znf326 Antibody

What is ZNF326 and why is it a target of interest for antibody-based research?

ZNF326 (Zinc finger protein 326) is a 582 amino acid protein belonging to the AKAP95 family that is primarily localized to the nuclear matrix. It functions as a transcriptional regulator with significant roles in neuronal differentiation during development. ZNF326 contains a Krüppel-type DNA-binding domain and a KRAB domain that interacts with KAP1, facilitating the recruitment of histone-modifying proteins essential for chromatin remodeling and gene expression regulation . Recent studies have identified ZNF326 as a core component of the DBIRD complex, which integrates transcript elongation with the regulation of alternative splicing, particularly affecting exons embedded in (A+T)-rich DNA regions . Its upregulation has been implicated in malignant phenotypes of glioma and breast cancer, making it a valuable target for oncology research .

How do I select the appropriate ZNF326 antibody for my experiment?

Selection depends on your experimental goals and detection methods:

For multi-species studies, verify cross-reactivity, as some ZNF326 antibodies detect protein from mouse, rat, and human origins , while others are species-specific. When studying specific isoforms, select antibodies targeting unique regions of the isoform of interest, as ZNF326 exists in at least two isoforms due to alternative splicing .

What are the optimal conditions for Western blot detection of ZNF326?

For optimal Western blot detection of ZNF326:

• Sample preparation: When preparing cell or tissue lysates, use RIPA buffer with protease inhibitors and phosphatase inhibitors if phosphorylation status is relevant.

• Gel electrophoresis: ZNF326 has a calculated molecular weight of 66 kDa but is typically observed at 66-70 kDa, likely due to post-translational modifications . Use 8-10% SDS-PAGE gels for optimal resolution.

• Transfer conditions: Transfer proteins to PVDF or nitrocellulose membranes at 100V for 60-90 minutes in standard transfer buffer (with 20% methanol).

• Blocking and antibody dilution:

  • Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

  • Dilute primary antibodies according to manufacturer specifications:

    • Polyclonal antibodies: typically 1:500-1:1000

    • Monoclonal antibodies: typically 1:500-1:2000

• Incubation and visualization: Incubate with primary antibody overnight at 4°C, followed by appropriate HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature. Develop using enhanced chemiluminescence.

Some ZNF326 antibodies are available as direct HRP conjugates, which can eliminate the need for secondary antibodies and reduce background .

How should I optimize immunohistochemistry protocols for ZNF326 detection in tissue samples?

Optimizing IHC for ZNF326 requires attention to several parameters:

• Fixation: Formalin-fixed paraffin-embedded (FFPE) tissues typically work well, but over-fixation can mask epitopes.

• Antigen retrieval: This is critical for ZNF326 detection:

  • For many ZNF326 antibodies, citrate buffer (pH 6.0) is recommended

  • Some antibodies perform better with TE buffer (pH 9.0)

  • Always verify the recommended retrieval method for your specific antibody

• Blocking and antibody dilution:

  • Block with serum matching the species of secondary antibody

  • Initial antibody dilutions typically range from 1:50-1:500

  • For monoclonal antibodies, start at 1:50 dilution and optimize

• Detection systems:

  • For brightfield microscopy: HRP/DAB-based detection

  • For fluorescent detection: fluorophore-conjugated secondary antibodies

• Scoring and evaluation: ZNF326 staining intensity can be scored as negative (-), weakly positive (+), moderately positive (++), or strongly positive (+++), based on both intensity and percentage of positive cells . This quantitative approach is particularly useful for correlating expression with clinical parameters.

What controls should be included when using ZNF326 antibodies?

Include these essential controls for reliable ZNF326 antibody experiments:

• Positive controls:

  • Cell lines with confirmed ZNF326 expression: Jurkat, HeLa, MCF-7, and PC-3 cells have been validated for Western blot

  • Tissue samples: Mouse liver tissue has been validated for IHC

• Negative controls:

  • No primary antibody control (secondary antibody only)

  • Isotype control (irrelevant antibody of same isotype)

  • Blocking peptide competition: Some manufacturers offer ZNF326 blocking peptides that can competitively inhibit specific antibody binding

  • ZNF326 knockdown/knockout samples: Cells treated with ZNF326-specific shRNA or CRISPR/Cas9 knockout

• Method-specific controls:

  • For immunoprecipitation: Input sample, IgG control IP

  • For ChIP assays: Input chromatin, IgG control, positive control genomic regions

How can ZNF326 antibodies be used to study protein-protein interactions in the DBIRD complex?

To investigate ZNF326 interactions within the DBIRD complex:

• Co-immunoprecipitation (Co-IP): Use ZNF326 antibodies to pull down associated proteins, followed by Western blot analysis for suspected interacting partners such as DBC1 (Deleted in Breast Cancer 1) or components of RNA polymerase II . Several ZNF326 antibodies have been specifically validated for IP applications .

• Proximity ligation assay (PLA): This technique can visualize protein-protein interactions in situ with single-molecule resolution. Use primary antibodies against ZNF326 and potential interacting partners from different species, followed by species-specific PLA probes.

• Chromatin immunoprecipitation (ChIP) assays: ZNF326 has been shown to bind to specific promoter regions via its transcriptional activation domain and zinc-finger structures . ChIP-seq analysis using ZNF326 antibodies can identify genome-wide binding sites and potential transcriptional targets. One study used ChIP-seq with H1299 cells overexpressing ZNF326 to identify binding to the HDAC7 promoter region .

• Mass spectrometry analysis of immunoprecipitated complexes: ZNF326 antibodies can be used to purify native protein complexes for mass spectrometry identification of novel interacting partners. This approach has revealed that ZNF326 undergoes symmetric dimethylation by PRMT5, which affects its function in regulating innate immune activation .

These approaches have revealed that ZNF326 interacts with DBC1 in breast cancer cells and that this interaction is critical for ZNF326's ability to promote proliferation and invasiveness .

How can I use ZNF326 antibodies to investigate its role in cancer progression?

To investigate ZNF326's role in cancer:

• Expression analysis in clinical samples:

  • IHC with tissue microarrays can establish ZNF326 expression patterns across tumor grades and stages

  • Correlate expression with patient outcomes and clinicopathological factors

  • Studies have shown ZNF326 is highly expressed in 60.9% of glioma samples and 54.1% of breast cancer specimens

• Mechanistic studies in cell models:

  • Combine ZNF326 antibodies with antibodies against EMT markers (E-cadherin, Snail, Slug) and cell cycle proteins (cyclinA2, cyclinB1) to analyze how ZNF326 affects these pathways

  • ChIP assays can identify direct transcriptional targets of ZNF326 that promote cancer progression

  • Use immunofluorescence to track nuclear localization and redistribution of β-catenin in the presence of overexpressed or knocked-down ZNF326

• Functional pathway analysis:

  • Combined immunoprecipitation and phosphorylation-specific antibodies can reveal how ZNF326 affects post-translational modifications of key signaling proteins

  • In glioma, ZNF326 activates the Wnt/β-catenin pathway by regulating β-catenin acetylation and phosphorylation

  • In breast cancer, ZNF326 interacts with DBC1 to promote malignant phenotypes

• In vivo tumor models:

  • Use antibodies to confirm ZNF326 expression in xenograft tumors derived from cells with manipulated ZNF326 levels

  • Correlate tumor growth rates with ZNF326 expression and localization

These approaches have revealed that ZNF326 expression correlates with advanced tumor grade, positive lymph node metastasis, and poor prognosis in patients with glioma and breast cancer .

Why might I observe multiple bands when probing for ZNF326 in Western blots?

Multiple bands in ZNF326 Western blots may occur due to:

• Isoforms: ZNF326 exists in at least two isoforms due to alternative splicing . Confirm which isoforms your antibody should detect based on the epitope location.

• Post-translational modifications: ZNF326 undergoes various modifications including:

  • Symmetric dimethylation by PRMT5

  • Potential phosphorylation that may alter migration patterns

  • Other modifications that may affect its apparent molecular weight (observed at 66-70 kDa despite calculated 66 kDa)

• Proteolytic degradation: ZNF326 may be sensitive to proteolysis during sample preparation. Ensure protease inhibitors are fresh and used at appropriate concentrations.

• Cross-reactivity: Some antibodies may cross-react with structurally similar zinc finger proteins. Verify specificity with:

  • ZNF326 knockdown/knockout controls

  • Blocking peptide competition assays

  • Multiple antibodies targeting different epitopes

If working with a new antibody, validate the detected bands by comparing with published literature where ZNF326 has been characterized with validated antibodies.

What are common pitfalls when performing immunoprecipitation with ZNF326 antibodies?

Common IP challenges with ZNF326 include:

• Inefficient extraction: As a nuclear matrix protein, ZNF326 may require specialized lysis conditions. Use nuclear extraction buffers containing 0.3-0.4M NaCl to efficiently release ZNF326 from chromatin.

• Antibody binding interference: The epitope recognized by the antibody may be masked by:

  • Protein-protein interactions (especially in the DBIRD complex)

  • Post-translational modifications

  • Protein conformation changes

• Non-specific binding: Use appropriate pre-clearing steps and stringent wash conditions. For critical experiments, consider:

  • Two-step IP validation (sequential IP with different antibodies)

  • IP-mass spectrometry to confirm pulled-down proteins

• Cross-linking considerations: If using formaldehyde cross-linking for chromatin IP:

  • Optimize cross-linking time (typically 10-15 minutes)

  • Ensure complete reversal of cross-links before SDS-PAGE

• Protein complex preservation: If studying ZNF326 interactions with DBC1 or other partners, avoid harsh detergents that might disrupt these interactions. Consider using digitonin or low concentrations of NP-40.

Successful ZNF326 IP has been performed in multiple studies to demonstrate its interactions with DBC1 and its role in the β-catenin pathway in glioma cells .

How should I interpret differences in ZNF326 subcellular localization in normal versus diseased tissues?

When analyzing ZNF326 localization patterns:

• Normal localization: ZNF326 is primarily localized to the nuclear matrix . In normal tissues, expect predominantly nuclear staining with some nucleolar enrichment.

• Pathological alterations:

  • Increased nuclear intensity may indicate upregulation of transcriptional activity

  • Cytoplasmic localization could suggest dysregulation of nuclear import/export

  • Changes in nuclear distribution patterns (diffuse vs. punctate) may reflect alterations in chromatin association

• Quantitative assessment:

  • Use digital image analysis to quantify nuclear vs. cytoplasmic ratios

  • Compare staining intensity with prognostic markers in serial sections

  • Correlate subcellular distribution with clinical outcomes

• Mechanistic implications:

  • Nuclear accumulation of ZNF326 in glioma tissues correlates with advanced tumor grade and activation of the Wnt/β-catenin pathway

  • Changes in ZNF326 localization may affect its interaction with binding partners like DBC1 in breast cancer

An immunohistochemistry scoring system that considers both intensity (0-2) and percentage of positive cells (1-4) has been successfully employed to correlate ZNF326 expression with clinicopathological factors in cancer studies .

How can I integrate ZNF326 antibody data with transcriptomic and genomic analyses?

To integrate multi-omics approaches with ZNF326 antibody data:

• Correlating protein with transcript levels:

  • Compare ZNF326 protein levels (Western blot/IHC) with mRNA expression (RT-qPCR/RNA-seq)

  • Discrepancies may indicate post-transcriptional regulation

  • In glioma studies, both protein and mRNA levels of ZNF326 target genes like HDAC7 were examined to confirm transcriptional activation

• ChIP-seq integration:

  • Use ZNF326 antibodies for ChIP-seq to identify genome-wide binding sites

  • Correlate binding sites with gene expression changes in ZNF326 knockdown/overexpression models

  • Integrate with histone modification ChIP-seq to understand chromatin context

  • One study used ChIP-seq to demonstrate that ZNF326 binds to the HDAC7 promoter region via its zinc-finger domains

• Protein-protein interaction networks:

  • Combine co-IP/mass spectrometry data with protein expression data

  • Map ZNF326 interactome changes in response to treatments or disease conditions

  • The ZNF326-DBC1 interaction in breast cancer represents a novel oncogenic pathway identified through such approaches

• CRISPR screening validation:

  • Recent genome-wide CRISPR screens have identified various zinc finger proteins, including ZNF326, as potential regulators of innate immune responses and viral infections

  • Antibody-based validation of these hits is essential for confirming their roles

This integrated approach has revealed that ZNF326 functions as a transcriptional activator of HDAC7, which subsequently affects β-catenin acetylation and activity in the Wnt signaling pathway in glioma .

What are the considerations when using ZNF326 antibodies for studying its role in RNA processing and alternative splicing?

When investigating ZNF326's role in RNA processing:

• Nuclear co-localization studies:

  • Use double immunofluorescence with antibodies against ZNF326 and spliceosome components

  • Analyze co-localization at the single-cell level in different cell states

  • ZNF326 is a component of the DBIRD complex that affects alternative splicing of exons embedded in (A+T)-rich DNA regions

• RNA-protein interaction analysis:

  • Combine ZNF326 immunoprecipitation with RNA sequencing (RIP-seq)

  • Use cross-linking immunoprecipitation (CLIP) methods to identify direct RNA binding sites

  • Compare binding patterns before and after cellular stress or differentiation

• Splicing outcome assessment:

  • Correlate ZNF326 levels (detected by antibodies) with splicing patterns of target genes

  • Use minigene splicing reporters to quantify the effect of ZNF326 on specific splicing events

  • The DBIRD complex containing ZNF326 affects local transcript elongation rates, which influences alternative splicing decisions

• Transcription-coupled splicing:

  • Use antibodies against phosphorylated RNA Polymerase II along with ZNF326 to study co-transcriptional splicing regulation

  • Analyze chromatin association patterns at alternatively spliced exons

These approaches can help elucidate how ZNF326 integrates transcript elongation with alternative splicing regulation, a function that may be dysregulated in cancer and other diseases.

How can ZNF326 antibodies be used to investigate its role in innate immune responses?

Recent research has identified a PRMT5-ZNF326 axis in innate immune activation :

• Post-translational modification analysis:

  • Use antibodies specific for symmetric dimethylarginine (SDMA) to immunoprecipitate modified ZNF326

  • Western blot analysis with ZNF326 antibodies can confirm enrichment of dimethylated ZNF326 upon treatments like hydroxyurea (HU)

  • Combine with site-specific mutation studies to identify critical methylation sites

• Regulation of interferon-stimulated genes (ISGs):

  • Use ChIP assays with ZNF326 antibodies to identify binding to ISG promoters

  • Correlate ZNF326 recruitment with ISG expression changes during immune activation

  • Compare wild-type vs. methylation-deficient ZNF326 in regulating ISG expression

• PRMT5-ZNF326 interaction studies:

  • Co-immunoprecipitation with ZNF326 antibodies can pull down associated PRMT5

  • Proximity ligation assays can visualize this interaction in intact cells

  • Study how various stimuli affect this interaction and subsequent innate immune responses

• Functional consequences in infection models:

  • Examine how ZNF326 levels, detected by antibodies, correlate with resistance to viral infection

  • Compare ISG induction in cells with wild-type vs. mutant ZNF326 unable to be methylated by PRMT5

These approaches can help understand how ZNF326 contributes to the regulation of innate immunity, a relatively unexplored function of this protein.

What methodological considerations are important when studying ZNF326 mutants or variants in disease contexts?

When investigating ZNF326 variants:

• Antibody epitope considerations:

  • Ensure your antibody recognizes regions that are not affected by the mutations/variants

  • For domain-specific mutations (e.g., ΔZn1, ΔZn2, ΔTAD), use antibodies targeting conserved regions

  • Consider using multiple antibodies targeting different epitopes to confirm results

• Expression system validation:

  • When studying ectopically expressed ZNF326 variants, use antibodies against both ZNF326 and any epitope tags

  • Compare expression levels of mutant and wild-type ZNF326 to ensure comparable expression

  • Studies with ZNF326 mutants lacking specific domains have revealed their importance in transcriptional activation

• Functional assays:

  • Use luciferase reporter assays to measure transcriptional activity of wild-type vs. mutant ZNF326

  • Perform ChIP assays to compare DNA binding properties

  • Assess protein-protein interactions via co-IP to determine if mutations affect complex formation

  • ZNF326 mutants lacking zinc-finger domains showed impaired binding to the HDAC7 promoter region

• Patient-derived samples:

  • When studying natural variants (such as His262Tyr in PLSCR1), compare antibody staining patterns between wild-type and variant-expressing cells

  • Consider heterozygous vs. homozygous contexts when interpreting results