ZNF366 Antibody

What is ZNF366 and what are its key cellular functions?

ZNF366, also known as DC-SCRIPT (Dendritic Cell-Specific Transcript), is an evolutionarily conserved zinc finger protein that functions primarily as a transcriptional corepressor. It plays a significant role in estrogen receptor-α (ERα) signaling by interacting with the DNA binding domain (DBD) of ERα. ZNF366 represses ERα activity through association with other corepressor proteins including RIP140, CtBP, and histone deacetylases . The protein has a calculated molecular weight of approximately 85 kDa, though it typically appears at 90-100 kDa in Western blots due to post-translational modifications .

ZNF366 inhibits the expression of estrogen-responsive genes in vivo, suggesting it plays an important regulatory role in estrogen signaling pathways . This function makes it particularly relevant for research in breast cancer and other estrogen-responsive tissues.

What applications are ZNF366 antibodies validated for?

ZNF366 antibodies have been validated for multiple research applications:

Different antibodies target various epitopes of ZNF366, including:

• C-terminal regions (e.g., ABIN6740640)

• Internal regions (AA 455-650, AA 501-600)

• Full-length protein recognition

The selection of the appropriate antibody should be guided by the specific research application and experimental conditions.

What species reactivity is available for ZNF366 antibodies?

ZNF366 antibodies demonstrate varied species reactivity profiles:

When selecting an antibody for a particular species, researchers should verify the validated reactivity and consider sequence homology if working with species not explicitly tested.

What is the recommended protocol for Western blotting with ZNF366 antibodies?

For Western blotting applications using ZNF366 antibodies, the following methodological approach is recommended:

• Sample Preparation: Prepare lysates from tissues (mouse heart and skeletal muscle have shown positive results) or cell lines expressing ZNF366 .

• Protein Loading: Load 20-50 μg total protein per lane, depending on expression levels.

• Separation: Use 8-10% SDS-PAGE gels for optimal resolution around the 90-100 kDa range where ZNF366 migrates .

• Antibody Dilution: For antibody 24340-1-AP, use a dilution range of 1:200-1:1000 in primary antibody buffer (typically 5% BSA or milk in TBST) .

• Detection: For chemiluminescent detection, ensure exposure times are optimized based on expression levels.

• Expected Band Size: Look for bands in the 90-100 kDa range, which corresponds to the observed molecular weight of ZNF366 .

It is recommended to include appropriate positive controls (mouse heart or skeletal muscle tissue) and negative controls in your experimental design to validate antibody specificity.

How should researchers store and handle ZNF366 antibodies for optimal performance?

Proper storage and handling are critical for maintaining antibody performance:

• Storage Temperature: Store ZNF366 antibodies at -20°C for long-term stability .

• Storage Buffer: Most ZNF366 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Stability: When properly stored, antibodies are typically stable for one year after shipment .

• Aliquoting: For 24340-1-AP, aliquoting is unnecessary for -20°C storage, though for other antibodies, aliquoting is generally recommended to avoid freeze-thaw cycles .

• Working Solution: When preparing working dilutions, maintain sterile conditions and use fresh buffers.

• Specific Considerations: Note that some formulations (20μl sizes) contain 0.1% BSA, which may affect certain applications .

Following these guidelines will help ensure consistent antibody performance across experiments.

How can ZNF366 antibodies be used to study estrogen receptor signaling pathways?

ZNF366 antibodies can be valuable tools for investigating estrogen receptor signaling through several methodological approaches:

• Co-Immunoprecipitation Experiments:

  • ZNF366 has been shown to interact with ERα, specifically through the zinc finger domains of both proteins .

  • Researchers can use anti-FLAG antibodies (for tagged ZNF366) or anti-ERα antibodies to precipitate protein complexes, followed by immunoblotting to detect interaction partners .

  • This methodology can reveal the dynamics of ZNF366 interaction with ERα under different hormonal conditions.

• Chromatin Immunoprecipitation (ChIP):

  • Since ZNF366 acts as a corepressor for ERα, ChIP experiments using ZNF366 antibodies can identify genomic regions where this repression occurs.

  • Combining ZNF366 ChIP with ERα ChIP can provide insight into how these factors cooperate at specific estrogen-responsive gene promoters.

• Gene Expression Analysis:

  • After manipulating ZNF366 levels (overexpression or knockdown), researchers can use antibodies to confirm protein levels while monitoring estrogen-responsive genes.

  • Published research has demonstrated that ZNF366 overexpression suppresses estrogen-induced expression of genes such as cathepsin D and progesterone receptor .

• Corepressor Complex Characterization:

  • ZNF366 forms complexes with other corepressors including RIP140, CtBP, and histone deacetylases .

  • Antibodies can be used in sequential immunoprecipitation experiments to isolate and characterize these multi-protein repressive complexes.

What approaches should researchers use to validate ZNF366 antibody specificity?

Rigorous validation of antibody specificity is crucial for reliable research findings. For ZNF366 antibodies, consider these validation strategies:

• Genetic Controls:

  • Perform experiments with ZNF366 knockdown (siRNA/shRNA) or knockout (CRISPR/Cas9) samples

  • The expected signal should decrease or disappear in these conditions

• Overexpression Controls:

  • Compare wild-type cells with those overexpressing tagged ZNF366

  • Signal intensity should increase proportionally to overexpression levels

• Peptide Competition Assays:

  • Pre-incubate the antibody with the immunizing peptide (where available)

  • This should abolish specific binding in subsequent applications

• Cross-Validation with Multiple Antibodies:

  • Use antibodies targeting different epitopes of ZNF366

  • Consistent results with different antibodies increase confidence in specificity

• RT-PCR Correlation:

  • Compare protein detection patterns with mRNA expression across tissues/cell lines

  • Published RT-PCR primers for ZNF366 include: 5′-CCCCATCCAGTACAACTGCT-3′ and 5′-CTTCACGTCAGAGTGGACGA-3′

How can researchers troubleshoot non-specific binding when using ZNF366 antibodies?

When encountering non-specific binding issues with ZNF366 antibodies, consider the following methodological approaches:

• Optimization of Blocking Conditions:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Increase blocking time or blocker concentration if background is high

• Antibody Dilution Optimization:

  • For Western blotting, test a broader range of dilutions (e.g., 1:100-1:2000)

  • The recommended range for 24340-1-AP is 1:200-1:1000, but optimal dilution may be sample-dependent

• Buffer Adjustments:

  • Increase salt concentration in wash buffers to reduce non-specific ionic interactions

  • Add mild detergents (0.05-0.1% Tween-20) to reduce hydrophobic interactions

• Cross-Reactivity Assessment:

  • If working with non-validated species, consider protein sequence homology

  • BLAST analysis has shown variable homology across species: Human (100%), Pig (85%), which may affect specificity

• Sample Preparation Refinement:

  • Ensure complete protein denaturation for Western blotting

  • For immunohistochemistry, optimize fixation and antigen retrieval methods

• Secondary Antibody Controls:

  • Include secondary-only controls to identify background from secondary antibodies

  • Consider switching to more specific secondary antibodies if background persists

What are the optimal methods for studying ZNF366 protein-protein interactions?

To effectively study ZNF366's interactions with other proteins such as ERα, RIP140, and CtBP, consider these methodological approaches:

• Co-Immunoprecipitation (Co-IP):

  • Use cleared cell lysates (2 mg) immunoprecipitated with anti-FLAG (for tagged ZNF366) or anti-ERα antibodies

  • Include appropriate controls (mouse IgG for non-specific binding)

  • Resolve precipitates by SDS-PAGE and immunoblot with antibodies against expected interaction partners

  • Previously validated antibody combinations for this approach include:

    • Anti-FLAG M2 for IP, anti-HA-HRP for detection (tagged proteins)

    • Anti-ERα (6F11) for IP, anti-ERα (HC20) for detection

    • Anti-CtBP (sc-17759) for IP, anti-CtBP (sc-11390) for detection

• Proximity Ligation Assay (PLA):

  • Allows visualization of protein interactions in situ

  • Requires primary antibodies from different host species against ZNF366 and its interacting partners

• Fluorescence Resonance Energy Transfer (FRET):

  • For studying dynamic interactions in living cells

  • Requires fluorophore-conjugated antibodies or fluorescent protein-tagged constructs

• Bimolecular Fluorescence Complementation (BiFC):

  • For visualizing interactions in living cells

  • Complementary to Co-IP for confirming interactions in their native cellular context

• Mammalian Two-Hybrid Assays:

  • Useful for mapping interaction domains

  • Previous work has shown the zinc finger domains of ZNF366 and ERα mediate their interaction

Which cell and tissue types show significant ZNF366 expression?

Understanding the expression pattern of ZNF366 across different tissues helps in experimental design:

• Tissue Expression Pattern:

  • Positive Western blot detection has been observed in:

    • Mouse heart tissue

    • Mouse skeletal muscle tissue

  • ZNF366 expression has been studied in breast cancer cell lines including:

    • MCF7 cells (estrogen-responsive)

    • MDA-MB-231 cells

• Expression Level Variations:

  • When overexpressed in MCF7 cells, ZNF366 has been shown to suppress estrogen-induced gene expression

  • Endogenous expression levels may vary significantly across tissues and cell types

• Subcellular Localization:

  • Primarily nuclear localization consistent with its role as a transcriptional regulator

  • Can be visualized using immunofluorescence with Alexa Fluor 488/594-conjugated secondary antibodies

Researchers should consider these expression patterns when selecting appropriate experimental systems and controls.

What are the considerations for immunofluorescence studies with ZNF366 antibodies?

For immunofluorescence applications to visualize ZNF366 in cells:

• Fixation and Permeabilization:

  • Recommended fixation with 4% paraformaldehyde followed by permeabilization with 0.2-0.5% Triton X-100

  • Over-fixation may mask epitopes, especially for antibodies targeting conformational epitopes

• Antibody Incubation:

  • Primary antibody incubation at optimal dilution (antibody-dependent)

  • Secondary detection using fluorophore-conjugated antibodies such as:

    • Alexa Fluor 488 goat anti-mouse immunoglobulins (green fluorescence)

    • Alexa Fluor 594 goat anti-rabbit immunoglobulins (red fluorescence)

  • Typical working dilution: 1:3000 for secondary antibodies

• Mounting and Visualization:

  • Mount using medium containing DAPI for nuclear counterstaining

  • Visualize using confocal microscopy for optimal resolution of nuclear structures

• Co-localization Studies:

  • ZNF366 can be co-stained with ERα to study their nuclear co-localization

  • Careful selection of primary antibodies from different host species is essential to avoid cross-reactivity

How can ZNF366 antibodies support functional gene expression studies?

ZNF366 antibodies are valuable tools for investigating the functional impact of this corepressor on gene expression:

• Monitoring ZNF366 Protein Levels:

  • In overexpression or knockdown experiments, antibodies allow verification of successful manipulation

  • Western blotting with anti-FLAG antibody can confirm expression of transfected ZNF366-FLAG constructs

• Correlating ZNF366 Levels with Target Gene Expression:

  • Published studies have used this approach to demonstrate ZNF366's repressive effect on estrogen-responsive genes

  • Estrogen-responsive genes such as cathepsin D and progesterone receptor show decreased expression when ZNF366 is overexpressed

  • RT-PCR for target genes (e.g., GREB1) can be combined with immunoblotting for ZNF366

• Growth Assay Correlation:

  • ZNF366 overexpression has been studied in cell proliferation assays in MCF7 and MDA-MB-231 cells

  • Antibodies confirm expression while cell counts determine functional impact

• ChIP-Seq Integration:

  • ZNF366 antibodies can be used in ChIP experiments followed by sequencing

  • This approach identifies genome-wide binding sites and, when integrated with RNA-Seq data, reveals the direct transcriptional impact

What controls should be included when interpreting ZNF366 antibody data?

Proper experimental controls are essential for accurate data interpretation:

• Loading Controls for Western Blots:

  • Include housekeeping proteins such as β-actin

  • For subcellular fractionation studies, include compartment-specific markers (e.g., Lamin A/C for nuclear fraction)

• Positive Tissue Controls:

  • Mouse heart and skeletal muscle tissues have been validated as positive controls for ZNF366 detection

  • Include these when testing new antibody lots or experimental conditions

• Negative Controls:

  • For immunoprecipitation, include isotype-matched IgG (e.g., mouse IgG for mouse monoclonal antibodies)

  • For immunofluorescence, include secondary-only controls to assess background

• Expression Validation:

  • Confirm protein detection with mRNA expression data

  • Validated RT-PCR primers: 5′-CCCCATCCAGTACAACTGCT-3′ and 5′-CTTCACGTCAGAGTGGACGA-3′ (ZNF366)

  • Reference gene primers: 5′-GCGTACGGCTCTCATCAACT-3′ and 5′-GACACTGGAGGCAGAAGAGC-3′ (Lamin A/C) and 5′-TCCCATCACCATCTTCCA-3′ and 5′-CATCACGCCACAGTTTCC-3′ (GAPDH)