RCOR3 Antibody

What is RCOR3 and what cellular functions does it perform?

RCOR3 (REST corepressor 3) functions as a component of a corepressor complex that represses transcription . It is also known as KIAA1343 and is part of the REST corepressor family . Understanding RCOR3's role in transcriptional regulation is essential for researchers investigating gene expression mechanisms, particularly in neurological and developmental studies. The protein typically has a calculated molecular weight of 48-56 kDa (from sequences of 436aa or 495aa), although it often appears at approximately 69 kDa in Western blot analysis due to post-translational modifications .

What species reactivity can be expected from commercially available RCOR3 antibodies?

Commercial RCOR3 antibodies demonstrate reactivity with multiple species, primarily human, mouse, and rat samples . Antibody 25970-1-AP has been validated with human, mouse, and rat samples in Western blot and ELISA applications . Similarly, the ab76921 antibody has confirmed reactivity with human and mouse samples in immunocytochemistry, immunoprecipitation, Western blot, and immunohistochemistry (paraffin-embedded sections) . Cross-species reactivity is an important consideration when designing experiments, especially for comparative studies or when working with animal models.

What are the most common applications for RCOR3 antibodies in research?

RCOR3 antibodies are validated for multiple research applications including:

• Western Blot (WB): For protein expression analysis and quantification

• Immunohistochemistry (IHC-P): For localization studies in formalin/PFA-fixed paraffin-embedded tissue sections

• Immunocytochemistry/Immunofluorescence (ICC-IF): For cellular localization studies

• Immunoprecipitation (IP): For protein-protein interaction studies

• ELISA: For quantitative detection of RCOR3 protein

Each application requires specific optimization of antibody dilution and experimental conditions to ensure reliable and reproducible results.

What dilutions are recommended for RCOR3 antibodies in Western blot applications?

How can epitope mapping inform the selection of RCOR3 antibodies for specific experimental applications?

Epitope mapping is crucial for selecting the most appropriate RCOR3 antibody for specific experimental applications. Different antibodies target distinct epitopes within the RCOR3 protein, which can affect their performance in various applications. For instance, some RCOR3 antibodies are generated using fusion protein immunogens (e.g., Ag23055) , while others use synthetic peptides within human RCOR3 . When selecting an antibody, researchers should consider whether the epitope is accessible in their specific experimental context, particularly for techniques like immunoprecipitation or immunohistochemistry where protein folding or fixation may mask certain epitopes.

Advanced techniques in antibody library design, such as those leveraging deep learning and multi-objective linear programming, can now predict the effects of mutations on antibody properties and may inform future development of more specific RCOR3 antibodies . These computational approaches can enhance antibody specificity and performance without requiring iterative feedback from wet laboratory experiments.

What strategies can be employed to validate RCOR3 antibody specificity and minimize cross-reactivity issues?

Validating antibody specificity is essential for ensuring reliable research outcomes. For RCOR3 antibodies, multiple validation strategies should be employed:

• Knockout/knockdown controls: Using CRISPR-Cas9 RCOR3 knockout cell lines or siRNA-mediated knockdown to confirm specificity.

• Competitive blocking: Pre-incubating the antibody with the immunogen peptide to confirm signal reduction.

• Multiple antibody validation: Using different antibodies targeting distinct RCOR3 epitopes to confirm consistent localization or expression patterns.

• Cross-species validation: Testing reactivity in multiple species to confirm conservation of epitope recognition.

• Molecular weight verification: Confirming detection at the expected molecular weight (calculated 48-56 kDa, though often observed at 69 kDa for RCOR3) .

Modern antibody validation approaches increasingly incorporate computational predictions and structural analyses to enhance specificity. For instance, recent advances in antibody design leverage sequence and structure-based deep learning to predict mutation effects on antibody properties , which could potentially be applied to enhance RCOR3 antibody specificity.

How do post-translational modifications of RCOR3 affect antibody recognition, and how can these be addressed experimentally?

Post-translational modifications (PTMs) of RCOR3 can significantly impact antibody recognition. This is evidenced by the discrepancy between the calculated molecular weight (48-56 kDa) and the observed molecular weight (69 kDa) in Western blot analyses . These modifications may include phosphorylation, glycosylation, or SUMOylation, which can alter epitope accessibility or protein mobility on gels.

To address PTM-related challenges experimentally:

• Use phosphatase or glycosidase treatments: Treating samples with these enzymes prior to Western blotting can help determine if phosphorylation or glycosylation accounts for the higher-than-expected molecular weight.

• Apply multiple antibodies targeting different epitopes: This helps ensure detection regardless of PTM status.

• Employ 2D gel electrophoresis: This technique separates proteins by both isoelectric point and molecular weight, helping to differentiate between modified forms.

• Combine immunoprecipitation with mass spectrometry: This approach can identify specific PTMs present on RCOR3 in different cellular contexts.

• Consider sample preparation conditions: Different lysis buffers and protease/phosphatase inhibitors may preserve different PTMs, affecting antibody recognition.

Understanding the impact of PTMs on RCOR3 function and antibody recognition is particularly important when studying this protein's role in transcriptional regulation under various cellular conditions.

What are the challenges in detecting RCOR3 isoforms, and how can these be overcome?

RCOR3 exists in multiple isoforms, including those with calculated molecular weights of 48 kDa (436aa) and 56 kDa (495aa) , presenting challenges for comprehensive detection and analysis. Researchers face several challenges when studying these isoforms:

• Isoform-specific detection: Different antibodies may preferentially detect certain isoforms based on epitope location.

• Tissue-specific expression: Isoform expression patterns may vary across tissues and cell types.

• Resolution limitations: Closely migrating isoforms may be difficult to distinguish using standard Western blotting techniques.

Strategies to overcome these challenges include:

• High-resolution gel systems: Using gradient gels or Phos-tag™ acrylamide for improved separation of closely migrating isoforms.

• Isoform-specific primers: Designing PCR primers targeting unique regions of each isoform for transcript-level analysis.

• Immunoprecipitation followed by mass spectrometry: This approach can identify and quantify specific isoforms present in a sample.

• Selection of appropriate antibodies: Using antibodies raised against regions common to all isoforms or isoform-specific epitopes, depending on research objectives.

• Complementary techniques: Combining protein and mRNA analysis methods to correlate isoform presence at both levels.

What are the optimal conditions for using RCOR3 antibodies in immunohistochemistry applications?

For successful immunohistochemistry (IHC) of RCOR3 in formalin/PFA-fixed paraffin-embedded tissues, researchers should consider the following methodological aspects:

• Antibody selection: Ab76921 has been validated for IHC-P applications on human tissues .

• Dilution optimization: A starting dilution of 1:200 has been successful for human testicular seminoma tissue .

• Antigen retrieval: Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) is typically recommended for optimal epitope exposure.

• Detection system: A sensitive detection system such as polymer-HRP should be employed for visualization.

• Controls: Inclusion of positive tissue controls (such as testicular seminoma) and negative controls (primary antibody omission) is essential for validating staining specificity.

• Counterstaining: Hematoxylin counterstaining provides cellular context for RCOR3 localization.

Researchers should optimize these conditions based on their specific tissue samples and experimental questions, as fixation time and tissue processing methods can impact antibody performance.

What protocol modifications are necessary when performing immunoprecipitation with RCOR3 antibodies?

Immunoprecipitation (IP) with RCOR3 antibodies requires careful optimization to ensure efficient and specific protein capture. Based on validated protocols , consider the following methodological aspects:

• Antibody amount: Use approximately 3μg of antibody per mg of cell lysate for optimal precipitation efficiency .

• Lysate preparation: Use a lysis buffer containing 1% NP-40 or similar non-denaturing detergent that preserves protein-protein interactions.

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

• Binding conditions: Allow adequate incubation time (4-16 hours) at 4°C with gentle rotation for antibody-protein binding.

• Wash stringency: Balance between removing non-specific interactions and maintaining specific interactions with appropriate salt concentration in wash buffers.

• Elution conditions: Use SDS sample buffer at 95°C for 5 minutes for complete elution.

• Loading control: Load approximately 20% of IP material per lane for Western blot detection .

Additional considerations include cross-linking the antibody to beads to prevent antibody co-elution and using appropriate negative controls such as non-immune IgG to assess non-specific binding.

How should Western blot protocols be optimized for clear detection of RCOR3?

Optimizing Western blot protocols for RCOR3 detection requires addressing several key methodological considerations:

• Sample preparation: Include phosphatase inhibitors in lysis buffers to preserve native phosphorylation states that may affect mobility and antibody recognition.

• Gel percentage selection: Use 8-10% acrylamide gels for optimal resolution around the observed 69 kDa molecular weight .

• Transfer conditions: Wet transfer is recommended for proteins of this size, using either PVDF or nitrocellulose membranes.

• Blocking agent: 5% non-fat dry milk in TBST is typically effective, though BSA may be required if phospho-specific detection is intended.

• Antibody dilution: Start with the recommended 1:500-1:1000 dilution range and adjust based on signal strength .

• Incubation temperature and time: Primary antibody incubation overnight at 4°C can improve signal-to-noise ratio.

• Detection system: Enhanced chemiluminescence with exposure times starting at 10 seconds has been successful .

• Positive control: Include samples known to express RCOR3, such as brain tissue from mice or rats .

What are the critical parameters for optimizing immunofluorescence with RCOR3 antibodies?

For successful immunofluorescence detection of RCOR3, researchers should optimize these critical parameters:

• Fixation method: 4% paraformaldehyde (NBF-fixed) has been successfully used with HeLa cells .

• Permeabilization: Use 0.1-0.3% Triton X-100 for adequate permeabilization without disrupting epitope integrity.

• Blocking solution: 5-10% normal serum from the same species as the secondary antibody helps reduce background.

• Primary antibody dilution: Start with 1:80 (2.5μg/ml) for ab76921 and optimize based on signal intensity .

• Secondary antibody selection: A DyLight® 488-conjugated goat anti-rabbit IgG (H+L) at 1:100 dilution has been validated .

• Nuclear counterstain: DAPI at 1μg/ml provides context for nuclear vs. cytoplasmic localization.

• Mounting medium: Use anti-fade mounting medium to prevent photobleaching during imaging.

• Controls: Include primary antibody omission controls and ideally a RCOR3 knockdown control for specificity verification.

The subcellular localization pattern of RCOR3 is primarily nuclear, consistent with its role in transcriptional regulation, but specific patterns may vary by cell type and physiological context.

How can researchers address inconsistent molecular weight observations for RCOR3 in Western blotting?

The discrepancy between calculated (48-56 kDa) and observed (69 kDa) molecular weights for RCOR3 can lead to uncertainty in data interpretation. To address this issue:

• Confirm antibody specificity: Use RCOR3 knockdown/knockout controls to verify that the observed band is indeed RCOR3.

• Evaluate post-translational modifications: Treat samples with phosphatases, glycosidases, or other demodifying enzymes to determine if PTMs contribute to altered mobility.

• Assess sample preparation effects: Different lysis buffers and denaturing conditions can affect protein migration patterns.

• Compare multiple antibodies: Use antibodies targeting different RCOR3 epitopes to confirm consistent molecular weight observation.

• Include positive controls: Use samples with well-characterized RCOR3 expression, such as brain tissue .

• Consider isoform expression: Different cell types may express different RCOR3 isoforms with varying molecular weights.

• Use gradient gels: These can provide better resolution and more accurate molecular weight determination.

When reporting results, researchers should acknowledge both the expected and observed molecular weights, with possible explanations for discrepancies.

What strategies can help differentiate between specific and non-specific binding in RCOR3 immunohistochemistry?

Differentiating specific from non-specific binding in RCOR3 immunohistochemistry requires rigorous controls and careful interpretation:

• Antibody titration: Perform a dilution series to identify the optimal concentration that maximizes specific signal while minimizing background.

• Blocking optimization: Test different blocking reagents (BSA, normal serum, commercial blockers) to reduce non-specific binding.

• Antigen competition: Pre-incubate the antibody with the immunizing peptide to confirm signal reduction.

• Tissue panel analysis: Compare staining patterns across tissues with known RCOR3 expression levels.

• Multiple antibody comparison: Use different antibodies targeting distinct RCOR3 epitopes to confirm consistent localization patterns.

• Technical controls: Include isotype controls and primary antibody omission controls.

• Biological validation: Where possible, correlate protein detection with mRNA expression data from the same tissues.

• Counter-staining: Use counter-stains to provide cellular context and help distinguish specific subcellular localization.

When interpreting staining patterns, consider that RCOR3's role as a transcriptional corepressor suggests primarily nuclear localization, though cytoplasmic staining may also be observed depending on cellular context and antibody specificity.

How can researchers interpret contradictory results when using different RCOR3 antibodies?

When faced with contradictory results from different RCOR3 antibodies, researchers should systematically evaluate several factors:

• Epitope differences: Different antibodies target distinct epitopes that may be differentially accessible depending on protein conformation, complex formation, or post-translational modifications.

• Validation status: Assess the validation data for each antibody, including knockout/knockdown controls and specificity tests.

• Application optimization: Ensure each antibody was used according to optimized protocols for the specific application (WB, IHC, IP, etc.).

• Isoform specificity: Determine if the antibodies recognize different RCOR3 isoforms, which may have distinct expression patterns or functions.

• Sample preparation effects: Different fixation methods, lysis buffers, or antigen retrieval approaches can affect epitope accessibility.

• Quantification methods: Standardize quantification approaches across experiments using different antibodies.

• Complementary techniques: Use orthogonal methods (e.g., mass spectrometry, RNA analysis) to resolve contradictions.

When reporting contradictory results, researchers should transparently describe the conditions under which each antibody was used and consider the biological implications of different detection patterns rather than simply dismissing one result as "incorrect."

What approaches can help resolve weak or absent signal when working with RCOR3 antibodies?

Researchers encountering weak or absent RCOR3 signal can systematically troubleshoot using these approaches:

• Antibody concentration: Increase primary antibody concentration, starting with a 2-fold increase from recommended dilutions.

• Incubation conditions: Extend primary antibody incubation time (e.g., overnight at 4°C) to enhance binding.

• Antigen retrieval optimization: For IHC applications, test different antigen retrieval methods (heat vs. enzymatic, different pH buffers).

• Signal amplification: Use more sensitive detection systems such as polymer-HRP or tyramide signal amplification.

• Sample enrichment: For low-abundance proteins, consider immunoprecipitation prior to Western blotting.

• Fresh antibody aliquots: Avoid repeated freeze-thaw cycles that can degrade antibody activity.

• Sample handling: Ensure samples are not degraded by including appropriate protease inhibitors.

• Blocking optimization: Excessive blocking can mask epitopes; reduce blocking agent concentration or time.

• Alternative antibody: Try antibodies targeting different RCOR3 epitopes, as some may be more accessible in certain applications.