RPS4A Antibody
Description
Overview of RPS4 Proteins and Antibodies
Ribosomal protein S4 isoforms (RPS4X and RPS4Y1) are structural components of the 40S ribosomal subunit. These proteins exhibit 93% amino acid sequence homology but differ in chromosomal origin and sex-specific expression:
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RPS4X: Expressed in all cells, located on the X chromosome (UniProt P62701).
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RPS4Y1: Y chromosome-linked, expressed in males (UniProt P22090) .
Antibodies targeting these proteins are critical for distinguishing male cells (expressing both isoforms) from female cells (expressing only RPS4X) and studying ribosome biology .
Development of RPS4Y1-Specific Antibodies
A 2021 study developed monoclonal antibodies against RPS4Y1 using peptide antigens from divergent regions (Table 1) :
| Antigen Region | Amino Acid Position | Unique Residues |
|---|---|---|
| Y1 | 68–84 | 2/17 |
| Y2 | 106–121 | 3/16 |
| Y3 | 155–177 | 4/23 |
Key findings:
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Only the Y3 peptide (155–177 aa) induced immunogenic antibodies.
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Antibodies validated via Western blot (WB) and immunohistochemistry (IHC) showed no cross-reactivity with RPS4X .
Validation and Applications of RPS4 Antibodies
RPS4X antibody (14799-1-AP):
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Host: Rabbit IgG.
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Applications: WB, IHC, IP, ELISA.
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Reactivity: Human, mouse, rat (validated in HepG2, HeLa, MCF-7 lysates) .
Performance metrics:
| Assay | Sample | Observed Band |
|---|---|---|
| WB | Human liver | 30 kDa |
| IP | HepG2 lysate | 30 kDa |
| IHC | Pancreatic cancer | Nuclear staining |
This antibody targets a fusion protein antigen (Ag6513) and shows no cross-reactivity with RPS4Y1 under optimized conditions .
Challenges in Antibody Specificity
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High homology: RPS4X and RPS4Y1 share 19 divergent residues across 263 amino acids, complicating epitope selection .
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Validation methods:
Functional Insights from RPS4 Studies
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Immune signaling: In plants, RPS4 partners with RRS1 to form a nuclear immune complex activating defense genes against pathogens like Pseudomonas .
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Antibody-mediated immunity: Intracellular antibodies recruit TRIM21 for pathogen degradation and immune activation via NF-κB and IRF pathways .
Future Directions
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
KEGG: sce:YHR203C
STRING: 4932.YJR145C
Q&A
Frequently Asked Questions on RPS4Y1 Antibody Applications in Academic Research
How can researchers validate the specificity of an anti-RPS4Y1 antibody for male cell identification?
To confirm antibody specificity, a multi-step validation pipeline is required. First, perform Western blotting on lysates from male (e.g., HepG2) and female (e.g., HEK293) cell lines. A single band at ~30 kDa (the molecular weight of RPS4Y1) should appear exclusively in male samples . Second, use immunofluorescence to visualize male-specific staining; anti-RPS4Y1 antibodies should label >75% of male cells (e.g., HepG2) with minimal background in female cells (<2%) . Third, conduct immunoprecipitation to verify antibody binding to the native protein. Male cell lysates incubated with the antibody should yield a 29.4 kDa band post-immunoprecipitation, absent in female samples . Include controls such as tubulin or RPS6 antibodies to ensure equal loading and rule out non-specific binding.
What experimental controls are critical when using RPS4Y1 antibodies in prenatal diagnostics?
Key controls include:
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Negative controls: Female-derived cell lines (e.g., HEK293) to confirm no cross-reactivity with RPS4X .
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Isotype controls: Non-specific IgG to assess background staining in fluorescence-based assays .
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Housekeeping protein controls: Antibodies against tubulin or RPS6 to normalize protein loading in Western blots .
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Peptide competition assays: Pre-incubate the antibody with the Y3 peptide (155–177 aa of RPS4Y1) to block antigen binding, which should abolish signal in male cells .
How can cross-reactivity between RPS4Y1 and its homolog RPS4X be minimized in immunoassays?
The high homology (~93%) between RPS4Y1 and RPS4X necessitates stringent validation. Strategies include:
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Epitope mapping: Target regions with maximal sequence divergence, such as the Y3 peptide (155–177 aa), which contains four male-specific amino acids .
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CRISPR-edited cell lines: Use RPS4X-knockout male cells to confirm antibody binding is Y1-specific .
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Dual validation: Pair antibody-based detection with RT-PCR for RPS4Y1 transcripts to corroborate protein expression .
What methodologies enhance the sensitivity of RPS4Y1 detection in rare fetal cell populations?
For fetal cells in maternal circulation (1–5 cells/mL blood), employ:
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Pre-enrichment protocols: Magnetic-activated cell sorting (MACS) with anti-CD71 or anti-HLA-G antibodies to isolate trophoblasts .
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Multi-modal imaging: Combine anti-RPS4Y1 immunofluorescence with Y-chromosome FISH to confirm fetal origin .
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Single-cell RNA-seq: Post-sorting, validate RPS4Y1 expression alongside placental biomarkers (e.g., CGA) .
How can RPS4Y1 antibody-based assays be integrated with genomic techniques for hemophilia diagnosis?
A hybrid workflow is recommended:
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Fetal cell isolation: Use anti-RPS4Y1 FACS to enrich male cells from maternal blood .
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Whole-genome amplification (WGA): Amplify DNA from isolated cells for downstream analysis.
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Targeted sequencing: Screen for F8 or F9 mutations linked to hemophilia .
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Data correlation: Cross-validate antibody-positive cells with Y-chromosome sequences and hemophilia-associated variants .
Methodological Challenges and Solutions
Data Interpretation Guidelines
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False positives: Eliminate by co-staining with female-specific markers (e.g., XIST RNA FISH) .
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Quantitative thresholds: Define positivity thresholds using receiver operating characteristic (ROC) curves based on male/female cell mixtures .
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Batch variability: Standardize antibody lots using ELISA reactivity to the Y3 peptide (OD >2.5 at 2 µg/mL) .
