NCOA5 Antibody

Basic Research Questions on NCOA5 Antibodies

For maintaining antibody integrity and performance:

• Store at -20°C as recommended by manufacturers

• Most preparations are stable for one year after shipment when stored properly

• Antibodies are typically provided in storage buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting is generally unnecessary for -20°C storage but may be considered for frequently used antibodies

• Some preparations (20μl sizes) may contain 0.1% BSA as a stabilizer

Avoid repeated freeze-thaw cycles and follow manufacturer-specific guidance for individual products.

How can researchers validate the specificity of NCOA5 antibodies in their experimental systems?

Establishing antibody specificity is critical for reliable results:

• Knockdown/Knockout Validation: Use siRNA-mediated knockdown or CRISPR/Cas9 knockout of NCOA5. For example, studies have used siRNA sequences such as:

  • siNCOA5 sense: 5′-AGGGAUCUUAGAGACUUUCGUTT-3′

  • siNCOA5 antisense: 5′-ACGAAAGUCUCUAAGAUCCCUTT-3′

• Cross-Reactivity Assessment: Test against recombinant protein fragments. Some antibodies have been validated against 364 human recombinant protein fragments

• Multiple Antibody Comparison: Use antibodies targeting different epitopes (C-terminal vs N-terminal)

• Predicted Reactivity Verification: Verify cross-reactivity with predicted species including mouse, orangutan, monkey, gorilla, chimpanzee, and various primates

• Western Blot Analysis: Confirm single band at expected molecular weight (66 kDa)

• Immunohistochemistry Controls: Include positive (tissues known to express NCOA5) and negative controls

What is known about NCOA5's role in cancer progression and how can researchers study this using antibodies?

NCOA5 has demonstrated significant roles in multiple cancer types:

How does NCOA5 function in inflammatory pathways and what antibody-based approaches can reveal its mechanisms?

NCOA5 plays a critical role in inflammatory signaling:

• LXR-NCOA5 Regulatory Complex:

  • NCOA5 functions as an LXR corepressor

  • It attenuates Abca1 expression which affects cholesterol efflux

  • TLR3-LXR signal crosstalk promotes recruitment of NCOA5 to the Abca1 promoter

• Experimental Approaches to Study This Mechanism:

  • Chromatin Immunoprecipitation (ChIP): Using NCOA5 antibodies to detect promoter occupancy at the Abca1 LXRE (LXR response element)

  • Co-Immunoprecipitation: Demonstrating direct interaction between NCOA5 and LXR in macrophage nuclear extracts

  • GST-Pulldown Assays: To map interaction domains using in vitro translated NCOA5 protein with GST-LXRα

• Methodological Insights:

  • NCOA5 binds to the Abca1 promoter in a ligand-dependent manner 18 hours post-treatment

  • In LXR-/- macrophages, NCOA5 shows ligand-independent occupancy, suggesting additional recruitment mechanisms

  • These interactions affect RNA polymerase II recruitment to target genes

What are the technical challenges in detecting NCOA5 in different cellular compartments?

NCOA5 detection presents several compartment-specific challenges:

• Nuclear Localization:

  • As a transcriptional coregulator, NCOA5 primarily localizes to the nucleus

  • Challenge: Nuclear extraction protocols may affect epitope accessibility

  • Solution: Use optimized nuclear extraction buffers containing appropriate protease inhibitors

  • Validation Method: Compare cytoplasmic and nuclear fractions using compartment-specific markers alongside NCOA5 detection

• Chromatin-Associated NCOA5:

  • Challenge: Cross-linking procedures in ChIP can affect antibody recognition

  • Solution: Test different cross-linking conditions (formaldehyde concentration and incubation time)

  • Optimization Strategy: Perform epitope retrieval steps when using antibodies for IHC on paraffin-embedded sections

• Detection in Different Cell Types:

  • NCOA5 expression varies significantly between cell types and tissues

  • Validation Controls: Use cell lines with known high expression (HEK-293, HeLa)

  • Sensitivity Considerations: For low-expressing samples, consider signal amplification methods or more sensitive detection systems

How can researchers effectively use NCOA5 antibodies to study protein-protein interactions?

To characterize NCOA5's diverse interaction network:

• Co-Immunoprecipitation (Co-IP) Strategies:

  • Use antibodies targeting different epitopes to avoid disrupting specific interactions

  • Both N-terminal (1-280aa) and C-terminal antibodies have been successfully used

  • For nuclear receptor interactions, studies have shown that the NH2-terminus of NCOA5 interacts with LXRα in a ligand-independent manner

• Mapping Interaction Domains:

  • Combine IP with deletion mutants to map interaction domains

  • Research shows that deletion of NH2-terminus (Δ1–280aa) prevents interaction with LXRα despite the presence of the canonical nuclear receptor interaction motif in the C-terminus

• Validating in Cellular Contexts:

  • Determine if interactions detected in vitro occur in living cells

  • Example: While NCOA5 interacts with LXR in vitro in both presence and absence of ligand, it only localizes to the Abca1 promoter following ligand stimulation in vivo

• Proximity Ligation Assays:

  • Can demonstrate protein-protein interactions in intact cells while preserving cellular architecture

  • Requires two antibodies from different species targeting the interacting proteins

How might NCOA5 antibodies contribute to developing cancer biomarkers and therapeutic targets?

The potential of NCOA5 as a biomarker is supported by several findings:

• Diagnostic Applications:

  • NCOA5 expression correlates significantly with established ovarian cancer markers CA125 and HE4

  • Expression is significantly higher in multiple cancer tissues compared to adjacent normal tissues

  Research Direction: Develop antibody-based diagnostic assays combining NCOA5 with existing markers for improved sensitivity and specificity

• Prognostic Value:

  • High NCOA5 expression correlates with poorer survival in ovarian cancer patients

  • Expression levels correlate with clinicopathological features including tumor length, lymph node staging and cancer staging in CRC

  Methodological Approach: Use validated antibodies in tissue microarrays to establish standardized scoring systems for clinical application

• Therapeutic Target Validation:

  • NCOA5 knockdown inhibits key cancer hallmarks including proliferation, migration, and invasion

  • It modulates critical signaling pathways including AKT signaling and EMT

  Experimental Strategy: Use antibodies to monitor NCOA5 expression and pathway modulation in response to potential therapeutic compounds

What are the emerging techniques for studying NCOA5's genomic binding sites using antibodies?

Advanced genomic techniques using NCOA5 antibodies:

• Chromatin Immunoprecipitation Sequencing (ChIP-seq):

  • Can identify genome-wide binding sites of NCOA5

  • Research has shown NCOA5 occupancy at the Abca1 promoter near the LXRE

  • Optimization Requirement: Antibodies must be highly specific and efficient in chromatin immunoprecipitation conditions

• CUT&RUN and CUT&Tag:

  • Emerging alternatives to ChIP-seq with improved signal-to-noise ratio

  • Require less starting material and can provide higher resolution

  • Antibody Consideration: These techniques often work with antibodies that perform well in IP applications

• Combinatorial Approaches:

  • ChIP-seq combined with RNA-seq after NCOA5 manipulation can identify direct vs. indirect transcriptional effects

  • Integrated Analysis: Correlate NCOA5 binding patterns with changes in RNA polymerase II recruitment and activation

• Single-Cell Applications:

  • Emerging techniques to study NCOA5 binding heterogeneity at single-cell resolution

  • Technical Challenge: Requires highly specific antibodies compatible with low-input material

How can researchers effectively use NCOA5 antibodies in multiplex detection systems?

Strategies for multiplexed detection using NCOA5 antibodies:

• Spectral Imaging:

  • Simultaneous detection of NCOA5 with interacting partners or pathway components

  • Panel Design Example: Combine NCOA5 with EMT markers (E-cadherin, N-cadherin, Vimentin) to study correlation with cancer progression

• Sequential Multiplex Immunohistochemistry:

  • Allows for detection of 5+ proteins on the same tissue section

  • Protocol Consideration: Requires complete stripping of previous antibodies or spectral unmixing capabilities

• Mass Cytometry (CyTOF):

  • Metal-tagged antibodies allow for highly multiplexed detection without spectral overlap

  • Application: Simultaneously measure NCOA5 with dozens of signaling proteins to place it in cellular signaling networks

• Antibody Validation for Multiplex Systems:

  • Verify antibody performance in multiplexed conditions, as steric hindrance may affect binding

  • Test for cross-reactivity with other primary or secondary antibodies in the panel

Troubleshooting NCOA5 Antibody Applications

Robust experimental design requires appropriate controls:

• Western Blot Controls:

  • Positive Control: Lysates from HEK-293 or HeLa cells with confirmed NCOA5 expression

  • Negative Control: Lysates from cells after NCOA5 knockdown using validated siRNA sequences

  • Loading Control: Detection of housekeeping proteins to normalize expression levels

• Immunohistochemistry Controls:

  • Positive Tissue Control: Human stomach tissue has been validated for NCOA5 detection

  • Negative Control: Primary antibody omission or isotype control

  • Absorption Control: Pre-incubation of antibody with immunizing peptide to demonstrate specificity

• ChIP Controls:

  • Positive Control Region: LXRE region of the Abca1 promoter

  • Negative Control Region: Upstream region lacking an LXRE

  • Input DNA: Unimmunoprecipitated chromatin to normalize enrichment

  • IgG Control: Non-specific IgG immunoprecipitation to establish background levels

• Functional Controls in Expression Studies:

  • Compare phenotypic effects after NCOA5 knockdown and rescue experiments

  • Use pharmacological inhibitors (e.g., PI3K inhibitor LY294002) to validate pathway connections

What are the emerging roles of NCOA5 in disease processes beyond cancer?

Research indicates NCOA5 involvement in:

• Inflammatory Disorders:

  • NCOA5 mediates crosstalk between pro-inflammatory and anti-inflammatory pathways

  • Polymorphisms in NCOA5 are associated with chronic inflammatory diseases

  • Research Application: Use antibodies to study NCOA5 expression in inflammatory tissue samples and correlate with disease severity

• Metabolic Diseases:

  • NCOA5 affects cholesterol efflux through regulation of Abca1

  • It's implicated in metabolic disease through genetic associations

  • Experimental Approach: Study NCOA5 expression and localization in metabolic tissues using tissue-specific protocols

• Stem Cell Biology:

  • NCOA5 has been identified as a conserved component of pluripotent stem cells

  • It may contribute to maintaining pluripotent stem cell populations

  • Research Direction: Use antibodies to track NCOA5 expression during stem cell differentiation

How can researchers integrate antibody-based detection with other molecular techniques for comprehensive NCOA5 studies?

Comprehensive research strategies combining multiple techniques:

• Multi-Omics Integration:

  • Correlate antibody-detected protein levels with transcriptome data

  • Example workflow:
    a) ChIP-seq to identify NCOA5 binding sites
    b) RNA-seq to measure expression changes after NCOA5 manipulation
    c) Antibody-based detection to confirm protein-level changes

• Live-Cell Imaging Approaches:

  • CRISPR-mediated tagging of endogenous NCOA5

  • Compare with antibody-based detection to validate localization patterns

  • Study dynamic changes in NCOA5 localization in response to stimuli

• Structural Biology Integration:

  • Use antibody-based mapping to guide structural studies

  • For example, the NH2-terminus (1-280aa) has been identified as critical for LXR interaction

  • This information can inform domain-focused structural analyses

• Systems Biology Approaches:

  • Use antibody data as validation for predicted protein interaction networks

  • Integrate NCOA5 protein expression data with pathway analyses to build comprehensive regulatory models