RERG Antibody

Basic Research Questions

• What is RERG and why is it important in research?

  RERG (Ras-related and estrogen-regulated growth inhibitor) is a GTPase that binds GDP/GTP and possesses intrinsic GTPase activity. It demonstrates higher affinity for GDP than for GTP. RERG has significant research importance because its overexpression leads to a reduction in the rate of proliferation, colony formation, and tumorigenic potential in cell lines . The protein functions as a tumor suppressor in several cancer types, particularly in estrogen-responsive tissues. Understanding RERG's role in cell growth regulation provides valuable insights into cancer development mechanisms and potential therapeutic targets for cancer treatment. RERG's expression patterns can serve as biomarkers for cancer prognosis and treatment response in certain contexts.

• What is the difference between RERG and ERG antibodies?

  Despite their similar abbreviations, RERG and ERG antibodies target distinct proteins with different cellular functions and significance in research:

  Characteristic	RERG Antibody	ERG Antibody
  Full Protein Name	Ras-related and estrogen-regulated growth inhibitor	ETS-related gene
  Protein Function	GTPase with growth inhibitory properties	Transcription factor
  Clinical Significance	Potential tumor suppressor	Oncogenic in prostate cancer (rearrangement marker)
  Expression Pattern	Regulated by estrogen	Expressed in endothelial cells and prostate cancer with TMPRSS2-ERG fusion
  Molecular Weight	23 kDa	Variable (most common: 54 kDa)
  Diagnostic Use	Limited	Established marker for prostate cancer diagnosis

  ERG antibodies have gained significant clinical application in prostate cancer diagnosis, with studies showing 95.7% sensitivity and 96.5% specificity for detecting ERG rearrangement-positive prostate cancers . RERG antibodies, while valuable in research settings, have not yet achieved the same level of clinical utility but remain important for investigating tumor suppression mechanisms.

Advanced Research Applications

• How can I validate the specificity of a RERG antibody for my experimental system?

  Validating RERG antibody specificity requires a multi-faceted approach to ensure reliable and reproducible results:

  1. Positive and negative control lysates: Use cell lines or tissues with known RERG expression levels. Mouse thymus and rat lung lysates have been validated as positive controls for RERG expression .

  2. Knockdown/knockout validation: Compare RERG detection in wild-type cells versus RERG-knockdown or knockout cells using siRNA or CRISPR-Cas9 technology.

  3. Recombinant protein competition: Pre-incubate the antibody with recombinant RERG protein before immunostaining or Western blotting. Specific binding will be blocked by the recombinant protein.

  4. Multiple antibody validation: Use at least two different RERG antibodies targeting distinct epitopes to confirm consistent detection patterns.

  5. Mass spectrometry correlation: Compare immunoprecipitation results with mass spectrometry analysis to confirm the identity of the detected protein.

  For Western blot validation specifically, RERG antibodies should detect bands at the predicted molecular weight of 23 kDa and potentially 37 kDa (depending on post-translational modifications) . Cross-reactivity with other Ras-family proteins should be minimal given the unique C-terminal sequence of RERG compared to other Ras proteins.

• What are the optimal conditions for immunohistochemistry with RERG antibodies?

  Achieving optimal RERG detection in tissue samples requires careful optimization of immunohistochemistry protocols:

  Detailed IHC-P Protocol for RERG Detection:

  1. Tissue preparation: Fix tissues in 10% neutral buffered formalin for 24-48 hours, followed by paraffin embedding. Cut sections at 4-5 μm thickness.

  2. Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95-100°C for 20 minutes provides optimal results for most RERG antibodies .

  3. Blocking: Use 5% normal goat serum in PBS with 0.1% Triton X-100 for 1 hour at room temperature.

  4. Primary antibody incubation: Apply RERG antibody at 1/100 dilution (for ab235439) or 1/100-1/300 dilution (for other antibodies) and incubate overnight at 4°C .

  5. Detection system: For most consistent results, use a polymer-based detection system rather than biotin-avidin systems to minimize background.

  6. Counterstaining: Light hematoxylin counterstaining for 8 minutes followed by bluing reagent for 4 minutes at 37°C .

  7. Controls: Include positive control tissues (breast tissue, thymus) and technical negative controls (primary antibody omission) in each staining run.

  The staining pattern for RERG is typically cytoplasmic with some nuclear localization, reflecting its role in cellular signaling and growth regulation. Careful optimization of antibody concentration is essential, as excess antibody can lead to nonspecific background staining.

• How can I troubleshoot weak or absent RERG signal in Western blot experiments?

  When encountering weak or absent RERG signals in Western blot experiments, implement this systematic troubleshooting approach:

  1. Sample preparation optimization:

     • Ensure efficient protein extraction using RIPA buffer supplemented with protease inhibitors

     • Avoid repeated freeze-thaw cycles of protein samples

     • Confirm protein concentration using BCA or Bradford assay

  2. Loading control verification:

     • Confirm equal loading with housekeeping proteins (β-actin, GAPDH)

     • Verify protein transfer efficiency with reversible staining (Ponceau S)

  3. Antibody-specific parameters:

     • Increase antibody concentration (try 1/300 dilution if 1/500 was unsuccessful)

     • Extend primary antibody incubation time (overnight at 4°C)

     • Use freshly prepared antibody dilutions

  4. Signal enhancement strategies:

     • Implement more sensitive detection systems (ECL-Plus)

     • Increase exposure time during imaging

     • Consider signal amplification systems

  5. Protein size considerations:

     • Use 8-10% gels for optimal resolution of RERG (predicted band sizes: 23 kDa, 37 kDa)

     • Include molecular weight markers covering low molecular weight ranges

  6. Membrane optimization:

     • Use PVDF membranes rather than nitrocellulose for better protein retention

     • Optimize blocking conditions (5% non-fat milk vs. BSA)

  If signals remain problematic after these steps, consider immunoprecipitation to concentrate RERG protein before Western blot analysis, particularly for samples with low endogenous expression levels.

• How do RERG expression patterns correlate with cancer progression and prognosis?

  RERG expression demonstrates significant correlations with cancer progression and prognosis across multiple tumor types:

  1. Breast cancer: RERG expression is estrogen-regulated and frequently downregulated in high-grade, estrogen receptor-negative breast cancers. Immunohistochemical detection of RERG in breast tissue samples correlates with better prognosis and response to hormonal therapies .

  2. Expression analysis in clinical samples: Paraffin-embedded human breast cancer tissue stained for RERG using RERG antibodies at 1/100 dilution shows variable expression patterns correlating with tumor grade and molecular subtype .

  3. Placental tissue: RERG demonstrates specific expression patterns in placental tissue, with potential implications for understanding trophoblast invasion and placenta-related disorders .

  4. Mechanistic basis: RERG's tumor-suppressive function is linked to its ability to inhibit cell proliferation and colony formation. The protein's intrinsic GTPase activity appears crucial for this function, as it regulates downstream signaling pathways involved in cell cycle progression.

  Researchers investigating RERG in cancer contexts should consider:

  • Correlating RERG protein expression with clinical outcomes

  • Examining RERG subcellular localization changes during cancer progression

  • Evaluating RERG expression in relation to other biomarkers

  • Investigating the regulatory mechanisms controlling RERG expression in different cancer types

• What are the best methodologies for quantifying RERG protein levels in complex biological samples?

  Accurate quantification of RERG protein levels in complex samples requires selecting appropriate methodologies based on research objectives:

  Method	Advantages	Limitations	Best Application
  Western Blot	Semi-quantitative, detects specific isoforms	Labor-intensive, limited sample throughput	Examining RERG in cell/tissue lysates
  ELISA	Highly quantitative, high throughput	Limited isoform discrimination	Population studies, screening
  Immunohistochemistry	Preserves spatial context, cell-type specific	Subjective scoring, semi-quantitative	Tissue distribution studies
  Mass Spectrometry	Absolute quantification, isoform detection	Complex sample preparation, specialized equipment	Detailed proteomic analysis

  Recommended Quantification Protocol:

  1. For Western blot quantification: Use recombinant RERG protein to create a standard curve (1-100 ng range) processed alongside samples. Normalize RERG signal to total protein (measured by stain-free technology) rather than housekeeping proteins for more accurate quantification. Recommended antibody dilution: 1/500 .

  2. For IHC quantification: Implement digital pathology approaches using color deconvolution algorithms to separate RERG-specific staining from counterstains. Score intensity (0-3+) and percentage of positive cells to generate H-scores (0-300) for statistical analysis. Recommended antibody dilution: 1/100 .

  3. For ELISA-based quantification: Commercial RERG ELISA kits are available with detection ranges typically between 0.1-10 ng/mL. For custom ELISA development, use RERG antibodies at 1:5000 dilution for coating .

  When comparing RERG expression across different sample types or experimental conditions, it is crucial to process all samples simultaneously with identical protocols to minimize technical variability.

Research Design and Interpretation

• How can RERG antibodies be incorporated into multiplexed immunofluorescence assays?

  Incorporating RERG antibodies into multiplexed immunofluorescence assays requires strategic planning to achieve optimal signal detection while minimizing cross-reactivity:

  1. Antibody panel design considerations:

     • Selection of compatible primary antibodies from different host species

     • Prioritizing rabbit polyclonal RERG antibodies based on validation data

     • Careful selection of fluorophore-conjugated secondary antibodies

  2. Optimized protocol elements:

     • Sequential staining with appropriate stripping or blocking between rounds

     • Tyramide signal amplification for enhancing RERG detection sensitivity

     • DAPI counterstaining for nuclear visualization

  3. Technical validation steps:

     • Single-color controls to establish spectral profiles

     • Fluorophore minus one (FMO) controls to assess bleed-through

     • Absorption controls using recombinant RERG protein

  4. Data analysis approaches:

     • Cell segmentation based on nuclear and membrane markers

     • Quantitative analysis of RERG colocalization with other proteins

     • Machine learning algorithms for pattern recognition

  When selecting fluorophores for RERG detection, consider that rabbit polyclonal RERG antibodies work well with secondary antibodies conjugated to Alexa Fluor 488, 594, or 647. For multiplexed panels including ERG (which is often detected with rabbit antibodies), choose a RERG antibody from a different host species to avoid cross-reactivity issues.

• What are the critical considerations when comparing results from different RERG antibody clones?

  When comparing results obtained using different RERG antibody clones, researchers must consider several factors that might influence data interpretation:

  1. Epitope differences and functional implications:

     • Different antibodies target distinct regions of the RERG protein

     • Ab235439 recognizes the full-length recombinant RERG protein

     • Other antibodies (PACO05553) target specific internal regions

     • Epitope accessibility may vary depending on RERG's conformational state or protein interactions

  2. Validation status comparison:

     • Cross-reference published validation data for each antibody

     • Consider performing side-by-side validation experiments

     • Evaluate knockout/knockdown validation status for each antibody

  3. Application-specific performance:

     • Some antibodies perform better in certain applications than others

     • Ab235439 is validated for WB and IHC-P with proven reactivity in multiple species

     • PACO05553 has been validated for ELISA and IHC applications

  4. Quantification standardization:

     • Establish standard curves using recombinant RERG with each antibody

     • Determine antibody-specific detection limits and linear ranges

     • Use consistent analysis methods across antibody comparisons

  To facilitate accurate comparisons, maintain detailed records of antibody catalog numbers, lot numbers, and dilutions used. When publishing, report these details to enable reproduction and comparison of results across studies. Consider using multiple antibodies targeting different RERG epitopes to strengthen confidence in observations, particularly for novel findings.

• How can I design experiments to distinguish between RERG and other Ras-family proteins?

  Designing experiments to specifically distinguish RERG from other Ras-family proteins requires multiple complementary approaches:

  1. Antibody selection strategy:

     • Choose RERG antibodies validated against a panel of related Ras proteins

     • Select antibodies targeting unique C-terminal regions of RERG

     • Confirm specificity through Western blot analysis of multiple Ras proteins

  2. Experimental controls:

     • Include recombinant RERG and related Ras proteins (H-Ras, K-Ras, N-Ras)

     • Utilize RERG-overexpressing and RERG-knockout cell lines

     • Employ siRNA knockdown of RERG with monitoring of other Ras proteins

  3. Functional assays exploiting RERG-specific properties:

     • RERG has higher affinity for GDP than GTP (unlike many Ras proteins)

     • RERG overexpression reduces proliferation and colony formation

     • Design nucleotide binding assays to distinguish based on these properties

  4. Advanced techniques for definitive identification:

     • Immunoprecipitation followed by mass spectrometry

     • Proximity ligation assays with RERG-specific binding partners

     • Analysis of subcellular localization patterns (which differ from classic Ras proteins)

  When analyzing RERG expression in tissue samples, complement antibody staining with RNA in situ hybridization using RERG-specific probes to confirm protein detection specificity. This multi-modal approach provides stronger evidence for RERG-specific detection versus other Ras-family members that may share some epitope similarities.

• What are the emerging applications of RERG antibodies in cancer research and diagnostics?

  RERG antibodies are finding expanding applications in cancer research and potential diagnostic developments:

  1. Biomarker development:

     • Tissue microarray studies correlating RERG expression with clinical outcomes

     • Evaluation of RERG as part of multi-marker prognostic panels

     • Exploration of RERG as a predictive marker for response to hormone therapy

  2. Therapeutic target validation:

     • Antibody-based verification of RERG modulation by candidate drugs

     • Monitoring RERG expression changes during treatment response

     • Identification of compounds that restore RERG expression in cancers

  3. Mechanistic research applications:

     • Investigation of RERG protein interactions using co-immunoprecipitation

     • ChIP-seq studies examining RERG-mediated transcriptional regulation

     • Analysis of RERG post-translational modifications affecting function

  4. Technological innovations:

     • Development of phospho-specific RERG antibodies for signaling studies

     • Creation of conformation-specific antibodies distinguishing active/inactive RERG

     • Application in single-cell proteomics for tumor heterogeneity assessment

  The integration of RERG antibodies with advanced deep learning-based analysis approaches, similar to those described for other antibody epitope repertoire analyses , represents a promising frontier. Such computational approaches could enhance the extraction of clinically relevant information from RERG expression patterns in complex tumor samples, potentially leading to improved patient stratification and personalized treatment decisions.