NCOR1 Antibody, HRP conjugated

What is NCOR1 and what cellular functions does it regulate?

NCOR1, also known as KIAA1047 and TRAC1, belongs to the N-CoR nuclear receptor corepressors family. This 270 kDa protein mediates transcriptional repression by certain nuclear receptors and is part of a complex that promotes histone deacetylation and repressive chromatin structure formation, impeding basal transcription factor access . In the liver, NCOR1 exhibits dual regulatory roles: repressing lipid synthesis during fasting while inhibiting PPARα activation upon feeding, which blunts ketogenesis . The protein contains multiple transcriptional repression domains (RDs) that recruit additional corepressor complex components including HDACs, mSin3, GPS2, and TBL1/TBLR1 . Its C-terminal portion contains nuclear receptor interaction domains with conserved CoRNR motifs that enable binding to unliganded nuclear hormone receptors such as thyroid hormone (THR) and retinoic acid (RAR) receptors .

What applications are validated for NCOR1 antibodies?

NCOR1 antibodies have been validated for multiple research applications:

When using HRP-conjugated NCOR1 antibodies specifically, the primary advantage is eliminating the need for secondary antibody incubation in applications like Western blotting and ELISA, which streamlines experimental workflows and potentially reduces non-specific background.

What is the expected molecular weight when detecting NCOR1?

While the calculated molecular weight of NCOR1 is 270 kDa based on amino acid sequence, the observed molecular weight in experimental conditions is often approximately 120 kDa . This discrepancy may result from post-translational modifications, alternative splicing variants, or protein degradation during sample preparation. When optimizing your experimental protocol, it's advisable to include positive control samples (such as HepG2 or K-562 cell lysates) to confirm the correct band identification .

How should NCOR1 antibodies be stored and handled?

For optimal performance and longevity, NCOR1 antibodies should be stored at -20°C, where they typically remain stable for one year after shipment . Most preparations are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Aliquoting is generally unnecessary for -20°C storage, especially for smaller quantities (20μl sizes that contain 0.1% BSA) . When working with HRP-conjugated antibodies specifically, avoid repeated freeze-thaw cycles and exposure to direct light during storage and handling to preserve enzymatic activity of the HRP moiety.

What are the critical controls for validating NCOR1 antibody specificity?

Validating NCOR1 antibody specificity requires multiple complementary approaches:

• Positive controls: Use cell lines with known NCOR1 expression such as HEK293T, K-562, or NIH 3T3 cells as confirmed through Western blotting .

• Knockout/knockdown validation: Include NCOR1 knockout or knockdown samples to confirm signal specificity. Published applications demonstrate successful use of NCOR1 antibodies in KD/KO experimental designs .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (residues 1525-1575 of human NCOR1 for some commercial antibodies) to block specific binding .

• Cross-validation: Compare results from different NCOR1 antibody clones targeting distinct epitopes. For example, compare antibodies that recognize the N-terminal region versus those recognizing the C-terminal domains containing the nuclear receptor interaction motifs .

• Species cross-reactivity confirmation: Validate reactivity across human, mouse, and rat samples if performing comparative studies, as most commercial antibodies show reactivity with these species .

For HRP-conjugated antibodies specifically, include an enzyme activity control to ensure the HRP component is functional by performing a standard curve detection with known concentrations of target protein.

How can NCOR1 phosphorylation be studied in relation to its functional regulation?

NCOR1 undergoes regulatory phosphorylation that modulates its activity and interactions. Research strategies include:

• Phospho-specific antibodies: Use antibodies that specifically detect phosphorylated NCOR1, such as those targeting phosphorylated serine 1460, which has been studied in relation to Akt1 kinase activity .

• In vitro kinase assays: Implement assays using recombinant Akt1 (5 ng) with GST-fusion proteins harboring NCOR1 peptide sequences (e.g., residues 1453-1466 and 2401-2415) . The reaction can be performed in kinase assay buffer containing 25 mM Tris pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na₃VO₄, 10 mM MgCl₂, and serine/threonine phosphatase inhibitors at 30°C for 75 minutes .

• Phosphorylation site analysis: Analyze potential phosphorylation using phospho-(Ser/Thr) Akt Substrate Antibodies for detection, followed by mass spectrometry confirmation of specific sites .

• Functional correlation: Correlate phosphorylation status with nuclear receptor binding ability through co-immunoprecipitation assays using FLAG-tagged constructs and anti-FLAG M2 Affinity Gel for precipitation .

• Pharmacological manipulation: Utilize LY294002 (PI3K/Akt pathway inhibitor) to modulate phosphorylation levels and assess functional consequences in cellular models .

How can ChIP experiments with NCOR1 antibodies be optimized?

Chromatin immunoprecipitation experiments with NCOR1 antibodies require optimization for maximum sensitivity and specificity:

• Cell number optimization: Start with approximately 10×10⁶ cells per ChIP experiment as validated in published protocols .

• Chromatin fragmentation: Optimize sonication conditions to generate DNA fragments of 200-500 bp for optimal resolution of binding sites.

• Antibody concentration: For ChIP applications, titrate antibody concentration starting from manufacturer recommendations. NCOR1 antibodies have been successfully used at 0.1-0.2 μg/ml for ChIP-seq applications .

• Positive control regions: Include primer sets for known NCOR1 binding regions such as:

  • Srebp1c LXRE (5'-AGG CTC TTT TCG GGG ATG G-3' and 5'- TGG GGT TAC TGG CGG TCA C-3')

  • Cpt1a PPRE (5'-CTT TCC TAC TGA GGC CCA GAT AG-3' and 5'-TAC AGC CTA GAA CCC TGA CTG C-3')

  • Sdhb ERRE (5'- CTT CCT GTA CAT TGG CTC GGA GAA ACC-3' and 5'-CTT CAA GGA GAC CCC GAC CGT CGC CGC-3')

• Nuclear receptor ligands: When studying NCOR1 in relation to nuclear receptors, include appropriate ligands such as GW3965 for LXR or Wy14643 for PPAR to observe displacement of NCOR1 from repressive complexes .

How do I interpret discrepancies between NCoR1 knockout effects and expected gene repression patterns?

Recent research has revealed unexpected complexity in NCoR1 function. While the classic model suggests NCoR1 is the primary mediator of unliganded nuclear receptor repression, experimental data shows NCoR1 deletion does not prevent all gene repression and histone deacetylation . When interpreting discrepant results:

• Consider redundant mechanisms: Despite NCoR1 deletion, strong repression of more than 43% of positive T3 targets was observed in hypothyroid mice, suggesting alternative repression mechanisms exist .

• Analyze histone modification patterns: Approximately half of genes repressed in the absence of NCoR1 showed decreased H3K27 acetylation, with nearly 80% of these regions containing bona fide TRβ1-binding sites .

• Validate receptor dependency: Use receptor-specific knockout models (e.g., liver-specific TRβ1-KO mice) to confirm whether observed changes in gene expression and histone acetylation truly depend on the nuclear receptor of interest .

• Consider isoform specificity: The NCoR1ΔID hypomorphic allele, which cannot interact with thyroid hormone receptor, didn't reverse all ligand-independent TR actions, suggesting isoform-specific or context-dependent functions .

• Evaluate paralog compensation: While NCoR1 plays a significant role in thyroid hormone signaling, its paralog SMRT has been found to play little role in this pathway, highlighting the importance of considering paralog-specific effects .

What methodological considerations are important when differentiating between direct and indirect NCOR1 genomic recruitment?

NCOR1 can be recruited to chromatin through direct interaction with nuclear receptors or indirectly through other transcription factors. To distinguish between these mechanisms:

• Sequential ChIP (Re-ChIP): Perform immunoprecipitation with an antibody against the nuclear receptor of interest, followed by a second immunoprecipitation with the NCOR1 antibody to identify regions where both proteins co-localize.

• Motif analysis: Analyze ChIP-seq data for enrichment of specific nuclear receptor binding motifs such as LXREs, PPREs, or ERREs at NCOR1 binding sites .

• Correlation with histone modifications: Compare NCOR1 binding with histone deacetylation patterns, particularly H3K27 acetylation, which has been linked to NCOR1 activity .

• Nuclear receptor ligand treatment: Treat samples with specific nuclear receptor ligands (GW3965, Wy14643) and observe displacement of NCOR1 from direct but not indirect binding sites .

• Mutation of interaction domains: Utilize cells expressing NCOR1 with mutations in specific CoRNR box motifs to disrupt interaction with particular nuclear receptors and identify receptor-specific recruitment sites.

How can I address weak or non-specific signals when using NCOR1 antibodies in Western blotting?

When troubleshooting Western blot experiments with NCOR1 antibodies:

• Antigen retrieval optimization: For tissue samples, test both TE buffer pH 9.0 and citrate buffer pH 6.0 for optimal antigen retrieval, as both have been validated for NCOR1 detection .

• Loading control selection: Due to NCOR1's high molecular weight (observed at 120 kDa), select appropriate loading controls that won't overlap with your target band.

• Sample preparation: Ensure complete protein denaturation and use freshly prepared samples, as NCOR1 can be susceptible to degradation.

• Blocking optimization: Test both BSA and non-fat dry milk as blocking agents, as some epitopes may be masked differently depending on the blocking agent used.

• Positive control inclusion: Always include proven positive control samples such as HepG2 or K-562 cell lysates when optimizing detection protocols .

For HRP-conjugated antibodies specifically, include an additional control of unconjugated primary antibody plus HRP-conjugated secondary antibody to compare signal quality and verify that conjugation hasn't compromised epitope recognition.

What strategies can address variability in NCOR1 detection across different tissue types?

Tissue-specific detection of NCOR1 can be challenging due to variable expression levels and potential interactions with tissue-specific factors:

• Tissue-specific optimization: Adjust antibody dilutions based on expression levels in different tissues. Start with 1:500-1:2000 for Western blotting and 1:20-1:200 for IHC applications, but optimize for each tissue type .

• Sample preparation modifications: For tissues with high lipid content (brain, adipose), modify extraction protocols to improve protein recovery and reduce lipid interference.

• Panel validation: Test antibody performance across a panel of tissues to establish baseline detection parameters before executing comparative studies.

• Isoform considerations: Consider that tissue-specific alternative splicing may affect epitope availability. Where possible, use antibodies targeting conserved regions.

• Context-dependent interactions: Be aware that NCOR1's interaction with nuclear receptors varies by tissue context. In liver, NCOR1 represses lipid synthesis during fasting while inhibiting PPARα upon feeding , which may affect complex formation and epitope accessibility.

How can I design experiments to study NCOR1's role in transcriptional repression?

To investigate NCOR1's function in transcriptional repression:

• Reporter gene assays: Utilize luciferase reporter constructs containing promoters with known nuclear receptor binding elements (LXREs, PPREs, ERREs) to quantitatively assess repression .

• Loss-of-function approaches: Implement NCoR1-knockout or knockdown systems, such as the NCoR1ΔID hypomorphic model that cannot interact with thyroid hormone receptor .

• Point mutations in interaction domains: Generate point mutations in the CoRNR box motifs to disrupt specific nuclear receptor interactions while maintaining other functions.

• Histone modification analysis: Monitor histone deacetylation, particularly H3K27 acetylation, as a functional readout of NCOR1-mediated repression using ChIP followed by qPCR or sequencing .

• Nuclear receptor ligand manipulation: Test the effects of adding or removing specific nuclear receptor ligands (T3 for thyroid hormone receptor, retinoic acid for RAR) to observe NCOR1 displacement and derepression .

What considerations are important when using NCOR1 antibodies to study protein-protein interactions?

When investigating NCOR1's interactions with other proteins:

• Epitope interference: Select antibodies whose epitopes don't overlap with key interaction domains. For nuclear receptor interactions, avoid antibodies targeting residues 1525-1575 if studying C-terminal interactions .

• Co-immunoprecipitation optimization: For co-IP studies, use 40 μL of anti-FLAG M2 Affinity Gel suspension for FLAG-tagged constructs, following established protocols for detecting NCOR1 interactions .

• Cross-linking considerations: For transient interactions, implement protein cross-linking (0.5-1% formaldehyde) before immunoprecipitation to capture dynamic complexes.

• Sequential immunoprecipitation: For complex multi-protein assemblies, design sequential IP protocols to isolate specific subcomplexes containing NCOR1.

• Native vs. denaturing conditions: Compare results under native conditions (to preserve interactions) and denaturing conditions (to confirm direct binding) when assessing the specificity of detected interactions.

How can NCOR1 antibodies be used to investigate tissue-specific metabolic regulation?

NCOR1 plays critical roles in metabolic regulation that vary by tissue context:

• Liver metabolism studies: Investigate NCOR1's dual role in repressing lipid synthesis during fasting and inhibiting PPARα activation during feeding using tissue-specific knockout models .

• ChIP-seq analysis: Perform genome-wide binding studies in different metabolic states (fed, fasted, disease models) to identify condition-specific binding patterns .

• Multi-omics integration: Combine ChIP-seq data with transcriptomics and metabolomics to correlate NCOR1 binding with functional metabolic outcomes.

• Phosphorylation status analysis: Assess how nutritional status affects NCOR1 phosphorylation through the Akt pathway, using phospho-specific antibodies to monitor Ser1460 phosphorylation .

• Interaction partner profiling: Use co-IP followed by mass spectrometry to identify tissue-specific NCOR1 interaction partners that may mediate context-dependent functions.

How can NCOR1 antibodies be utilized in studying nuclear receptor-independent functions?

Recent research suggests NCOR1 has functions beyond classic nuclear receptor corepression:

• Global ChIP-seq analysis: Perform unbiased genome-wide binding studies to identify binding sites lacking canonical nuclear receptor motifs .

• Protein interactome studies: Use NCOR1 antibodies for immunoprecipitation followed by mass spectrometry to identify novel interaction partners beyond nuclear receptors.

• Non-canonical repression mechanisms: Investigate NCOR1's role in repression not prevented by NCoR1ΔID expression, suggesting alternative mechanisms exist .

• Developmental biology applications: Study the essential developmental roles of NCOR1 that cannot be explained solely through nuclear receptor interactions.

• Post-translational modification mapping: Identify novel modifications beyond phosphorylation that might regulate NCOR1's non-canonical functions using immunoprecipitation followed by modification-specific proteomic analysis.

What are the methodological approaches for studying NCOR1 in the context of metabolic disease models?

NCOR1 plays significant roles in metabolic regulation that are relevant to disease states:

• Diet-induced obesity models: Compare NCOR1 binding and function between normal and high-fat diet conditions to identify dysregulated pathways.

• Diabetes model applications: Investigate NCOR1's connection to diabetes, as recent research indicates "WTAP boosts lipid oxidation and induces diabetic cardiac fibrosis by enhancing AR methylation" .

• Circadian rhythm integration: Study NCOR1's role in "transcriptional programming of lipid and amino acid metabolism by the skeletal muscle circadian clock" .

• Tissue cross-talk analysis: Implement tissue-specific knockout models (liver, adipose, muscle) to dissect tissue-specific contributions to systemic metabolic regulation.

• Therapeutic target validation: Use NCOR1 antibodies to confirm target engagement and pathway modulation in preclinical studies of metabolic disease therapeutics targeting nuclear receptor pathways.