RPS19 Antibody

What applications are supported by commercially available RPS19 antibodies?

RPS19 antibodies have been validated for multiple research applications, primarily Western blotting (WB), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) . When selecting an antibody, researchers should verify application-specific validation data and optimize protocols for their particular experimental system. For quantitative studies of RPS19 expression levels, Western blotting remains the gold standard, as demonstrated in studies examining protein levels in RPS19-deficient cell models .

What species reactivity can be expected from RPS19 antibodies?

Many commercially available RPS19 antibodies demonstrate cross-reactivity with human, mouse, and rat proteins . This cross-reactivity is facilitated by the high level of sequence conservation between species. When working with less common experimental models, researchers should conduct preliminary validation experiments to confirm reactivity with their target species. Published studies have successfully employed RPS19 antibodies in human cell lines and patient-derived samples, as well as in mouse models of DBA .

What are the optimal storage and handling conditions for RPS19 antibodies?

To maintain antibody functionality, RPS19 antibodies should be stored at -20°C for long-term preservation . For frequent use within one month, storage at 4°C is acceptable but requires careful handling to prevent microbial contamination. Repeated freeze-thaw cycles significantly reduce antibody performance and should be avoided by preparing small working aliquots upon initial thawing. Most commercial RPS19 antibodies are supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide as preservatives .

What positive controls should be used when validating a new RPS19 antibody?

For appropriate validation, researchers should include lysates from cell lines known to express RPS19 at detectable levels. The TF-1-B cell line has been extensively characterized for RPS19 expression and serves as an excellent positive control . When evaluating antibody specificity, comparing wild-type cells with those subjected to siRNA-mediated knockdown of RPS19 provides a robust control system. In published studies, RPS19 protein typically presents as a distinct band at approximately 16 kDa on Western blots .

How can RPS19 antibodies be employed to study Diamond-Blackfan anemia mechanisms?

RPS19 antibodies have proven invaluable for investigating the molecular pathogenesis of Diamond-Blackfan anemia, particularly in cases associated with RPS19 mutations. Researchers can establish experimental systems using patient-derived lymphoblastoid cell lines (LCLs) harboring different RPS19 mutations, such as splice site mutations (c.411+1G>A), start codon mutations (c.1A>G), gene deletions, or insertions (c.104_105insA) . By quantifying RPS19 protein levels and comparing them with other small and large subunit ribosomal proteins, researchers can assess the impact of RPS19 haploinsufficiency on ribosome biogenesis. Western blot analysis using specific antibodies against RPS19 and other ribosomal proteins (such as RPS20, RPS21, RPS24, RPL3, RPL9, RPL30, and RPL38) enables the detection of coordinated down-regulation of small subunit proteins, which is a characteristic feature of RPS19 deficiency .

What methodological approaches can detect interactions between RPS19 and other proteins?

To identify and characterize protein-protein interactions involving RPS19, researchers can employ several complementary techniques:

• Co-immunoprecipitation (co-IP): This approach has successfully identified RPS19 as an interaction partner of Macrophage Migration Inhibitory Factor (MIF). By using antibodies against MIF for immunoprecipitation followed by Western blotting with RPS19 antibodies, researchers detected a specific interaction between these proteins .

• In vivo biotin-tagging: This technique involves expressing a tagged RPS19 fusion protein that can be biotinylated in vivo by bacterial birA biotin ligase co-expressed in the same cells. The biotinylated RPS19 and its interaction partners can then be purified using streptavidin-agarose beads and identified by mass spectrometry .

• Surface plasmon resonance: This biophysical technique can quantify binding affinities between RPS19 and potential interaction partners. Previous studies have determined the dissociation constant (KD) for RPS19-MIF interaction to be approximately 1.3 × 10-6 M .

How can researchers analyze the differential expression of small versus large ribosomal subunit proteins?

To investigate the coordinated regulation of ribosomal proteins in response to RPS19 deficiency, researchers should implement a multi-parameter analytical approach:

• Antibody panel selection: Utilize antibodies against multiple ribosomal proteins from both small (e.g., RPS20, RPS21, RPS24) and large (e.g., RPL3, RPL9, RPL30, RPL38) subunits .

• Protein quantification: Perform Western blotting with densitometric analysis to measure relative protein levels. Normalize each ribosomal protein signal to a loading control such as fibrillarin, which remains unchanged in RPS19-deficient cells .

• SSU/LSU ratio calculation: Calculate the ratio of small subunit to large subunit ribosomal proteins to detect subunit-specific effects. In RPS19-deficient cells, this ratio is significantly reduced compared to control cells .

• Statistical analysis: Apply appropriate statistical tests (e.g., two-tailed Student's t-test) to compare expression levels between experimental and control groups .

What experimental strategies can differentiate between transcriptional and post-transcriptional regulation of RPS19?

To distinguish between transcriptional and post-transcriptional regulation mechanisms affecting RPS19 expression, researchers should implement parallel analyses of both mRNA and protein levels:

• mRNA quantification: Employ quantitative RT-PCR to measure transcript levels of RPS19 and other ribosomal proteins. In cells with siRNA-mediated RPS19 knockdown, mRNA levels for other ribosomal proteins typically remain unchanged despite reduced protein levels, indicating post-transcriptional regulation .

• rRNA analysis: Quantify 18S rRNA (small subunit) and 28S rRNA (large subunit) levels. A decreased 18S/28S ratio correlates with reduced small subunit ribosomal protein levels in RPS19-deficient cells .

• Polysome profiling: Analyze ribosome assembly by sucrose gradient ultracentrifugation to detect accumulation of pre-ribosomal particles and assess translation efficiency.

• Protein stability assessment: Conduct pulse-chase experiments with cycloheximide to determine whether reduced protein levels result from increased degradation or decreased synthesis.

What optimization strategies improve Western blot detection of RPS19?

When optimizing Western blot protocols for RPS19 detection, researchers should consider:

• Protein extraction: Use RIPA buffer supplemented with protease inhibitors for efficient extraction of ribosomal proteins. For membrane-rich samples, include 1% Triton X-100 to enhance solubilization.

• Gel selection: Utilize 15-16% polyacrylamide gels to achieve optimal separation of low molecular weight proteins like RPS19 (~16 kDa) .

• Transfer conditions: Implement semi-dry transfer with methanol-containing buffer at 15-20V for 30-45 minutes to efficiently transfer small proteins.

• Blocking optimization: Use 5% non-fat dry milk in TBS-T for 1 hour at room temperature to minimize background without interfering with antibody binding.

• Antibody dilution: Titrate primary RPS19 antibodies (typically 1:500 to 1:2000) to determine optimal concentration for specific detection while minimizing background .

• Detection method: Enhanced chemiluminescence (ECL) provides sufficient sensitivity for most applications, though fluorescent secondary antibodies offer advantages for multiplex detection and quantification.

How should researchers address variability in RPS19 expression between cell lines?

When comparing RPS19 expression across different cell lines or patient samples, researchers should implement standardization measures:

• Multiple biological replicates: Analyze at least three independent samples to account for biological variation. Patient-derived lymphoblastoid cell lines (LCLs) can show considerable variability in ribosomal protein expression .

• Pooled analysis: When comparing groups (e.g., patient vs. control), pool data from small subunit and large subunit ribosomal proteins separately before calculating ratios to minimize individual protein variations .

• Internal controls: Include cell lines with characterized RPS19 expression levels in each experiment as reference standards.

• Normalization strategy: Beyond standard loading controls, normalize RPS19 levels to the average of multiple housekeeping proteins to compensate for potential biological variation.

• Statistical approaches: Apply appropriate statistical methods to detect significant differences despite variability. The Mann-Whitney U test may be preferable to parametric tests when sample sizes are small or distributions non-normal .

What methods ensure specificity in RPS19 immunoprecipitation experiments?

To ensure specificity in RPS19 immunoprecipitation experiments, researchers should implement rigorous controls:

• Antibody validation: Confirm antibody specificity through Western blotting of input samples and knockdown controls before proceeding with immunoprecipitation .

• Isotype controls: Include matched isotype control antibodies processed identically to RPS19 antibodies to identify non-specific binding .

• Pre-clearing step: Pre-clear lysates with protein A/G beads to reduce non-specific interactions.

• Washing optimization: Determine optimal washing stringency to remove non-specific binders while preserving genuine interactions.

• Reciprocal co-IP: Confirm interactions by performing reverse immunoprecipitation with antibodies against the putative binding partner (e.g., MIF) followed by RPS19 detection .

• Mass spectrometry verification: Identify co-precipitated proteins by mass spectrometry to confirm specific interactions, as demonstrated in the identification of RPS19-MIF interaction .

How can RPS19 antibodies help characterize haploinsufficiency in Diamond-Blackfan anemia?

RPS19 antibodies serve as critical tools for characterizing the molecular consequences of haploinsufficiency in Diamond-Blackfan anemia (DBA):

• Mutation impact assessment: Different mutations in the RPS19 gene result in varying degrees of protein deficiency. Western blot analysis with RPS19 antibodies can quantify protein levels in patient-derived cells carrying distinct mutations, including splice site mutations (c.411+1G>A), start codon mutations (c.1A>G), gene deletions, or insertions (c.104_105insA) .

• Haploinsufficiency verification: In DBA patient-derived lymphoblastoid cell lines (LCLs), RPS19 protein levels may show partial compensation rather than the expected 50% reduction. RPS19 antibodies can detect these subtle variations in protein expression .

• Ribosome biogenesis analysis: By comparing the levels of small subunit ribosomal proteins (using antibodies against RPS19, RPS20, RPS21, and RPS24) to large subunit proteins (using antibodies against RPL3, RPL9, RPL30, and RPL38), researchers can determine how RPS19 haploinsufficiency affects the balance between subunits .

• Therapeutic efficacy monitoring: RPS19 antibodies can assess the effectiveness of experimental therapies aimed at increasing RPS19 expression or compensating for its deficiency.

What experimental approaches can investigate the interaction between RPS19 and MIF (Macrophage Migration Inhibitory Factor)?

The interaction between RPS19 and Macrophage Migration Inhibitory Factor (MIF) has significant implications for inflammatory responses. Researchers can investigate this interaction using:

• Direct binding assays: Surface plasmon resonance experiments have demonstrated direct physical interaction between RPS19 and MIF with a dissociation constant (KD) of 1.3 × 10-6 M .

• Functional interference assays: RPS19 has been shown to inhibit MIF-CD74 interaction at low doses, suggesting a regulatory role. Researchers can design competitive binding assays to investigate this mechanism .

• Flow chamber experiments: RPS19 significantly compromises CXCR2-dependent MIF-triggered adhesion of monocytes to endothelial cells under flow conditions, providing a functional readout for RPS19-MIF interaction .

• Structure-function studies: Using wild-type and mutant MIF proteins in pulldown experiments with RPS19 can identify critical interaction domains and residues .

• Inflammatory response models: In models of infection, sepsis, or autoimmune disease where MIF levels are elevated, researchers can investigate whether RPS19 released from apoptotic cells acts as an extracellular negative regulator of MIF .

How do RPS19 antibodies facilitate studies of coordinated regulation of ribosomal proteins?

RPS19 antibodies enable researchers to investigate the mechanisms underlying coordinated regulation of ribosomal proteins:

• Subunit-specific effects: In RPS19-deficient cells, Western blot analysis with antibodies against multiple ribosomal proteins reveals that other small subunit proteins (RPS20, RPS21, RPS24) are significantly reduced while large subunit proteins (RPL3, RPL9, RPL30, RPL38) remain relatively unchanged .

• Post-transcriptional regulation: By comparing protein levels (detected with antibodies) with mRNA levels (measured by qRT-PCR), researchers have determined that the coordinated down-regulation of small subunit ribosomal proteins occurs post-transcriptionally, as mRNA levels remain relatively unchanged despite reduced protein expression .

• rRNA correlation: The reduction in small subunit ribosomal proteins correlates with decreased 18S rRNA levels relative to 28S rRNA, suggesting that subunit assembly is a major mechanism in the co-regulation of ribosomal protein levels .

• Compensatory mechanisms: In cells with heterozygous RPS19 mutations, the effects on the small-to-large subunit protein ratio are less pronounced than in siRNA knockdown models, suggesting the existence of compensatory mechanisms that can be investigated using RPS19 antibodies .