RREB1 Antibody

What is RREB1 and what is its biological significance?

RREB1 is a zinc finger transcription factor that binds specifically to RAS-responsive elements (RRE) of gene promoters. It contains fifteen C2H2-type zinc fingers and localizes primarily to nuclear speckles. RREB1 plays a dual role in transcriptional regulation, functioning as both an activator and repressor depending on the genomic context. It potentiates the transcriptional activity of NEUROD1 and promotes brown adipocyte differentiation .

The biological significance of RREB1 stems from its involvement in Ras/Raf-mediated cell differentiation pathways. It enhances calcitonin expression and regulates multiple genes critical for cellular development and function. Genes induced by RREB1 include those encoding calcitonin, FSH, MT-IIA, p53, and secretin; conversely, it represses genes encoding angiotensinogen, HLA-G, hZIP1, p16, and PSA . RREB1 also interacts with nuclear proteins including CtBP, NeuroD, and androgen receptor (AR), suggesting its role as a transcriptional cofactor in various cellular contexts .

What are the known isoforms of RREB1 and how are they detected?

RREB1 exists in multiple splice variants resulting from alternative splicing, with five currently identified isoforms:

Research has shown that the expression patterns of these isoforms vary across tissue types and between normal and cancer tissues. RREB1α and RREB1β appear to be the predominant isoforms, with others expressed at lower levels . Specifically designed isoform-specific primers targeting unique exon junctions are essential for accurate mRNA quantification of individual variants. For protein detection, antibodies recognizing shared or unique epitopes must be carefully selected based on experimental requirements.

What applications are RREB1 antibodies validated for?

RREB1 antibodies have been validated for multiple research applications, with varying specifications depending on the antibody clone and manufacturer:

When selecting an antibody for a specific application, researchers should review manufacturer datasheets for validated applications and optimal conditions. Cross-referencing with published literature utilizing the same antibody is highly recommended for experimental planning .

What should researchers expect when detecting RREB1 by Western blotting?

When performing Western blotting for RREB1, researchers should be aware of several technical considerations:

The calculated molecular weight of RREB1 is 181 kDa, but observed molecular weights typically range between 160-181 kDa, with some reports noting a band at approximately 250 kDa . This variability may result from post-translational modifications, alternative splicing, or cell-type specific differences in protein processing.

Multiple bands in Western blots may indicate:

• Detection of different RREB1 isoforms

• Truncated protein products

• Degradation products

• Post-translational modifications

For optimal detection, researchers should:

• Use appropriate positive controls (BxPC-3, SH-SY5Y, or HeLa cells have been validated)

• Prepare proper nuclear extracts using kits like NE-PER extraction kit (Thermo Scientific)

• Include nuclear loading controls such as TBP/TFIID

• Optimize protein loading (typically 20-40 μg of nuclear extract)

• Test a range of antibody dilutions (generally starting with 1:500-1:1000)

How should researchers optimize RREB1 antibody conditions for IHC applications?

Optimizing RREB1 antibody conditions for immunohistochemistry requires systematic approach to antigen retrieval, blocking, and antibody incubation:

For antigen retrieval, Proteintech recommends TE buffer pH 9.0 as the primary method, with citrate buffer pH 6.0 as an alternative . Optimal retrieval conditions should be determined empirically for each tissue type:

For validation, human stomach tissue and human breast cancer tissue have been confirmed as positive controls for RREB1 staining . Always include appropriate negative controls (primary antibody omission, isotype control) to assess background and non-specific staining.

What are the critical considerations for RREB1 antibody validation in experimental systems?

Rigorous validation of RREB1 antibodies is essential for ensuring experimental reliability. Researchers should implement the following validation strategies:

• Genetic approaches: Perform RREB1 knockdown or knockout experiments using siRNA, shRNA, or CRISPR-Cas9, and confirm reduced signal in Western blot, IF, or IHC .

• Multiple antibody approach: Use antibodies targeting different epitopes of RREB1 to confirm consistent detection patterns.

• Positive controls: Test antibodies on cell lines with confirmed RREB1 expression (BxPC-3, SH-SY5Y, HeLa cells) .

• Peptide competition: Perform blocking experiments with the immunizing peptide to confirm specificity (neutralizing peptides may be available, e.g., sc-515600 P) .

• Recombinant protein testing: Compare antibody detection of recombinant RREB1 with known concentrations.

• Western blot molecular weight confirmation: Verify that detected bands correspond to expected molecular weights for RREB1 (160-181 kDa, sometimes 250 kDa) .

• Cross-reactivity assessment: Test for potential cross-reactivity with related zinc finger proteins.

• Subcellular localization verification: Confirm nuclear localization consistent with RREB1's known function as a transcription factor.

Implementing multiple validation approaches substantially increases confidence in antibody specificity and reliability across experimental applications.

How should researchers design experiments to study RREB1 isoform expression in cancer?

Designing experiments to study RREB1 isoform expression in cancer requires a multi-faceted approach that integrates mRNA and protein detection methods:

mRNA Analysis:

• Design isoform-specific primers spanning unique exon junctions for each RREB1 variant.

• Perform RT-qPCR on matched normal and tumor samples using validated reference genes.

• Calculate relative expression using the 2^-ΔΔCt method.

• Consider RNAseq or NanoString analysis for comprehensive isoform profiling.

Protein Analysis:

• Select antibodies capable of detecting total RREB1 or specific isoforms based on epitope mapping.

• Perform Western blotting on nuclear extracts from matched normal and tumor samples.

• Quantify bands using densitometry and normalize to appropriate nuclear loading controls (TBP/TFIID) .

• Conduct IHC on tissue microarrays containing multiple patient samples for higher throughput analysis.

Experimental Design Considerations:

• Include sufficient biological replicates (minimum n=3 for cell lines, larger cohorts for patient samples).

• Use appropriate statistical analyses to compare expression levels.

• Correlate RREB1 isoform expression with clinical parameters and outcomes.

• Consider functional assays to determine the biological significance of altered isoform expression.

Recent research has shown that total and isoform-specific RREB1 mRNA expression is lower in most but not all tumors compared with normal tissues, highlighting the importance of comprehensive analysis across multiple cancer types .

What methods are recommended for studying RREB1's role in transcriptional regulation?

To investigate RREB1's role in transcriptional regulation, researchers should consider the following methodological approaches:

Chromatin Immunoprecipitation (ChIP):

• Use validated ChIP-grade RREB1 antibodies (e.g., 20280-1-AP) .

• Optimize crosslinking conditions (typically 1% formaldehyde for 10 minutes).

• Sonicate chromatin to 200-500 bp fragments.

• Immunoprecipitate with 2-5 μg RREB1 antibody per reaction.

• Analyze enrichment at known RREB1 binding sites (RAS-responsive elements) by qPCR.

• Perform ChIP-seq for genome-wide binding analysis.

Reporter Gene Assays:

• Clone promoters containing RAS-responsive elements upstream of luciferase reporter.

• Co-transfect with RREB1 expression constructs (wild-type or isoform-specific).

• Measure transcriptional activation or repression relative to control.

• Include promoters of known RREB1 target genes (calcitonin, angiotensinogen, etc.) .

Protein Interaction Studies:

• Perform co-immunoprecipitation using RREB1 antibodies to identify interacting partners.

• Verify interactions with known partners (CtBP, NeuroD, AR) .

• Map interaction domains using deletion constructs.

• Assess functional consequences of interactions using reporter assays.

RREB1 Modulation:

• Perform isoform-specific knockdown using siRNA/shRNA.

• Create CRISPR-Cas9 knockout models.

• Analyze effects on target gene expression by RT-qPCR and Western blotting.

• Conduct RNA-seq to identify global transcriptional changes.

These approaches, individually or in combination, provide comprehensive insights into RREB1's transcriptional regulatory functions in various cellular contexts.

How can researchers effectively study RREB1's involvement in Ras/Raf signaling pathways?

To investigate RREB1's role in Ras/Raf signaling, researchers should implement experimental strategies that link pathway activation to RREB1 function:

Cellular Models:

• Use cell lines with constitutively active Ras mutations (e.g., KRAS G12V).

• Employ inducible Ras or Raf expression systems for temporal control.

• Apply pathway inhibitors (MEK inhibitors, RAF inhibitors) to assess RREB1 dependency on pathway activation.

• Compare RREB1 function in isogenic cell lines differing only in Ras/Raf status.

Signaling Analysis:

• Monitor RREB1 phosphorylation status following pathway activation.

• Assess RREB1 nuclear localization in response to pathway modulation.

• Examine RREB1 binding to target promoters after Ras/Raf activation using ChIP.

• Analyze expression of RREB1 target genes in response to pathway activation/inhibition.

Functional Assays:

• Perform RREB1 knockdown in Ras-mutant cells to assess phenotypic rescue.

• Create phosphorylation-deficient RREB1 mutants to identify regulatory sites.

• Use reporter gene assays with RAS-responsive elements to measure RREB1 activity.

• Assess cell differentiation markers in the context of RREB1 manipulation.

This multi-faceted approach can elucidate RREB1's position within Ras/Raf signaling cascades and its contribution to downstream cellular responses. The initial study of RREB1 found that it bound the calcitonin promoter in medullary thyroid carcinomas in response to Ras activation, providing a foundational model for further investigation .

What approaches should be used for selective knockdown of specific RREB1 isoforms?

Selective knockdown of specific RREB1 isoforms requires careful design of targeting strategies that exploit unique sequences in each variant:

siRNA/shRNA Design:

• Target unique exon junctions or exons specific to individual isoforms.

• Design multiple siRNAs/shRNAs per isoform to ensure robust knockdown.

• Perform BLAST analysis to confirm specificity and minimize off-target effects.

• Include non-targeting controls with similar GC content.

CRISPR-Cas9 Approaches:

• Design guide RNAs targeting isoform-specific exons.

• Use paired nickase approaches for enhanced specificity.

• Consider inducible CRISPR systems for temporal control.

• For complex splicing patterns, target the splice donor/acceptor sites.

Validation Strategy:

• Design isoform-specific qRT-PCR primers spanning unique exon junctions.

• Perform Western blotting with antibodies capable of detecting specific isoforms.

• Quantify knockdown efficiency at both mRNA and protein levels.

• Assess potential compensatory upregulation of other isoforms.

Research has demonstrated successful selective knockdown of RREB1 isoforms, providing established protocols in the literature . This approach is critical for dissecting the specific functions of individual variants in different biological contexts.

What are the critical troubleshooting steps for unsuccessful RREB1 Western blotting?

When encountering difficulties with RREB1 Western blotting, researchers should systematically address potential issues in sample preparation, antibody selection, and detection conditions:

Sample Preparation Issues:

• Inadequate nuclear extraction: RREB1 is predominantly nuclear; ensure proper nuclear extraction using validated methods (e.g., NE-PER kit) .

• Protein degradation: Include fresh protease inhibitors in all buffers and keep samples cold.

• Insufficient protein: Load 20-40 μg of nuclear extract; adjust based on RREB1 expression levels.

• Incomplete denaturation: Ensure thorough heating of samples (95°C for 5 minutes) in appropriate buffer.

Antibody and Detection Optimization:

• Antibody concentration: Test a range of dilutions (1:100-1:1000) to find optimal conditions .

• Incubation conditions: Try both overnight at 4°C and room temperature for 1-2 hours.

• Blocking optimization: Test different blocking agents (5% milk, 5% BSA) to reduce background.

• Secondary antibody selection: Ensure compatible species and isotype (e.g., anti-rabbit IgG for rabbit polyclonal antibodies).

Transfer and Detection Parameters:

• Transfer efficiency: Optimize transfer time for high molecular weight proteins (90-181 kDa).

• Membrane selection: PVDF membranes may provide better results than nitrocellulose for RREB1.

• Detection method: For low abundance, consider high-sensitivity ECL substrates or fluorescent secondaries.

• Exposure time: Try multiple exposure times to capture optimal signal-to-noise ratio.

If problems persist, consider validating the antibody using positive control samples (BxPC-3, SH-SY5Y, or HeLa cells) that have been confirmed to express RREB1 . Additionally, trying alternative antibodies targeting different epitopes may help identify the most reliable detection method for your experimental system.

How can researchers quantitatively assess RREB1 expression in clinical specimens?

Quantitative assessment of RREB1 expression in clinical specimens requires rigorous methodological approaches and appropriate controls:

Immunohistochemistry Quantification:

• Use standardized staining protocols with validated RREB1 antibody dilutions (1:50-1:500) .

• Employ digital pathology software to quantify:

  • Staining intensity (0, 1+, 2+, 3+)

  • Percentage of positive cells

  • Subcellular localization (nuclear vs. cytoplasmic)

• Calculate H-scores (0-300) by multiplying intensity by percentage of positive cells.

• Include multiple fields per sample (minimum 3-5) to account for tumor heterogeneity.

Tissue Processing Considerations:

• Standardize fixation protocols (typically 10% neutral buffered formalin for 24 hours).

• Use positive control tissues (human stomach, breast cancer) in each staining batch.

• Include on-slide negative controls (primary antibody omission).

• Process all comparative samples (normal vs. tumor) using identical protocols.

mRNA Analysis in Clinical Samples:

• Assess RNA quality using RIN values (aim for RIN > 7).

• Use isoform-specific primers for RT-qPCR analysis.

• Employ multiple reference genes validated for the tissue type.

• Calculate relative expression using 2^-ΔΔCt method.

• Consider RNAscope for in situ visualization with cellular resolution.

Statistical Analysis:

• Use appropriate statistical tests based on data distribution.

• Account for multiple testing when analyzing multiple isoforms.

• Correlate RREB1 expression with clinical parameters and outcomes.

• Consider multivariate analysis to identify independent prognostic value.

What cell fractionation procedures are optimal for studying RREB1 subcellular distribution?

Given RREB1's role as a nuclear transcription factor, proper cellular fractionation is critical for accurate analysis of its subcellular distribution:

Commercial Kit Method:

• The NE-PER extraction kit (Thermo Scientific) has been successfully used in published RREB1 research .

• Follow manufacturer's protocol precisely, adjusting cell numbers based on expression levels.

• Include PMSF and protease inhibitor cocktail in all buffers.

• Process samples quickly at 4°C to prevent protein degradation.

Differential Centrifugation Protocol:

• Harvest cells and wash twice with ice-cold PBS.

• Resuspend in hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT).

• Incubate on ice for 10 minutes to allow cell swelling.

• Add NP-40 to 0.5% final concentration and vortex briefly.

• Centrifuge at 3,000 g for 10 minutes at 4°C.

• Collect supernatant (cytoplasmic fraction).

• Resuspend nuclear pellet in high-salt extraction buffer (20 mM HEPES pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT).

• Incubate with rotation for 30 minutes at 4°C.

• Centrifuge at 18,000 g for 20 minutes.

• Collect supernatant (nuclear extract).

Fraction Quality Control:

• Verify compartment separation using established markers:

  • Cytoplasmic: α-tubulin, GAPDH

  • Nuclear: TBP/TFIID (as mentioned in search results) , lamin B

• Calculate enrichment ratios between fractions.

• Assess purity by immunoblotting for markers of potential contaminating organelles.

Analysis Considerations:

• Load equal amounts of protein from each fraction.

• Include loading controls specific to each cellular compartment.

• Assess RREB1 distribution across fractions under various conditions:

  • Cell cycle stages

  • Growth factor stimulation

  • Ras pathway activation/inhibition

  • Cellular differentiation states

This systematic approach allows for reliable assessment of RREB1's subcellular distribution and potential shuttling under different physiological or pathological conditions.

How can researchers integrate RREB1 antibody-based approaches with genomic technologies?

Integrating RREB1 antibody-based approaches with genomic technologies enables comprehensive analysis of RREB1's regulatory functions:

ChIP-seq Applications:

• Perform chromatin immunoprecipitation with validated RREB1 antibodies followed by next-generation sequencing.

• Identify genome-wide binding sites of RREB1 in different cell types or conditions.

• Use motif analysis to characterize RREB1 binding preferences beyond canonical RREs.

• Integrate with transcriptomic data to correlate binding with gene expression changes.

CUT&RUN or CUT&Tag Approaches:

• Apply these newer, more sensitive chromatin profiling methods using RREB1 antibodies.

• Achieve higher resolution mapping of RREB1 binding sites with lower background.

• Require fewer cells than traditional ChIP-seq, enabling analysis of rare cell populations.

HiChIP or PLAC-seq:

• Combine chromatin immunoprecipitation with proximity ligation to identify RREB1-mediated chromatin interactions.

• Map 3D chromatin architecture at RREB1-bound regulatory elements.

• Discover long-range gene regulatory interactions mediated by RREB1.

Proteomics Integration:

• Perform RREB1 immunoprecipitation followed by mass spectrometry (IP-MS).

• Identify novel RREB1 interacting partners in different cellular contexts.

• Characterize post-translational modifications of RREB1 and their functional significance.

Single-cell Approaches:

• Use RREB1 antibodies for CyTOF or CITE-seq to analyze protein expression at single-cell resolution.

• Combine with single-cell transcriptomics to correlate RREB1 levels with gene expression patterns.

• Apply spatial transcriptomics to map RREB1 activity in tissue contexts.

These integrated approaches provide systems-level insights into RREB1's role in transcriptional regulation and its contribution to normal development and disease states.

What considerations are important when using RREB1 antibodies in cancer biomarker studies?

When evaluating RREB1 as a potential cancer biomarker, researchers should consider several critical factors in antibody-based detection approaches:

Antibody Validation for Biomarker Studies:

• Verify antibody specificity using multiple validation approaches (genetic knockdown, peptide competition).

• Test antibody performance across multiple patient samples to assess reproducibility.

• Determine optimal conditions for each cancer type, as fixation and processing may affect epitope accessibility.

• Compare multiple antibodies targeting different RREB1 epitopes to confirm consistent patterns.

Standardization and Controls:

• Develop standardized scoring systems for IHC (H-score, Allred score) with clear cutoff values.

• Include tissue microarray controls with known RREB1 expression levels in each analysis batch.

• Implement digital pathology for objective quantification where possible.

• Ensure blinded scoring by multiple pathologists to minimize observer bias.

Clinical Correlation Approaches:

• Collect comprehensive clinical data to correlate with RREB1 expression.

• Perform survival analyses (Kaplan-Meier, Cox regression) to assess prognostic significance.

• Conduct multivariate analyses to determine if RREB1 is an independent prognostic factor.

• Validate findings in independent patient cohorts.

What are the best practices for long-term storage and handling of RREB1 antibodies?

Proper storage and handling of RREB1 antibodies is critical for maintaining their performance over time:

Storage Recommendations:

• Store antibodies at -20°C for long-term preservation as indicated in product information .

• Prepare small aliquots (e.g., 10-20 μl) upon receipt to minimize freeze-thaw cycles.

• Some antibodies contain 50% glycerol and can be stored at -20°C without aliquoting .

• For working stocks, store at 4°C for up to 2 weeks (check manufacturer recommendations).

• Protect antibodies from light, especially those conjugated to fluorophores.

Handling Best Practices:

• Avoid repeated freeze-thaw cycles (limit to <5 for optimal performance).

• Allow antibodies to equilibrate to room temperature before opening to prevent condensation.

• Mix gently by inversion or brief centrifugation; avoid vortexing which can damage antibodies.

• Use clean pipette tips and tubes to prevent contamination.

• Always include carrier protein (BSA) when diluting to very low concentrations.

Stability Assessment:

• Monitor antibody performance over time by regularly testing against positive controls.

• Document lot numbers and performance characteristics for reproducibility.

• If performance decreases, validate new antibody lots before applying to experimental samples.

• Consider antibody labeling with stable tags for long-term studies requiring consistent detection.

Documentation Practices:

• Maintain detailed records of antibody source, catalog number, and lot number.

• Document optimal working dilutions for each application.

• Record any modifications to manufacturer protocols and resulting performance.

• Include complete antibody information in publications for reproducibility.

Following these guidelines will help ensure consistent RREB1 antibody performance across experiments and maximize the shelf-life of valuable research reagents.

How can researchers effectively compare results from different RREB1 antibodies?

When using multiple RREB1 antibodies or comparing results between studies using different antibodies, researchers should implement systematic comparison strategies:

Side-by-Side Validation:

• Test all antibodies simultaneously on the same samples under identical conditions.

• Use positive controls (BxPC-3, SH-SY5Y, HeLa cells) and negative controls (knockdown/knockout samples).

• Compare detection sensitivity, specificity, and signal-to-noise ratio.

• Document banding patterns in Western blot and staining patterns in IHC/IF.

Epitope Mapping Considerations:

• Identify the specific epitopes recognized by each antibody:

  • ab113287: Synthetic Peptide within Human RREB1 aa 1350-1450

  • sc-515600 (B-7): Epitope mapping information may be available from manufacturer

  • 20280-1-AP: Peptide immunogen

• Consider whether antibodies detect all or specific RREB1 isoforms based on epitope location.

• Select antibodies recognizing different regions for confirmation of specificity.

Cross-Platform Comparison:

• Evaluate concordance between detection methods (e.g., WB vs. IHC vs. IF).

• Assess whether antibodies yield consistent results across different applications.

• Document application-specific optimization requirements for each antibody.

Reporting Guidelines:

• In publications, clearly specify antibody details:

  • Manufacturer and catalog number

  • Clone designation (for monoclonals)

  • Host species and antibody type

  • Working dilution and incubation conditions

  • Validation methods employed

• Include representative images showing detection patterns.

• Address any discrepancies between different antibodies in your discussion.

This systematic approach enables reliable comparison between studies using different RREB1 antibodies and helps establish consensus regarding RREB1 expression patterns and functions across different experimental systems.

What novel applications are emerging for RREB1 antibodies in translational research?

RREB1 antibodies are finding innovative applications in translational research beyond traditional laboratory techniques:

Liquid Biopsy Development:

• Explore RREB1 detection in circulating tumor cells (CTCs) using immunocapture approaches.

• Investigate RREB1 as a potential biomarker in extracellular vesicles or tumor-derived exosomes.

• Develop sensitive detection methods for monitoring treatment response or disease recurrence.

Therapeutic Target Validation:

• Use RREB1 antibodies to assess target engagement in drug development.

• Employ proximity ligation assays to study RREB1 interactions with therapeutic targets.

• Monitor RREB1 expression/localization as a pharmacodynamic marker in preclinical models.

Precision Medicine Applications:

• Develop RREB1 isoform-specific IHC for patient stratification in clinical trials.

• Correlate RREB1 expression patterns with treatment response.

• Integrate with genomic profiling to identify patients with alterations in RREB1 or related pathways.

Emerging Technologies:

• Adapt RREB1 antibodies for multiplexed tissue imaging (e.g., CODEX, Imaging Mass Cytometry).

• Develop RREB1-targeting antibody-drug conjugates for research applications.

• Create RREB1 proximity labeling tools (BioID, APEX) for spatially-resolved interactome analysis.

Functional Screening:

• Use RREB1 antibodies in high-content imaging screens to identify modulators of expression or localization.

• Develop FRET-based biosensors incorporating RREB1 antibody fragments for live-cell studies.

• Create RREB1 activity reporters for monitoring transcriptional function in real-time.

These emerging applications demonstrate the evolving utility of RREB1 antibodies beyond traditional research methods, providing new avenues for translational research and potential clinical applications.