NCOR1 Antibody, FITC conjugated

What is NCOR1 and why is it important in cellular function?

NCOR1 functions primarily as a transcriptional corepressor that was initially discovered through its interactions with nuclear receptors. Since then, research has revealed that NCOR1 interacts with numerous transcription factors involved in lymphocyte development, including STAT5, NF-κB, AP-1, and NUR77 . The significance of NCOR1 lies in its role as a scaffold for recruiting histone deacetylases, particularly HDAC3, to DNA-bound transcription factors . This recruitment is essential for silencing gene expression through histone deacetylation, which decreases chromatin accessibility. NCOR1 has demonstrated critical functions in thymocyte development and survival, making it an important target for immunological research .

What are the typical applications for FITC-conjugated NCOR1 antibodies?

FITC-conjugated NCOR1 antibodies are primarily used in fluorescence-based detection techniques. Based on the product specifications from multiple suppliers, these antibodies are suitable for:

• Immunofluorescence (IF) applications, including paraffin-embedded tissue sections (IHC-P)

• Immunofluorescence on frozen tissue sections (IHC-F)

• Immunocytochemistry (ICC)

• Flow cytometry for detecting NCOR1 in cell populations

• Fluorescence microscopy for visualizing subcellular localization of NCOR1

These applications allow researchers to visualize the expression and localization patterns of NCOR1 in various experimental systems without requiring secondary antibody incubation steps .

What species reactivity can I expect from commercially available NCOR1-FITC antibodies?

Commercial NCOR1 antibodies conjugated to FITC vary in their species reactivity profiles. Based on the search results:

*Note: NB100-58824 is listed as unconjugated but included for species reactivity reference .

When selecting an appropriate antibody for your experiments, verify the specific species reactivity needed for your research model and check if the reactivity has been experimentally validated rather than just predicted .

How should I design experiments to validate NCOR1 antibody specificity?

Validating antibody specificity is crucial for obtaining reliable results. For NCOR1-FITC antibodies, consider these methodological approaches:

• Western blot validation: Though not directly using the FITC-conjugate, perform parallel Western blot experiments with the unconjugated version of the same antibody clone to confirm it recognizes a protein of the expected molecular weight (~270 kDa for NCOR1). This provides confidence in the specificity of the conjugated version .

• Knockout controls: Include NCOR1 knockout or knockdown samples as negative controls. The CRISPR-Cas9 approach used for NCOR1 deletion in human CD4+ T cells described in the literature could serve as a methodology for generating such controls .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (if available from the manufacturer) before application to confirm signal specificity.

• Cross-validation: Compare staining patterns using different antibodies targeting distinct NCOR1 epitopes to confirm consistent localization patterns .

• Immunoprecipitation followed by mass spectrometry: This can verify that the antibody is truly capturing the NCOR1 protein rather than cross-reacting with other proteins .

What controls should I include when using NCOR1-FITC antibodies in immunofluorescence experiments?

For robust immunofluorescence experiments with NCOR1-FITC antibodies, include these essential controls:

• Isotype control: Use a FITC-conjugated rabbit IgG (matching the host species and isotype of your NCOR1 antibody) at the same concentration to assess non-specific binding .

• Negative tissue/cell control: Include samples known to express very low or undetectable levels of NCOR1 to establish background fluorescence levels.

• FITC auto-fluorescence control: Examine unstained samples to determine natural tissue/cell autofluorescence in the FITC channel.

• NCOR1 knockdown/knockout control: If available, include NCOR1-deficient samples as the gold standard negative control .

• Subcellular localization control: Include nuclear counterstains (e.g., DAPI) to verify the expected nuclear localization of NCOR1, which is described as predominantly nuclear in the literature .

• Fixation control: Compare different fixation methods to ensure the epitope recognized by your antibody remains accessible after processing.

What are the optimal storage conditions and shelf life for NCOR1-FITC conjugated antibodies?

FITC-conjugated antibodies require specific storage conditions to maintain their fluorescent properties and binding capacity:

• Storage temperature: Store at -20°C as recommended by manufacturers. Avoid storing at 4°C for extended periods as this can lead to gradual loss of fluorescence intensity .

• Buffer composition: Most FITC-conjugated NCOR1 antibodies are supplied in buffers containing glycerol (typically 50%), which prevents freezing at -20°C and protects antibody structure during freeze-thaw cycles .

• Aliquoting: Divide the stock antibody into small aliquots upon receipt to minimize repeated freeze-thaw cycles, which can degrade both the antibody protein and the FITC fluorophore .

• Light protection: FITC is sensitive to photobleaching. Store in amber tubes or wrap containers in aluminum foil to protect from light exposure .

• Shelf life: While specific shelf life information for NCOR1-FITC antibodies is not provided in the search results, fluorophore-conjugated antibodies typically maintain optimal activity for 6-12 months when properly stored. Monitor for decreasing signal intensity over time as an indicator of degradation.

How can I troubleshoot weak or absent signal when using NCOR1-FITC antibodies?

When encountering weak or absent signals with NCOR1-FITC antibodies, consider these methodological approaches:

• Antigen retrieval optimization: NCOR1 is a nuclear protein that may require robust antigen retrieval for epitope exposure. Test different antigen retrieval methods (heat-induced epitope retrieval with citrate buffer pH 6.0 or EDTA buffer pH 9.0) to improve accessibility .

• Antibody concentration: The recommended dilution ranges for IF applications are typically 1:50-1:200 . If signal is weak, titrate the antibody using serial dilutions to determine optimal concentration for your specific samples.

• Fixation method assessment: Overfixation can mask epitopes. Compare paraformaldehyde, methanol, and acetone fixation to determine which best preserves the NCOR1 epitope recognized by your antibody.

• Permeabilization optimization: For intracellular/nuclear antigens like NCOR1, ensure adequate permeabilization with appropriate detergents (0.1-0.5% Triton X-100 or 0.1% Saponin).

• Expression level verification: Confirm NCOR1 expression in your sample type through alternative methods such as RT-PCR or Western blotting using unconjugated antibodies against NCOR1 .

• Photobleaching prevention: FITC is prone to photobleaching. Use anti-fade mounting media containing protective agents and minimize exposure to excitation light during imaging.

What is the optimal concentration for NCOR1-FITC antibodies in different applications?

Optimal antibody concentration varies by application and must often be empirically determined:

For all applications, researchers should perform a titration experiment with serial dilutions (e.g., 1:50, 1:100, 1:200, 1:400) to determine the concentration that provides maximum specific signal with minimal background. The antibody concentration information provided by manufacturers (typically 1μg/μl for bs-0224R-FITC ) can be used as a starting point for calculating appropriate dilutions.

How can NCOR1-FITC antibodies be used to investigate NCOR1's role in T cell development?

NCOR1 plays critical roles in T cell development and regulatory T cell function, as evidenced by several studies in the search results . To investigate these functions using NCOR1-FITC antibodies:

• Flow cytometry analysis of thymocyte populations: Use NCOR1-FITC antibodies in combination with T cell developmental markers (CD4, CD8, CD25, CD44) to assess NCOR1 expression levels across different thymocyte developmental stages. Research has shown significant impairment of T cell development when both NCOR1 and NCOR2 are deleted, with marked increases in DP cells and decreases in both CD4 SP and CD8 SP populations .

• Confocal microscopy of thymic sections: Apply NCOR1-FITC antibodies to thymic tissue sections to visualize the spatial distribution of NCOR1 expression in relation to thymic architecture and cellular niches.

• Chromatin binding dynamics: Combine NCOR1-FITC immunofluorescence with DNA FISH (Fluorescence In Situ Hybridization) to visualize NCOR1 association with specific genomic loci known to be important in T cell development, such as the Bcl2l11 gene mentioned in the literature .

• Co-localization with transcription factors: Perform dual immunofluorescence with NCOR1-FITC and antibodies against transcription factors known to interact with NCOR1 (STAT5, NF-κB, AP-1, NUR77) to examine their spatial relationships in developing T cells .

• T cell receptor signaling: Use NCOR1-FITC antibodies to monitor NCOR1 localization changes during TCR stimulation, as research has shown that combined NCOR1/2 deletion results in increased signaling through the T cell receptor .

How can I use NCOR1-FITC antibodies to study interactions between NCOR1 and its binding partners?

To investigate NCOR1's interactions with its binding partners using FITC-conjugated antibodies:

• Proximity Ligation Assay (PLA): Although FITC-conjugated antibodies aren't typically used directly for PLA, the search results mention that the unconjugated NB100-58824 antibody can be used as a primary antibody in PLA assays with complementary antibodies like NB200-347 . This methodology allows visualization of protein-protein interactions at the single-molecule level.

• FRET (Förster Resonance Energy Transfer): Pair NCOR1-FITC (donor) with antibodies against interaction partners conjugated to appropriate acceptor fluorophores (e.g., HDAC3-TRITC) to detect molecular proximity (<10nm) indicative of protein-protein interactions.

• Co-immunoprecipitation followed by fluorescence detection: Use unconjugated NCOR1 antibodies for immunoprecipitation (as validated in source ), then detect co-precipitated proteins using fluorescently-labeled antibodies including FITC conjugates.

• ChIP-seq correlation: The NB100-58824 antibody has been validated for Chromatin Immunoprecipitation Sequencing . While this specific application wouldn't utilize the FITC conjugate directly, correlating ChIP-seq data with immunofluorescence patterns using NCOR1-FITC antibodies could provide insights into functional interactions.

• Live-cell imaging: For certain cell types and with careful antibody delivery methods (such as cell-penetrating peptide conjugation), NCOR1-FITC antibodies might be used to track dynamic interactions in living cells, though this is technically challenging for nuclear proteins.

What considerations are important when using NCOR1-FITC antibodies to study regulatory T cell biology?

When investigating regulatory T cell (Treg) biology with NCOR1-FITC antibodies, consider these specialized approaches:

• Treg subset characterization: Research shows NCOR1 controls naïve and effector Treg cell states . Use NCOR1-FITC antibodies in combination with markers that distinguish naïve (CD45RA+/CD45RO- in humans) from effector (CD45RA-/CD45RO+) Treg cells to correlate NCOR1 expression levels with Treg functional states .

• FOXP3 co-staining: Since FOXP3 is the master regulator of Treg cells, combine NCOR1-FITC staining with FOXP3 detection (using a different fluorophore) to investigate their relationship. Research indicates NCOR1 deletion affects FOXP3+ cell frequencies but is dispensable for maintaining FOXP3 expression in in vitro generated Treg cells .

• Functional correlation analysis: Correlate NCOR1 expression levels (as detected by FITC intensity) with suppressive capacity of Treg cells in in vitro suppression assays. This is relevant given the finding that NCOR1-deficient Treg cells show impaired suppressive function in CD4+ T cell transfer colitis models .

• Chromatin state assessment: While not directly using FITC conjugates, combine NCOR1-FITC immunofluorescence with assays that detect histone modifications (particularly acetylation states) to investigate NCOR1's role in epigenetic regulation of Treg-specific gene expression programs .

• Cross-species validation: Consider the species differences in NCOR1 functions, as the research demonstrates that NCOR1 deletion enhances the generation of FOXP3+ T cells in human systems, showing conservation of NCOR1 function in regulating naïve and effector Treg cell subset differentiation across species .

How should I interpret variability in NCOR1 staining intensity across different cell types?

When encountering variable NCOR1 staining intensity:

• Expression level differences: NCOR1 expression varies naturally across cell types and developmental stages. In T cell development, for instance, NCOR1 plays stage-specific roles that may correlate with expression levels .

• Nuclear architecture considerations: As a nuclear protein , NCOR1 detection may vary with nuclear morphology and chromatin compaction states. Cells with more condensed chromatin may show different staining patterns than those with open chromatin configurations.

• Epitope accessibility: The specific NCOR1 epitope targeted by your antibody may be differentially accessible depending on protein complex formation. For example, antibodies targeting amino acids 1770-1947 or 2301-2400/2453 regions may show different staining patterns based on NCOR1's interaction partners in different cell types.

• Standardization approach: To compare NCOR1 expression across cell types:

  • Use digital image analysis to quantify fluorescence intensity

  • Normalize to nuclear area

  • Include calibration standards in each experiment

  • Report relative rather than absolute expression levels

• Validation with multiple detection methods: Confirm unusual expression patterns using alternative techniques such as Western blotting, RT-PCR, or mass spectrometry to rule out antibody-specific artifacts .

How can I reconcile contradictory results between NCOR1 antibody staining and functional studies?

When faced with discrepancies between NCOR1 antibody staining patterns and functional data:

• Epitope-specific effects: Different NCOR1 antibodies target distinct regions of this large protein (2440+ amino acids). The functional domain affected in your study may not correspond to the epitope recognized by your antibody. Compare the immunogen ranges: the FITC-conjugated antibodies in the search results target different regions (amino acids 1770-1947 versus 2301-2400/2453 ).

• Post-translational modifications: NCOR1 function is regulated by modifications that may alter antibody binding without affecting protein levels. Consider using modification-specific antibodies alongside total NCOR1 detection.

• Protein complex formation: NCOR1 functions within multi-protein complexes including HDAC3 . Complex formation may mask epitopes while preserving function, or vice versa.

• Threshold effects: Functional impairment may require near-complete NCOR1 depletion, while antibody staining may detect residual protein. Note that in mouse studies, single knockout of NCOR1 showed less dramatic phenotypes than combined NCOR1/NCOR2 deletion .

• Temporal dynamics: Consider whether your functional assays and antibody staining are capturing the same temporal window. NCOR1's roles in processes like T cell development occur across multiple time points and developmental stages .

What considerations are important when quantifying NCOR1 expression using FITC-conjugated antibodies?

When quantifying NCOR1 expression using FITC-conjugated antibodies:

• FITC-specific technical limitations:

  • Photobleaching: FITC fluorescence decreases with exposure to excitation light. Standardize exposure times and minimize sample illumination before image capture.

  • pH sensitivity: FITC fluorescence is optimal at pH 8.0 and decreases at lower pH. Ensure consistent buffer conditions across samples.

  • Spectral overlap: FITC emission overlaps with cellular autofluorescence. Use appropriate filters and autofluorescence controls.

• Standardization procedures:

  • Include calibration standards such as MESF (Molecules of Equivalent Soluble Fluorochrome) beads in flow cytometry experiments.

  • For microscopy, use reference slides with known fluorophore concentrations.

  • Process all comparative samples in parallel with identical staining, imaging, and analysis parameters.

• Quantification methods:

  • Flow cytometry: Report median fluorescence intensity rather than mean to minimize the impact of outliers.

  • Microscopy: Define clear nuclear ROIs (Regions of Interest) for quantification, as NCOR1 is predominantly nuclear .

  • Consider three-dimensional analysis for thick specimens to account for total protein content.

• Signal saturation: Ensure signal intensity falls within the linear range of your detection system to avoid underestimating differences between high-expressing samples.

• Background correction: Implement rigorous background subtraction using matched isotype controls and unstained samples to account for autofluorescence, particularly in tissues with high intrinsic fluorescence.