • OGFOD1 knockout or knockdown systems provide a source of non-hydroxylated RPS23

  • Analysis of OGFOD1 D157A mutant (catalytically inactive) effects can help distinguish enzymatic from scaffolding functions

• Technical approaches:

  • Mass spectrometry is the gold standard for confirming hydroxylation status

  • Intact protein MS or peptide-based MS can quantify hydroxylation levels

  • Consider hydroxylation-specific antibodies if available

• Functional analyses:

  • Investigate effects on translational fidelity

  • Examine polysome profiles to assess ribosomal assembly

  • Assess effects on cellular stress responses, including stress granule formation

Research indicates that OGFOD1 siRNA suppresses RPS23 hydroxylation to similar extents in different cell types, but the physiological consequences may vary between cell lines . This suggests context-dependent functions that warrant careful experimental design.

What emerging applications of RPS23 antibodies should researchers be aware of?

As our understanding of RPS23 biology expands, several emerging applications deserve consideration:

• Ribosome heterogeneity studies:

  • Investigating how RPS23 hydroxylation contributes to specialized ribosomes

  • Examining tissue-specific variations in RPS23 modification patterns

  • Exploring translational regulation under different cellular conditions

• Cancer research applications:

  • Assessing RPS23 expression and modification in various tumor types

  • Investigating correlations with patient outcomes or therapeutic responses

  • Exploring potential as a biomarker for specific cancer subtypes

• Neurodegenerative disease connections:

  • Further characterizing the relationship between RPS23RG proteins and Alzheimer's pathology

  • Investigating potential roles in other neurodegenerative conditions

  • Exploring therapeutic strategies based on modulating RPS23RG function

• Technological advancements:

  • Development of hydroxylation-specific antibodies

  • Application of proximity labeling approaches to identify RPS23 interactors

  • Implementation of ribosome profiling techniques to assess translational impacts

The continued development and characterization of specific, well-validated RPS23 antibodies will be essential to advancing these research directions.

How should researchers approach experimental design when studying both RPS23 and its retroposed gene products?

When designing experiments to investigate both RPS23 and its retroposed gene products:

• Antibody selection strategy:

  • Use antibodies targeting unique regions that can differentiate between protein family members

  • Validate specificity using overexpression and knockdown approaches

  • Consider epitope-tagged constructs for unambiguous identification

• Functional differentiation approaches:

  • Exploit known differences in protein interactions (e.g., RPS23RG proteins interact with adenylate cyclases)

  • Utilize cellular localization differences when present

  • Implement selective knockdown of individual family members

• Comprehensive analysis workflow:

  • Begin with individual protein characterization

  • Progress to comparative analyses

  • Culminate with functional interaction studies