RPS10 Antibody Pair

What is RPS10 and what is its significance in molecular research?

RPS10 is a component of the 40S ribosomal subunit, playing a crucial role in protein synthesis. As part of the ribosomal machinery, it functions within a large ribonucleoprotein complex responsible for translating mRNA into proteins. RPS10 interacts with other ribosomal proteins such as RPS12 and RPS14 to maintain cellular homeostasis and facilitate responses to environmental stimuli. The calculated molecular weight of RPS10 is 19 kDa, and this protein is highly conserved across human, mouse, and rat species, making it an important target for cross-species research applications.

What are the optimal applications for RPS10 antibody pairs?

RPS10 antibody pairs can be effectively utilized across multiple experimental platforms as detailed in the following table:

For optimal results, antibody dilutions should be empirically determined for each experimental system and sample type.

How should researchers select between monoclonal and polyclonal RPS10 antibodies?

The selection between monoclonal and polyclonal RPS10 antibodies depends on specific experimental requirements:

Monoclonal RPS10 Antibodies:

• Provide high specificity to a single epitope of RPS10

• Ensure consistent results between experimental batches

• Ideal for applications requiring reproducibility across multiple experiments

• Example: Mouse monoclonal antibody (67609-1-Ig) shows excellent specificity with a single band corresponding to RPS10's molecular weight

Polyclonal RPS10 Antibodies:

• Recognize multiple epitopes across the RPS10 protein

• Often provide stronger detection signals due to multiple binding sites

• Better for detecting proteins with low expression levels

• Example: Rabbit polyclonal antibody (14894-1-AP) effectively detects RPS10 across multiple applications

For dual-detection systems, pairs consisting of one monoclonal and one polyclonal antibody from different host species provide optimal results, particularly in sandwich ELISA or co-immunoprecipitation experiments.

What positive controls are recommended for validating RPS10 antibodies?

Based on empirical validation data, the following biological samples serve as reliable positive controls for RPS10 antibody experiments:

Cell Lines for Western Blot:

• Raji cells (human B lymphocyte)

• HepG2 cells (human liver carcinoma)

• Jurkat cells (human T lymphocyte)

• K-562 cells (human myelogenous leukemia)

• HSC-T6 cells (rat hepatic stellate cells)

• NIH/3T3 cells (mouse fibroblast)

Tissue Samples for Immunohistochemistry:

• Human lymphoma tissue (with TE buffer pH 9.0 for antigen retrieval)

• Rat stomach tissue (with Tris-EDTA buffer pH 9.0)

When developing new experimental protocols, including at least one validated positive control enables proper assessment of antibody performance and helps establish optimal working conditions.

How can researchers optimize co-immunoprecipitation protocols for studying RPS10 interactions?

Optimizing co-immunoprecipitation (co-IP) protocols for RPS10 interactions requires careful attention to several critical parameters:

Antibody Selection and Sample Preparation:

• Select antibodies with validated IP capabilities (e.g., ABIN7118578 at 1:20-1:50 dilution)

• Use gentle lysis conditions (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40) supplemented with protease inhibitors

• Include RNase inhibitors when studying RNA-dependent interactions

• Pre-clear lysates with protein A/G beads to minimize non-specific binding

Crosslinking Strategy for Transient Interactions:

• For capturing dynamic RPS10 interactions, implement mild crosslinking with 0.5-1% formaldehyde for 10 minutes

• Quench crosslinking with glycine (final concentration 125 mM)

• Perform sonication to disrupt cellular structures while preserving protein complexes

Optimized Immunoprecipitation Protocol:

• Incubate pre-cleared lysates with RPS10 antibody overnight at 4°C (using 2-5 μg antibody per mg protein lysate)

• Add protein A/G magnetic beads and rotate for 1-2 hours at 4°C

• Perform sequential washes with decreasing salt concentrations to preserve interactions while removing contaminants

• Elute complexes under native conditions for functional studies or denaturing conditions for interaction analysis

Detection Strategies:

• Analyze by western blot using antibodies against suspected interacting partners

• For comprehensive analysis, employ mass spectrometry of immunoprecipitated complexes

FLAG-tagged RPS10 constructs have proven effective for studying RPS10 interactions, particularly when examining ubiquitination status and interactions with ribosomal assembly factors.

What methodological approaches are recommended for studying RPS10 post-translational modifications?

Based on recent research findings, RPS10 undergoes important post-translational modifications, particularly ubiquitination, which can be studied using the following approaches:

Enrichment and Detection Strategies:

• Express tagged versions of RPS10 (FLAG-tagged) for affinity purification

• Employ ribosome purification protocols using sucrose gradient ultracentrifugation

• Consider overexpression of Znf598 (E3 ligase) to maximize ubiquitination levels

• Detect modified forms via immunoblotting with anti-RPS10 antibodies to identify molecular weight shifts

Site-Specific Modification Analysis:

• Generate site-directed mutants targeting lysine residues (particularly K139/K140)

• Compare wild-type and mutant RPS10 behavior using western blot analysis

• Employ mass spectrometry to identify peptides with di-glycine remnants at modification sites

Temporal Analysis Protocol:

• Collect samples at defined time points (e.g., developmental stages or treatment intervals)

• Purify ribosomes using established protocols

• Perform western blotting with anti-RPS10 antibodies

• Quantify the ratio of modified to unmodified RPS10 using densitometry

• Correlate modification patterns with biological processes or cellular responses

Research has demonstrated that Znf598-mediated RPS10/eS10 ubiquitination at lysine 139 increases during development from 0 to 24 hours post-fertilization, highlighting the importance of temporal analysis when studying these modifications.

What are the optimal protocols for analyzing RPS10's role in ribosome biogenesis?

To comprehensively analyze RPS10's contribution to ribosome biogenesis, researchers should implement the following methodological approaches:

RPS10 Manipulation Strategies:

• Employ siRNA/shRNA knockdown with validated sequences targeting RPS10

• Use CRISPR-Cas9 for generating knockout or knockin cell lines

• Complement genetic approaches with pharmacological inhibitors of ribosome biogenesis

Ribosomal Assembly Analysis:

• Perform sucrose gradient centrifugation (10-50% gradients) to examine 40S subunit formation

• Analyze fractions by western blotting using anti-RPS10 antibodies (1:500-1:2000 dilution)

• Quantify pre-rRNA processing intermediates via northern blotting

• Track rRNA maturation using pulse-chase labeling with [³H]-uridine

Subcellular Localization Protocol:

• Fix cells with 4% paraformaldehyde (15 minutes at room temperature)

• Permeabilize with 0.2% Triton X-100 (10 minutes)

• Block with 5% normal serum (1 hour)

• Incubate with anti-RPS10 antibody (1:20-1:50 dilution)

• Counterstain with markers for nucleolus, nucleoplasm, and cytoplasm

• Analyze co-localization patterns using confocal microscopy

RNA-Protein Interaction Analysis:

• Perform RNA immunoprecipitation (RIP) using validated RPS10 antibodies

• Identify RPS10-associated RNAs through RT-PCR or sequencing

• Quantify binding affinities using filter binding assays

These approaches can effectively delineate RPS10's specific contributions to ribosome biogenesis, particularly when combined with structural analyses of resulting ribosomes using techniques such as cryo-electron microscopy.

How can researchers design ELISA-based quantification systems for RPS10?

Designing robust ELISA systems for RPS10 quantification requires careful optimization of multiple parameters:

ELISA Format Selection:

• Competition ELISA format shows excellent sensitivity for RPS10 detection

• Sandwich ELISA provides higher specificity but requires two non-competing antibodies

• Direct ELISA offers simplicity but may have lower sensitivity

Sample Preparation Protocol:

• For tissue homogenates: Homogenize in PBS with protease inhibitors (10 μl/mg tissue)

• Centrifuge at 10,000 × g for 10 minutes at 4°C

• Standardize protein concentration (typically 1-5 mg/ml)

• Filter samples through 0.45 μm filters to remove particulates

Standard Curve Optimization:

• Prepare fresh standards using recombinant RPS10 protein

• Create a minimum 7-point standard curve (typically 0.1-100 ng/ml)

• Include blank controls and quality control samples

• Perform quadruplicate measurements for critical samples

Assay Protocol Refinement:

• Optimize coating concentration and buffer (typically 1-10 μg/ml in carbonate buffer)

• Determine optimal blocking agent (BSA vs. non-fat milk)

• Test various incubation times and temperatures

• Validate washing protocols to minimize background while preserving signal

Data Analysis and Validation:

• Use 4-parameter logistic regression for standard curve fitting

• Calculate assay sensitivity, working range, and precision

• Perform spike-recovery tests to assess matrix effects

• Validate through comparison with other quantitative methods

Commercial ELISA kits for RPS10 are available with reported sensitivities of 0.1 ng/ml, suitable for various sample types including cell culture supernatant, plasma, serum, and tissue homogenates.

What strategies should be employed when investigating RPS10 mutations in disease models?

Investigating RPS10 mutations in disease contexts requires comprehensive molecular and functional approaches:

Mutation Identification and Characterization:

• Sequence RPS10 in disease samples using targeted approaches

• Employ bioinformatic analyses to predict functional consequences

• Validate expression of mutant proteins using antibodies with epitopes preserved in mutants

Model System Development:

• Generate cell lines expressing wild-type or mutant RPS10 variants

• Create isogenic lines using CRISPR-Cas9 to introduce specific mutations

• Establish patient-derived cell models when available

• Consider transgenic animal models for in vivo studies

Functional Assessment Protocol:

• Evaluate ribosome assembly using polysome profiling

• Analyze global translation rates with metabolic labeling (35S-methionine incorporation)

• Assess transcript-specific translation using luciferase reporters

• Examine ribosome quality control mechanisms

Biochemical Characterization:

• Express and purify recombinant wild-type and mutant RPS10 proteins

• Perform binding assays to assess interactions with other ribosomal components

• Evaluate structural alterations using limited proteolysis and circular dichroism

• Analyze post-translational modification patterns, especially ubiquitination status

Therapeutic Strategy Evaluation:

• Test compounds that promote read-through of premature termination codons

• Evaluate antisense oligonucleotides for splicing modulation

• Assess gene therapy approaches for severe loss-of-function mutations

These strategies can be effectively implemented using the range of RPS10 antibodies described in the search results, selecting those with epitopes preserved in the specific mutations under investigation.

What are the critical storage parameters for maintaining RPS10 antibody performance?

Proper storage is essential for maintaining RPS10 antibody functionality and experimental reproducibility:

Storage Conditions:

• Store most RPS10 antibodies at -20°C for long-term stability

• Maintain in appropriate storage buffers (typically PBS with 0.02% sodium azide and 50% glycerol at pH 7.3)

• Small volume preparations (20μl) often contain 0.1% BSA as stabilizer

• Antibodies remain stable for approximately one year when stored properly

Handling Recommendations:

• Avoid repeated freeze-thaw cycles by preparing appropriate working aliquots

• Allow antibodies to equilibrate to room temperature before opening

• Centrifuge briefly before use to collect solution at the bottom of the vial

• Return to -20°C promptly after use

Conjugated Antibody Considerations:

• Store fluorophore-conjugated antibodies (like Alexa Fluor 568 Anti-RPS10) protected from light

• Enzyme-conjugated antibodies (such as AP or HRP conjugates) require special attention to avoid denaturation

• Follow specific recommendations for each conjugate type

For maximum shelf life and performance, aliquoting is generally unnecessary for -20°C storage of RPS10 antibodies, according to manufacturer recommendations.

How can researchers troubleshoot common issues with RPS10 antibody applications?

When working with RPS10 antibodies, researchers may encounter various technical challenges that can be addressed through systematic troubleshooting:

Western Blot Issues:

• Multiple bands: Optimize primary antibody concentration (1:1000-1:2000), check sample preparation, confirm specificity with blocking peptide

• Weak signal: Increase antibody concentration, extend incubation time, enhance detection system sensitivity

• High background: Increase blocking stringency, optimize washing steps, reduce secondary antibody concentration

Immunohistochemistry Challenges:

• Poor staining: Optimize antigen retrieval (TE buffer pH 9.0 recommended for RPS10), adjust antibody concentration (1:50-1:200)

• Non-specific staining: Include appropriate blocking steps, titrate antibody concentration, include absorption controls

• Variable results: Standardize fixation protocols, control incubation times and temperatures

Immunofluorescence Optimization:

• Low signal: Use recommended dilution (1:20-1:50), increase exposure time, employ signal amplification systems

• High background: Increase blocking stringency, optimize washing steps, use highly cross-adsorbed secondary antibodies

• Photobleaching: Minimize exposure to light, use antifade mounting media, consider directly conjugated antibodies

ELISA Troubleshooting:

• Poor standard curve: Prepare fresh standards, optimize coating conditions, ensure proper blocking

• Sample matrix effects: Perform spike-recovery tests, optimize sample dilution, consider sample pre-treatment

• Inconsistent replicates: Standardize pipetting techniques, maintain consistent incubation times, use calibrated equipment

A verified customer review noted that the mouse monoclonal antibody (67609-1-Ig) showed excellent specificity with only one band corresponding to RPS10's molecular weight, suggesting this antibody may be particularly valuable for applications requiring high specificity.

What optimization strategies are recommended for dual detection of RPS10 and interacting proteins?

For effective dual detection of RPS10 and its interacting proteins, researchers should implement these optimization strategies:

Antibody Selection for Dual Detection:

• Choose antibodies raised in different host species (e.g., rabbit anti-RPS10 and mouse anti-interacting protein)

• Ensure antibodies recognize non-overlapping epitopes

• Consider directly conjugated primary antibodies to eliminate cross-reactivity issues

• Validate each antibody individually before dual-labeling experiments

Sequential Immunostaining Protocol:

• Incubate with first primary antibody (anti-RPS10 at 1:20-1:50 dilution)

• Apply corresponding secondary antibody

• Block with excess unconjugated host IgG from second primary species

• Incubate with second primary antibody

• Apply corresponding secondary antibody with distinct fluorophore

Simultaneous Staining Approach:

• Mix compatible primary antibodies at optimal dilutions

• Incubate simultaneously when antibodies require identical conditions

• Apply mixture of secondary antibodies with distinct fluorophores

• Include appropriate controls to assess cross-reactivity

Controls for Dual Detection:

• Single primary antibody controls to evaluate bleed-through

• Absorption controls using blocking peptides

• Isotype controls to assess non-specific binding

• Cross-reactivity controls by omitting one primary antibody

Imaging Optimization:

• Use sequential scanning for confocal microscopy to minimize spectral overlap

• Implement spectral unmixing algorithms when using closely related fluorophores

• Optimize detector settings for each fluorophore independently

• Employ appropriate co-localization analysis tools

Pre-conjugated antibodies, such as Alexa Fluor 568 Anti-RPS10 antibody [EPR8545] (ab312401), simplify dual staining protocols by eliminating secondary antibody cross-reactivity concerns.

What emerging technologies show promise for advanced RPS10 research?

Several cutting-edge technologies are poised to advance RPS10 research significantly:

Single-Molecule Approaches:

• PALM/STORM super-resolution microscopy for nanoscale localization of RPS10 within ribosomes

• Single-molecule FRET to analyze dynamic interactions with other ribosomal components

• Optical tweezers to study mechanical properties of ribosomes containing wild-type versus mutant RPS10

Advanced Proteomics:

• Proximity labeling methods (BioID, APEX) to identify transient RPS10 interactors

• Cross-linking mass spectrometry for structural mapping of RPS10 interfaces

• Top-down proteomics for comprehensive post-translational modification analysis

Genome Editing Applications:

• CRISPR base editing for precise introduction of disease-associated mutations

• CRISPR activation/interference systems to modulate RPS10 expression

• Scarless tagging for visualization and purification of endogenous RPS10

Translation Dynamics Analysis:

• Ribosome profiling with sub-codon resolution

• Live-cell translation biosensors incorporating RPS10 to monitor ribosome dynamics

• Cryo-electron tomography of ribosomes in cellular contexts

These emerging technologies will benefit from the development of specialized RPS10 antibodies optimized for particular applications, including those with enhanced sensitivity, specificity for modified forms, and compatibility with new methodological approaches.

How can RPS10 research contribute to understanding ribosomopathies and cancer biology?

RPS10 research holds significant potential for advancing our understanding of ribosomopathies and cancer:

Ribosomopathies:

• Investigating RPS10 mutations in Diamond-Blackfan anemia and other congenital disorders

• Characterizing differential effects of RPS10 alterations on specific mRNA translation

• Developing targeted therapies based on ribosome specialized functions

Cancer Biology Applications:

• Analyzing RPS10 post-translational modifications in transformed cells

• Investigating ribosome heterogeneity and specialized ribosomes in cancer progression

• Developing diagnostic markers based on RPS10 modification patterns

Therapeutic Target Exploration:

• Exploiting RPS10 interactions for small molecule development

• Targeting RPS10 ubiquitination pathways to modulate translation

• Identifying synthetic lethal interactions in RPS10-mutated cancers

Diagnostic Development:

• Creating modification-specific antibodies for cancer classification

• Developing ELISA-based detection systems for modified RPS10 in patient samples

• Establishing RPS10 alteration patterns as prognostic indicators