RPS3 Antibody

What is RPS3 and why is it an important research target?

Ribosomal protein S3 (RPS3) is a 26.7 kDa protein component of the 40S small ribosomal subunit with multifunctional characteristics beyond protein synthesis. RPS3 exhibits various extra-ribosomal functions including DNA repair endonuclease activity, transcriptional regulation through NF-κB complexes, and involvement in apoptotic pathways . Recent research has identified RPS3 as being secreted from cancer cells, suggesting its potential utility as a cancer biomarker . Additionally, RPS3 plays critical roles in DNA damage recognition with high binding affinity for 8-oxoguanine residues and interactions with DNA repair proteins such as OGG1 and Ref-1 . Its multiple cellular functions make it a significant target for studies in cancer biology, DNA damage response, and ribosomal function.

How should I choose between monoclonal and polyclonal RPS3 antibodies for my research?

The selection between monoclonal and polyclonal RPS3 antibodies depends on your experimental objectives:

Monoclonal antibodies:

• Offer higher specificity targeting single epitopes (e.g., mAb M7 targets amino acids 213-221 )

• Provide consistent lot-to-lot reproducibility for longitudinal studies

• Preferable for applications requiring precise epitope recognition

• Examples: RPS3 Mouse Monoclonal antibody (NovoPro) or Anti-RPS3 antibody [EPR7807] (Abcam)

Polyclonal antibodies:

• Recognize multiple epitopes, providing stronger signal in applications like Western blot

• Better for detecting denatured proteins or proteins with post-translational modifications

• Useful when protein conformation might vary between samples

• Examples: Rabbit anti-RPS3 polyclonal antibody (R2) recognizing amino acids 203-230

For critical applications requiring validation of results, using both antibody types can provide complementary data and increase confidence in your findings .

What species reactivity should I consider when selecting an RPS3 antibody?

When selecting an RPS3 antibody, consider the evolutionary conservation of RPS3 across species and match your experimental model with the antibody's validated reactivity. Most commercial RPS3 antibodies demonstrate reactivity across human, mouse, and rat samples due to high sequence homology . For example:

For less common experimental models, consider antibodies with broader reactivity profiles. Some RPS3 antibodies report cross-reactivity with additional species such as canine, porcine, and yeast orthologs based on gene homology . When working with uncommon model organisms, epitope sequence alignment analysis is recommended before antibody selection .

What are the optimal dilutions and conditions for using RPS3 antibodies in Western blot applications?

Optimal dilution ratios for RPS3 antibodies in Western blot vary by manufacturer and antibody type. Based on the search results, here are recommended guidelines:

For optimal results:

• Use PVDF or nitrocellulose membranes (0.45 μm pore size)

• Block with 5% non-fat milk or BSA in PBST for 1-2 hours

• Incubate primary antibody at 4°C overnight for best signal-to-noise ratio

• Include positive controls from validated sources such as HeLa cells, HEK-293 cells, or brain tissue

• Strip and reprobe with ribosomal housekeeping protein antibodies for normalization

Note that observed molecular weights may vary slightly (26.7-33 kDa) depending on the antibody used and experimental conditions .

How should I optimize immunohistochemistry protocols for RPS3 antibodies?

To optimize immunohistochemistry (IHC) protocols with RPS3 antibodies, consider these methodology recommendations:

Antigen Retrieval:

• For formalin-fixed paraffin-embedded tissues, use TE buffer at pH 9.0 as the preferred method

• Alternative: citrate buffer at pH 6.0 with heat-induced epitope retrieval (95-100°C for 15-20 minutes)

Antibody Dilutions:

• Proteintech 11990-1-AP: 1:500-1:2000

• Proteintech 15198-1-AP: 1:200-1:800

• Proteintech 66046-1-Ig: 1:50-1:500

• NovoPro RPS3 mAb: 1:20-1:200

Validated Tissue Samples:

• Human pancreas tissue shows positive staining with RPS3 antibodies

• Human colon cancer tissue demonstrates strong RPS3 expression

• Rat liver tissue has been validated as a positive control

Protocol Optimization:

• Use appropriate positive controls alongside experimental samples

• Include negative controls (omitting primary antibody)

• Titrate antibody concentration to determine optimal signal-to-background ratio

• Consider signal amplification systems for lower abundance targets

• Develop sections using DAB and counterstain with hematoxylin

• For multiplexing with other markers, sequential staining with appropriate blockers between steps is recommended

Validation of staining patterns is crucial, as RPS3 typically shows both cytoplasmic (ribosomal) and nuclear (DNA repair) localization depending on cellular conditions .

What are the best practices for immunoprecipitation using RPS3 antibodies?

For successful immunoprecipitation (IP) of RPS3, follow these best practices:

Antibody Selection and Amounts:

• Use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate

• For Proteintech antibodies (11990-1-AP, 15198-1-AP, 66046-1-Ig), 2-3 μg per sample is typically optimal

• NovoPro RPS3 monoclonal antibody has been validated at 1:1000-1:10000 dilution for IP

Validated IP Protocol:

• Prepare cell lysates in non-denaturing lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate with protease inhibitors)

• Pre-clear lysate with Protein A/G beads for 1 hour at 4°C

• Incubate pre-cleared lysate with RPS3 antibody overnight at 4°C

• Add fresh Protein A/G beads and incubate for 2-4 hours at 4°C

• Wash 4-5 times with lysis buffer containing reduced detergent

• Elute with 2X SDS sample buffer and analyze by Western blot

Validated Cell Lines:

• HEK-293 cells show consistent results for RPS3 IP

• Mouse testis tissue has been validated for successful IP with 11990-1-AP

• Mouse brain tissue works well with 15198-1-AP

Technical Considerations:

• Cross-linking antibodies to beads may reduce background from heavy/light chains

• For co-IP experiments investigating RPS3 interactions with other proteins (e.g., TRIP13 or NF-κB components), mild lysis conditions are essential to preserve protein complexes

• Include an isotype control antibody IP as a negative control to confirm specificity

When investigating post-translational modifications of RPS3, such as phosphorylation mediated by PKCδ, modified buffer conditions may be required to preserve these modifications .

Why might I observe different molecular weights for RPS3 in Western blots?

The discrepancy in observed molecular weights for RPS3 in Western blots is a common issue that can be attributed to several factors:

Expected vs. Observed Molecular Weights:

• Theoretical molecular weight: 26.7 kDa based on amino acid sequence

• Commonly observed weights: 27-33 kDa

Causes for Molecular Weight Variations:

• Post-translational modifications: Phosphorylation of RPS3 by PKCδ can increase apparent molecular weight

• Technical variations: Different running buffer compositions and gel percentages affect migration patterns

• Antibody specificity: Different antibodies may recognize forms of RPS3 with specific modifications

• Sample preparation: Heat-induced aggregation or incomplete denaturation can affect migration

• SDS-binding variation: Highly basic proteins like RPS3 can bind SDS irregularly

For example, Proteintech reports:

• 11990-1-AP detects RPS3 at 26.7 kDa

• 15198-1-AP and 66046-1-Ig detect RPS3 at 33 kDa

To address this issue:

• Include positive control lysates (e.g., HeLa cells) with known RPS3 expression

• Consider using gradient gels (4-20%) for better resolution

• When reporting results, always specify the antibody used and observed molecular weight

• For publication-quality blots, validate with a second RPS3 antibody recognizing a different epitope

How can I verify the specificity of an RPS3 antibody?

Verifying RPS3 antibody specificity is crucial for reliable research outcomes. Multiple validation approaches should be employed:

1. Genetic Validation:

• RPS3 knockout/knockdown experiments: Test antibody on RPS3 knockout samples to confirm signal loss

• Haematologica 2023 study demonstrated RPS3 antibody specificity using knockout MM cells

• Overexpression systems: Use cells transfected with tagged RPS3 to confirm co-localization with antibody signal

2. Epitope Competition:

• Block antibody with immunizing peptide prior to application

• Decreasing or eliminated signal indicates specific epitope binding

• For antibodies with mapped epitopes (e.g., mAb M7 binding aa 213-221), synthetic peptides can be used

3. Cross-Validation with Multiple Antibodies:

• Test multiple antibodies targeting different epitopes of RPS3

• Concordant results increase confidence in specificity

• Example antibody combinations:

  • Polyclonal (Proteintech 11990-1-AP) + Monoclonal (Proteintech 66046-1-Ig)

  • N-terminal epitope antibody + C-terminal epitope antibody

4. Western Blot Analysis:

• Verify single band at expected molecular weight (26.7-33 kDa)

• Test across multiple cell types with known RPS3 expression

• Validated cell lines: HeLa, HEK-293, COLO 320, NIH/3T3

5. Mass Spectrometry Confirmation:

• Perform IP with RPS3 antibody followed by mass spectrometry analysis

• Confirms presence of RPS3 peptides in immunoprecipitated material

For research applications requiring absolute certainty of specificity, combining at least two validation methods is recommended .

What potential cross-reactivity issues should I be aware of when using RPS3 antibodies?

When using RPS3 antibodies, researchers should be aware of several potential cross-reactivity issues:

1. Homologous Ribosomal Proteins:

• The ribosome contains multiple proteins with structural similarities

• RPS3 antibodies may cross-react with other members of the RPS family

• Particularly concerning: RPS3A, which shares sequence homology with RPS3

2. Species-Specific Considerations:

• Despite high conservation, species-specific variations in RPS3 sequence exist

• Antibodies raised against human RPS3 may show different affinities for orthologs

• Testing antibody reactivity with recombinant RPS3 from your experimental species is recommended

3. Isoform Specificity:

• RPS3 has multiple splice variants and isoforms

• Not all antibodies detect all isoforms equally

• Epitope location within the protein determines which isoforms are recognized

4. Epitope Masking in Protein Complexes:

• RPS3 functions within ribosomal complexes and in association with other proteins

• Epitopes may be masked in certain protein-protein interactions

• Different antibodies may perform differently depending on RPS3's association state

5. Mitochondrial vs. Cytoplasmic Forms:

• RPS3 is present in both mitochondria and cytoplasm

• Post-translational modifications differ between locations

• Antibody selection should consider the subcellular compartment of interest

To minimize cross-reactivity issues:

• Validate antibodies in multiple applications

• Include appropriate controls (knockout/knockdown)

• Consider using antibodies with mapped epitopes (e.g., pAb R2, mAb M7, mAb M8)

• When absolute specificity is required, use multiple antibodies targeting different RPS3 epitopes

How can RPS3 antibodies be utilized to study DNA damage repair mechanisms?

RPS3 antibodies provide valuable tools for investigating DNA damage repair mechanisms due to RPS3's dual role in ribosomes and DNA repair:

Experimental Approaches:

• Chromatin Immunoprecipitation (ChIP):

  • Use RPS3 antibodies (e.g., Proteintech 66046-1-Ig) for ChIP to identify RPS3 binding to damaged DNA regions

  • ChIP-seq can map genome-wide RPS3 recruitment to DNA damage sites

• Co-immunoprecipitation Studies:

  • RPS3 antibodies can pull down complexes containing DNA repair proteins

  • Validated interactions include OGG1, UNG1, and APEX1 DNA repair enzymes

  • Protocol: Cross-link cells, lyse in mild conditions, immunoprecipitate with RPS3 antibody, analyze interacting proteins

• Translocation Analysis:

  • Track RPS3 movement from cytoplasm to nucleus following DNA damage

  • Immunofluorescence with RPS3 antibodies at different timepoints after damage induction

  • Quantify nuclear/cytoplasmic ratio changes using fluorescence intensity measurements

DNA Lesion-Specific Applications:

• RPS3 has high affinity for 8-oxoguanine lesions caused by oxidative stress

• Study protocol: Create 8-oxoG lesions with H₂O₂ treatment, analyze RPS3 recruitment using immunofluorescence

• Co-staining with γH2AX confirms DNA damage site colocalization

DNA Repair Kinetics Analysis:

• Induce DNA damage with radiation or chemical agents

• Fix cells at various timepoints post-damage

• Co-stain with RPS3 antibody and DNA repair markers

• Quantify recruitment/dissociation kinetics through high-content imaging

Research has demonstrated that RPS3 stimulates base excision repair processes by enhancing the activities of OGG1 and APEX1, making RPS3 antibodies essential tools for mechanistic studies of these repair pathways .

What methodologies can be used to study RPS3's role in NF-κB signaling with RPS3 antibodies?

RPS3 functions as a non-Rel subunit of NF-κB complexes, making it a critical factor in NF-κB signaling. Here are methodologies utilizing RPS3 antibodies to investigate this relationship:

1. Sequential ChIP (ChIP-reChIP) Analysis:

• First ChIP: Use antibodies against NF-κB subunits (p65/RelA)

• Second ChIP: Re-immunoprecipitate with RPS3 antibody

• This identifies genomic loci where both proteins co-localize

• Protocol specifics: Crosslink cells with 1% formaldehyde, sonicate chromatin to 200-500bp fragments, perform sequential immunoprecipitations with validated antibody combinations

2. Co-immunoprecipitation of NF-κB Complexes:

• Immunoprecipitate with RPS3 antibodies (e.g., Proteintech 66046-1-Ig)

• Western blot for NF-κB components (p65, p50)

• Analyze how stimuli affect complex formation

• Example stimuli: TNF-α treatment, LPS activation, or oxidative stress

3. RPS3 Phosphorylation Analysis:

• RPS3 phosphorylation status affects its NF-κB-related functions

• Use phospho-specific antibodies after immunoprecipitation with RPS3 antibodies

• Research from Haematologica (2023) revealed TRIP13-mediated RPS3 phosphorylation via PKCδ activates canonical NF-κB signaling

4. Proximity Ligation Assay (PLA):

• Visualize and quantify RPS3-NF-κB interactions in situ

• Use RPS3 antibody paired with p65/RelA antibody

• PLA signal indicates close proximity (<40nm) between proteins

• Quantify interaction events per cell under different conditions

5. Gel Shift/EMSA with Supershift:

• Prepare nuclear extracts from cells

• Perform EMSA with labeled NF-κB consensus probes

• Add RPS3 antibody for supershift

• Presence of supershift confirms RPS3 in DNA-binding complex

6. Transcriptional Reporter Assays:

• Transfect cells with NF-κB-responsive luciferase reporter

• Manipulate RPS3 levels (overexpression/knockdown)

• Measure reporter activity with/without RPS3 antibody microinjection

• This approach demonstrates functional consequences of RPS3-NF-κB interaction

These methodologies have revealed that RPS3 enhances p65 binding to DNA and promotes transcription of specific NF-κB target genes, particularly in contexts like inflammation and cancer .

How can epitope mapping information enhance experimental design with RPS3 antibodies?

Epitope mapping information provides crucial insights for optimizing experimental design with RPS3 antibodies:

1. Strategic Selection Based on Functional Domains:

• RPS3 has distinct functional domains:

  • N-terminal region: Ribosomal function

  • KH domain: RNA/DNA binding

  • C-terminal region: Protein-protein interactions

• Select antibodies based on which domain is relevant to your research question

• Example: For DNA repair studies, antibodies targeting the KH domain (aa 116-148) are ideal

2. Avoiding Epitope Masking:
Known mapped epitopes from research:

• pAb R2: epitope from aa 203-230

• mAb M7: epitope from aa 213-221

• mAb M8: epitope from aa 197-219

When studying RPS3 in protein complexes, epitope accessibility is critical. If an interaction partner binds to the same region as your antibody epitope, signal detection may be hindered. Multiple antibodies targeting different epitopes can overcome this limitation.

3. Post-translational Modification Studies:

• RPS3 undergoes various modifications including phosphorylation and acetylation

• Choose antibodies with epitopes distant from modification sites when studying modified forms

• Example: When studying PKCδ-mediated phosphorylation of RPS3, avoid antibodies whose epitopes include the phosphorylation sites

4. Detection of Specific Conformational States:

• RPS3 adopts different conformations in ribosomes versus during DNA repair

• Antibodies recognizing conformation-specific epitopes can distinguish these states

• Linear epitope antibodies (e.g., mAb M7) versus conformational epitope antibodies provide complementary information

5. Multi-antibody Approaches:
Design experiments using antibody combinations targeting different epitopes:

• Combine N-terminal and C-terminal targeting antibodies for co-localization studies

• Use antibodies with non-overlapping epitopes for sandwich ELISA development

• For precipitation of intact complexes, select antibodies whose epitopes remain accessible

6. Translational Research Applications:
For cancer biomarker studies, epitope information is essential:

• Secreted RPS3 from cancer cells may expose different epitopes than intracellular RPS3

• Antibodies targeting epitopes retained in secreted forms maximize detection sensitivity

Understanding the precise epitope locations has enabled researchers to develop sophisticated experimental strategies, as demonstrated in the 2021 study mapping rpS3 antibody epitopes through peptide scanning techniques .

How can RPS3 antibodies be utilized for cancer biomarker studies?

RPS3 antibodies have emerging potential for cancer biomarker applications based on several key research findings:

Research Foundation:

• RPS3 is overexpressed in multiple cancers, including colorectal cancer

• RPS3 is secreted from cancer cells, making it detectable in liquid biopsies

• In multiple myeloma, RPS3 mediates drug resistance and is linked to poor prognosis

Methodological Approaches:

• Tissue Microarray (TMA) Analysis:

  • Use RPS3 antibodies (e.g., Proteintech 66046-1-Ig, 1:50-1:500 dilution) for IHC of cancer tissue microarrays

  • Validated in human colon cancer and ovarian tumor tissues

  • Quantify expression levels using H-score or digital image analysis

  • Correlate with clinical outcomes for prognostic marker development

• Liquid Biopsy Development:

  • Sandwich ELISA protocols using two antibodies targeting different RPS3 epitopes

  • Example pair: mAb M8 (aa 197-219) as capture antibody and pAb R2 (aa 203-230) as detection antibody

  • Apply to serum/plasma samples from cancer patients versus healthy controls

  • Establish normal range and disease-associated thresholds

• Multiplex Immunofluorescence Panels:

  • Combine RPS3 antibodies with other cancer markers

  • Use in FFPE tumor samples to analyze co-expression patterns

  • Quantify cellular localization changes (nuclear vs. cytoplasmic RPS3)

  • Nuclear RPS3 correlates with NF-κB activity and therapy resistance

• Therapy Response Monitoring:

  • Recent research demonstrated RPS3's role in proteasome inhibitor resistance in multiple myeloma

  • Monitor RPS3 levels before and during treatment using validated antibodies

  • Protocol: Serial sampling of bone marrow aspirates with subsequent IF/IHC

  • Decreasing RPS3 levels correlate with therapeutic response

Validation Requirements:

• Clinical-grade antibody validation includes:

  • Reproducibility testing across multiple lots

  • Standardized staining protocols with controls

  • Blinded observer scoring systems

  • Comparison with established prognostic markers

The 2023 Haematologica study provides compelling evidence for RPS3 as both a biomarker and therapeutic target, particularly when combined with assessment of its interaction partner TRIP13 in multiple myeloma .

What methods can be used to develop an RPS3-targeted sandwich ELISA for research applications?

Developing a sandwich ELISA for RPS3 detection requires careful antibody selection and protocol optimization:

Antibody Pair Selection Strategy:

• Choose antibodies recognizing non-overlapping epitopes:

  • Capture antibody: mAb M8 (epitope: aa 197-219)

  • Detection antibody: pAb R2 (epitope: aa 203-230) or commercial antibody with different epitope

  • Alternatively, use antibodies from different species (e.g., mouse monoclonal capture + rabbit polyclonal detection)

• Validated antibody combinations from research:

  • Mouse anti-rpS3 monoclonal (MyBioSource) paired with rabbit anti-rpS3 polyclonal (lab-produced)

  • For commercial options: Proteintech 66046-1-Ig (mouse mAb) paired with 11990-1-AP (rabbit pAb)

Optimized ELISA Protocol:

• Plate Coating:

  • Coat high-binding 96-well plates with capture antibody (10 μg/mL in carbonate buffer, pH 9.6)

  • Incubate at 4°C overnight

  • Wash 4× with PBST (PBS + 0.1% Tween-20)

• Blocking and Sample Addition:

  • Block with 4% BSA in PBST for 1-1.5 hours at 37°C

  • Wash 4× with PBST

  • Add samples and standards (recombinant RPS3 protein, 0-1000 ng/mL)

  • Incubate at 37°C for 1.5-2 hours

• Detection:

  • Wash 4× with PBST

  • Add detection antibody (0.5-1 μg/mL in 1% BSA-PBST)

  • Incubate at 37°C for 1-1.5 hours

  • Wash 4× with PBST

  • Add HRP-conjugated secondary antibody (1:5000 in 1% BSA-PBST)

  • Incubate at 37°C for 1 hour

• Visualization:

  • Wash 4× with PBST

  • Add TMB substrate (100 μL/well)

  • Monitor color development

  • Stop reaction with 50 μL 2N H₂SO₄

  • Read absorbance at 450 nm

Performance Optimization:

• Determine limit of detection (typically 5-10 ng/mL for optimized RPS3 ELISA)

• Establish standard curve using purified recombinant RPS3

• Validate linearity in relevant sample matrices (cell lysates, serum, etc.)

• Test specificity by spiking related proteins (RPS3A, other ribosomal proteins)

This methodology has been successfully applied to detect RPS3 in cancer cell supernatants and patient samples, as demonstrated in epitope mapping studies published in 2021 .

What are the critical considerations for investigating RPS3 in drug resistance mechanisms?

Investigating RPS3's role in drug resistance mechanisms requires specific methodological approaches and careful experimental design:

1. Expression Analysis in Resistant vs. Sensitive Cells:

• Western blot protocol: Compare RPS3 levels in paired sensitive/resistant cell lines

  • Use Proteintech 66046-1-Ig (1:20000-1:100000 dilution) for highest sensitivity

  • Include appropriate loading controls (β-actin, GAPDH)

  • Quantify expression differences using densitometry

• Immunofluorescence approach:

  • Fix cells with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100

  • Stain with RPS3 antibody (1:50-1:500 dilution)

  • Analyze subcellular localization differences between sensitive/resistant cells

  • Nuclear accumulation of RPS3 correlates with drug resistance

2. Functional Studies:

• RPS3 knockdown/knockout experiments:

  • Generate RPS3 knockout cell lines using CRISPR-Cas9

  • Measure drug sensitivity shifts after RPS3 depletion

  • The 2023 Haematologica study demonstrated RPS3 knockout inhibited MM cell growth and induced apoptosis

• Overexpression studies:

  • Transfect cells with RPS3 expression constructs

  • Test drug sensitivity changes

  • RPS3 overexpression has been shown to mediate proteasome inhibitor resistance

3. Pathway Analysis:

• NF-κB signaling assessment:

  • Measure canonical NF-κB activity in drug-resistant cells

  • Co-immunoprecipitate RPS3 with NF-κB components

  • Use ChIP to analyze RPS3 recruitment to NF-κB target genes

  • TRIP13-mediated phosphorylation of RPS3 activates NF-κB signaling in drug-resistant MM

4. Therapeutic Targeting Strategy:

• Combined inhibition approach:

  • Target RPS3-TRIP13 interaction with small molecule inhibitors

  • DCZ0415 (TRIP13 inhibitor) combined with bortezomib showed synergistic cytotoxicity

  • Measure RPS3 phosphorylation status to monitor efficacy

5. Clinical Correlation:

• Patient sample analysis:

  • Compare RPS3 expression in responders vs. non-responders

  • Use IHC with Proteintech 66046-1-Ig (1:50-1:500) on patient biopsies

  • Correlate RPS3 levels with survival outcomes

  • RPS3 overexpression shortened survival of MM tumor-bearing animals

These methodologies collectively provide a comprehensive framework for investigating RPS3 in drug resistance, as demonstrated in the 2023 Haematologica study on multiple myeloma .

What storage and handling practices maximize RPS3 antibody performance and shelf-life?

Proper storage and handling of RPS3 antibodies are critical for maintaining their performance and extending shelf-life:

Optimal Storage Conditions:

Handling Recommendations:

• Allow antibody to warm to room temperature before opening vial to prevent condensation

• Briefly centrifuge before opening to collect solution at the bottom

• Use clean, RNase/DNase-free pipette tips for each withdrawal

• Return to -20°C immediately after use

• Transport on ice when moving between laboratories

Stability Indicators:

• Typical shelf-life: One year after shipment when stored properly at -20°C

• Visual inspection: Solution should remain clear; cloudiness may indicate protein denaturation

• Performance validation: Periodically test on positive control samples (e.g., HeLa or HEK-293 cell lysates)

Application-Specific Considerations:

• For IHC applications: Diluted working solutions can be stored at 4°C for up to one week

• For WB applications: Diluted antibody in 5% BSA/TBST can typically be reused 2-3 times when stored at 4°C with 0.02% sodium azide

• For IP applications: Avoid repeated freeze-thaw of antibody-bead conjugates

Monitoring Recommendations:

• Document lot numbers and expiration dates

• Maintain consistency in storage conditions

• Include positive controls in each experiment to monitor antibody performance over time

• Record signal-to-noise ratios to track potential degradation

Following these practices will help ensure consistent, reproducible results when working with RPS3 antibodies across various applications.

How should I validate RPS3 antibodies for reproducible research?

A comprehensive validation protocol for RPS3 antibodies ensures reproducible research results:

Multi-tier Validation Framework:

Tier 1: Basic Characterization

• Western blot assessment using positive control samples:

  • Human: HeLa, HEK-293, COLO 320 cells

  • Mouse: NIH/3T3 cells, brain tissue

  • Rat: PC-12 cells, brain tissue

• Verify molecular weight (26.7-33 kDa depending on antibody)

• Test lot-to-lot consistency with standardized lysates

Tier 2: Genetic Validation

• RPS3 knockdown/knockout controls:

  • siRNA knockdown (transient)

  • shRNA knockdown (stable)

  • CRISPR-Cas9 knockout (complete)

• Signal reduction/elimination confirms specificity

• Overexpression of tagged RPS3 should show co-localization

Tier 3: Orthogonal Method Validation

• Compare results across multiple applications (WB, IF, IHC, IP)

• Use different antibodies targeting distinct epitopes:

  • mAb M7 (epitope: aa 213-221)

  • mAb M8 (epitope: aa 197-219)

  • pAb R2 (epitope: aa 203-230)

• Concordant results across methods increase confidence

Tier 4: Cross-laboratory Validation

• Implement standardized protocols across research groups

• Establish common positive controls and quantification methods

• Document batch effects and antibody performance metrics

Application-Specific Validation:

• For Western blot: Include gradient loading to establish linearity of detection

• For IHC/IF: Include known positive/negative tissues/cells in each run

• For IP: Confirm pull-down specificity by mass spectrometry

• For ChIP: Include IgG control and known RPS3-binding regions

Documentation Standards:

• Record complete antibody information:

  • Supplier and catalog number

  • Clone designation for monoclonals

  • Lot number

  • RRID (Research Resource Identifier)

• Example RRIDs: AB_2180758 (Proteintech 11990-1-AP), AB_2253905 (Proteintech 15198-1-AP), AB_11182493 (Proteintech 66046-1-Ig)

This validation framework aligns with current reproducibility initiatives in antibody-based research and has been successfully applied in RPS3 studies as demonstrated in the 2021 epitope mapping publication .

What controls should be included when using RPS3 antibodies for quantitative applications?

For quantitative applications using RPS3 antibodies, a robust set of controls is essential to ensure accuracy and reliability:

1. Sample-Related Controls:

Positive Controls:

• Cell lines with validated RPS3 expression:

  • HeLa cells, HEK-293 cells, COLO 320 cells for human samples

  • NIH/3T3 cells for mouse samples

  • PC-12 cells for rat samples

• Recombinant RPS3 protein at known concentrations for standard curves

Negative Controls:

• RPS3 knockdown/knockout samples (CRISPR, siRNA, or shRNA)

• Species-mismatched samples when testing specificity

• Isotype controls for monoclonal antibodies

2. Technical Controls:

Loading Controls for Western Blot:

• Total protein normalization using stain-free gels or REVERT total protein stain

• Housekeeping proteins (with caution):

  • β-actin, GAPDH for cytoplasmic normalization

  • Histone H3 for nuclear fraction normalization

  • Other ribosomal proteins (e.g., RPLP0) for ribosomal fraction comparison

Dilution Controls:

• Serial dilution of sample to confirm linearity of signal

• Example: 5-point 2-fold dilution series to establish quantification range

• Important for verifying antibody is used within linear dynamic range

3. Antibody-Specific Controls:

Primary Antibody Controls:

• Omission of primary antibody to assess secondary antibody background

• Blocking peptide competition to verify epitope specificity

• For monoclonals: isotype-matched irrelevant antibody control

• For polyclonals: pre-immune serum control (when available)

Secondary Detection Controls:

• Secondary-only controls to assess non-specific binding

• System suitability controls (e.g., HeLa lysate) analyzed on every experimental run

• Internal reference sample analyzed across multiple experiments for inter-assay normalization

4. Quantification Method Controls:

Standard Curve:

• For absolute quantification: Purified recombinant RPS3 standard curve

• For relative quantification: Common reference sample included in each experiment

• Quality control samples at low, medium, and high concentrations

Inter-Assay Controls:

• Common reference sample run on each experimental day

• Calculate coefficient of variation across runs (should be <15% for robust assays)

• Control charts to monitor assay performance over time

5. Application-Specific Controls:

For IHC/IF:

• Tissue microarrays with known RPS3 expression patterns

• Multiplexed staining with organelle markers to confirm subcellular localization

• Digital image analysis with standardized acquisition parameters

For ELISA:

• Spike-and-recovery controls to assess matrix effects

• Parallelism testing to verify antibody performance in different sample types