NCOA1 Antibody

Basic Research Questions

• What is NCOA1 and why is it important in research?

  NCOA1 (Nuclear receptor coactivator 1), also known as steroid receptor coactivator-1 (SRC-1), is a 157 kDa transcriptional coregulatory protein that binds directly to nuclear receptors, thereby stimulating transcriptional activities . NCOA1 plays a crucial role in gene regulation through interaction with multiple nuclear receptors, including those for steroids (PGR, GR, and ER), retinoids (RXRs), thyroid hormone (TRs), and prostanoids (PPARs) . It displays histone acetyltransferase activity toward H3 and H4, facilitating chromatin remodeling and recruitment of general transcription factors . NCOA1's significance in research stems from its overexpression in various cancers, particularly breast cancer, where it correlates with disease recurrence, metastasis, and therapy resistance .

• What types of NCOA1 antibodies are available for research applications?

  Research-grade NCOA1 antibodies are available in several formats to accommodate different experimental needs:

  Antibody Type	Format	Host Species	Applications	Examples
  Polyclonal	Purified IgG	Rabbit	Western Blot, ELISA	Rabbit anti-Human NCOA1
  Monoclonal	Purified IgG	Mouse	Western Blot, IP, Microarray	Mouse anti-Human NCOA1 (clone C02/13A7) , PCRP-NCOA1-1B11
  Conjugated	HRP-conjugated	Human	ELISA	NCOA1 Antibody, HRP conjugated
  Conjugated	FITC-conjugated	Human	Immunofluorescence	NCOA1 Antibody, FITC conjugated
  Conjugated	Biotin-conjugated	Human	ELISA	NCOA1 Antibody, Biotin conjugated

  Selection depends on experimental requirements, with monoclonal antibodies offering higher specificity and polyclonal antibodies providing broader epitope recognition .

• What are the recommended applications for NCOA1 antibodies?

  NCOA1 antibodies have been validated for multiple research applications:

  • Western Blotting: Detects NCOA1 protein bands at approximately 185 kDa in cell lysates, with recommended dilutions between 1:2,000-1:5,000

  • Immunohistochemistry (IHC): Effective for detecting NCOA1 in fixed tissue samples and tissue microarrays, particularly useful for cancer research

  • Immunoprecipitation (IP): Used for pulling down NCOA1 and its associated protein complexes in interaction studies

  • Chromatin Immunoprecipitation (ChIP): Employed to study NCOA1 recruitment to specific genomic regions, particularly promoters of target genes like VEGFa

  • Microarray Analysis: Used in protein expression profiling studies

  • ELISA: For quantitative detection of NCOA1 protein levels

  Method selection should be based on specific research questions and experimental design requirements.

• How should NCOA1 antibodies be stored and handled for optimal performance?

  Proper storage and handling are critical for maintaining antibody functionality:

  • Short-term storage: Maintain at 4°C for up to two weeks for immediate use

  • Long-term storage: Divide into small aliquots (≥20 μl) and store at -20°C or -80°C

  • Avoid freeze-thaw cycles: Repeated freezing and thawing can denature the antibody and reduce activity

  • Buffer considerations: Most NCOA1 antibodies are supplied in phosphate-buffered saline (PBS) with preservatives like sodium azide (typically 0.035-0.09%)

  • Working dilutions: Prepare fresh working dilutions on the day of experiment for optimal results

  Following these guidelines ensures antibody stability and experimental reproducibility.

Advanced Research Questions

• How can I validate the specificity of NCOA1 antibodies in my experimental system?

  Comprehensive validation ensures reliable experimental outcomes:

  • Positive controls: Use cell lines known to express high levels of NCOA1 (e.g., HEK293, MDA-MB-231 for human samples)

  • Negative controls: Utilize NCOA1 knockout (Ncoa1-/-) cells or tissues where available, or cells with NCOA1 knockdown using siRNA/shRNA

  • Rescue experiments: Reintroduce NCOA1 expression in knockout cells and confirm antibody detection, as demonstrated in studies showing restored VEGFa expression when NCOA1 was reintroduced in knockout cells

  • Peptide competition: Pre-incubate antibody with immunizing peptide before application to confirm signal specificity

  • Multiple antibody comparison: Use antibodies from different sources targeting different epitopes to verify consistent detection patterns

  • Size verification: Confirm detection at the expected molecular weight (~157-185 kDa)

  These validation steps are essential for avoiding false positive results and misinterpretation of data.

• What are the optimal conditions for detecting NCOA1 using Western blotting?

  Successful Western blotting for NCOA1 requires specific optimization:

  • Lysate preparation: Use strong lysis buffers containing protease inhibitors to efficiently extract nuclear proteins

  • Protein loading: Load 30-50 μg of total protein per lane for cell lines; higher amounts may be needed for tissue samples

  • Gel percentage: Use 6-8% SDS-PAGE gels to properly resolve the high molecular weight NCOA1 protein (~157-185 kDa)

  • Transfer conditions: Employ overnight transfer at low voltage (30V) or semi-dry transfer systems optimized for large proteins

  • Blocking: 5% non-fat dry milk or BSA in TBS-T for 1-2 hours at room temperature

  • Antibody dilution: Typically 1:2,000 for primary antibody incubation overnight at 4°C

  • Washing: Extensive washing (4-5 times, 5-10 minutes each) with TBS-T between antibody incubations

  • Detection: Enhanced chemiluminescence with extended exposure times (1-5 minutes) may be necessary

  Researchers should note that NCOA1 detection in Western blots requires careful optimization due to its high molecular weight and potential for degradation.

• How can I use NCOA1 antibodies for studying protein-protein interactions in ChIP assays?

  ChIP assays reveal NCOA1's role in transcriptional regulation:

  • Cross-linking conditions: Optimize formaldehyde concentration (1-1.5%) and cross-linking time (10-15 minutes) for nuclear proteins

  • Sonication parameters: Adjust to generate DNA fragments of 200-500 bp for optimal resolution

  • Antibody selection: Use ChIP-validated NCOA1 antibodies with demonstrated specificity

  • Input controls: Include chromatin samples before immunoprecipitation (5-10% of IP material)

  • IP conditions: Overnight incubation at 4°C with 2-5 μg of antibody per ChIP reaction

  • Washing stringency: Use increasingly stringent wash buffers to reduce background

  • Target regions: Design primers for potential NCOA1 binding sites, such as the VEGFa promoter region B where NCOA1 has been shown to be recruited by HIF1α and c-Fos

  • Sequential ChIP: For studying co-occupancy, perform sequential ChIP with NCOA1 antibody followed by antibodies against suspected interaction partners (e.g., c-Fos, HIF1α, NF-κB)

  Research has revealed that NCOA1 is efficiently recruited to the chromatin Region B proximal to the VEGFa promoter, with this recruitment being dependent on HIF1α and c-Fos but not NF-κB .

• What are the methodological considerations for using NCOA1 antibodies in cancer research?

  NCOA1 antibodies serve crucial functions in cancer studies:

  • Tissue microarrays (TMAs): Utilize standardized scoring systems (e.g., Allred scoring) for NCOA1 immunostaining as used in studies correlating NCOA1 levels with disease outcomes

  • Correlative studies: Combine NCOA1 staining with other markers (e.g., CD31 for angiogenesis, F4/80 for macrophage infiltration) to study multiple parameters simultaneously

  • Metastasis models: Use NCOA1 antibodies to track expression in primary tumors versus metastatic sites

  • Knockdown validation: Confirm NCOA1 silencing efficiency in functional studies using antibody-based detection methods

  • Multiplex immunofluorescence: Combine NCOA1 staining with other markers to study co-localization and pathway activation

  • Patient stratification: Develop standardized NCOA1 staining and scoring protocols for potential prognostic applications, as NCOA1 levels correlate with recurrence, higher tumor grade, and poor prognosis in breast cancer

  Comprehensive studies have shown that NCOA1 and CSF1 levels positively correlate with disease recurrence, higher tumor grade, and poor prognosis in a cohort of 453 human breast tumors .

• How does NCOA1 contribute to breast cancer progression and metastasis?

  Mechanistic studies using NCOA1 antibodies have revealed:

  • Angiogenesis regulation: NCOA1 promotes tumor angiogenesis by upregulating VEGFa expression through cooperation with HIF1α and c-Fos transcription factors

  • Macrophage recruitment: NCOA1 directly targets the CSF1 promoter through interaction with c-Fos at a functional AP-1 site, enhancing macrophage recruitment to tumors

  • Metastatic potential: Transgenic mouse models overexpressing NCOA1 show increased circulating tumor cells and lung metastasis efficiency without affecting primary tumor formation

  • Microvascular density: NCOA1 knockout reduces tumor microvascular density by 60-70%, while NCOA1 overexpression significantly increases it

  Experimental data from multiple mouse models demonstrates that NCOA1 promotes metastasis through distinct mechanisms including angiogenesis and immune cell recruitment rather than primary tumor growth .

• What controls should be included when using NCOA1 antibodies for immunohistochemistry?

  Robust controls ensure reliable IHC results:

  • Positive tissue controls: Include breast cancer tissues known to overexpress NCOA1

  • Negative tissue controls: Use paired adjacent non-tumor tissues (ANT) as comparative controls

  • Antibody controls: Include isotype control antibodies matched to the NCOA1 antibody class and concentration

  • Absorption controls: Pre-absorb antibody with immunizing peptide to confirm specificity

  • Cell line controls: Consider including sections from cell line pellets with known NCOA1 expression levels (e.g., ESCC cell lines vs. normal esophageal epithelial cells)

  • Scoring system: Implement standardized scoring methods such as the Allred scoring system for consistent evaluation

  Studies demonstrate significantly higher NCOA1 levels in ESCC tissues compared to paired adjacent non-tumor tissues, highlighting the importance of appropriate controls .

• How can I design experiments to study NCOA1's role in different cancer types?

  Comprehensive experimental approaches include:

  • Expression profiling: Use NCOA1 antibodies to screen tissue microarrays across different cancer types and correlate with clinicopathological parameters

  • Genetic manipulation: Create knockout/knockdown and overexpression models to study functional consequences

  • Pathway analysis: Couple NCOA1 detection with downstream targets (VEGFa, CSF1) to elucidate regulatory networks

  • In vivo imaging: Utilize NCOA1 antibodies for multiplex imaging of tumor microenvironment

  • Patient-derived xenografts: Compare NCOA1 expression in models with different metastatic potentials

  • Drug response studies: Correlate NCOA1 levels with therapeutic resistance patterns

  Research has established NCOA1 as a potential biomarker in esophageal squamous cell carcinoma (ESCC), where elevated levels associate with aggressive clinicopathological parameters and poorer survival .

• What are the technical challenges in detecting NCOA1 in different sample types?

  Sample-specific considerations are essential:

  • Fresh tissues: Process rapidly to minimize protein degradation; flash-freeze for optimal preservation

  • FFPE samples: Optimize antigen retrieval methods (high-pressure, extended time) for nuclear proteins

  • Cell fractionation: Ensure efficient nuclear extraction to capture the predominantly nuclear NCOA1

  • Background reduction: Implement specialized blocking protocols to minimize non-specific binding

  • Signal amplification: Consider tyramide signal amplification for low-abundance detection

  • Multiplexing challenges: When combining NCOA1 with other markers, carefully design antibody panels to avoid cross-reactivity

  Research protocols have successfully employed NCOA1 antibodies in various sample types, from cell lines to complex tissue microarrays with hundreds of patient samples .