ZNF365 Antibody, Biotin conjugated

What is ZNF365 and what are its known functions in cellular processes?

ZNF365 (zinc finger protein 365) is a multifunctional protein that plays critical roles in several cellular processes, most notably in DNA damage response pathways and genomic stability. Research has demonstrated that ZNF365 is involved in the recovery of stalled replication forks, which is essential for maintaining genome integrity . Additionally, ZNF365 has been implicated in BRCA-dependent DNA repair mechanisms, with the ZNF365 locus being associated with breast cancer risk in carriers of mutated BRCA1 and BRCA2 . The protein has several isoforms with distinct expression patterns and functions. ZNF365 has also been linked to colorectal cancer progression, where its expression correlates with phosphorylated p53 (Ser15) protein expression .

What are the key specifications of commercially available ZNF365 antibodies?

ZNF365 antibodies are available in several configurations with the following specifications:

All antibodies are generated against human recombinant protein fragment corresponding to amino acids 108-407 of human ZNF365 (NP_055766) produced in E.coli .

What is the significance of biotin conjugation for ZNF365 antibodies?

Biotin conjugation provides significant advantages in research applications due to the strong affinity between biotin and streptavidin/avidin. This conjugation enables signal amplification in detection systems, making it particularly valuable for low-abundance proteins. For ZNF365 research, biotin-conjugated antibodies offer several benefits:

• Enhanced sensitivity for detecting endogenous ZNF365 expression, which may be low in certain cell types

• Compatibility with a wide range of streptavidin-conjugated detection systems (fluorescent, enzymatic, nanoparticle-based)

• Flexibility in multi-color flow cytometry experiments through streptavidin-fluorophore combinations

• Capability for antibody layering in complex immunohistochemical studies of ZNF365 expression in tissue samples

When designing experiments, researchers should consider that biotinylated antibodies may require additional blocking steps to prevent non-specific binding, particularly in tissues with high endogenous biotin .

How should ZNF365 antibodies be optimized for Western blot applications?

Optimization of ZNF365 antibody for Western blot applications requires attention to several technical parameters:

What protocols are recommended for immunohistochemistry with ZNF365 antibodies?

Based on published methodologies, the following protocol is recommended for immunohistochemistry with ZNF365 antibodies:

• Tissue preparation: Fix tissues in 10% formalin at room temperature for 24 hours, embed in paraffin, and section into 4-μm thick slices .

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes.

• Blocking: Block endogenous peroxidase activity with 3% H₂O₂ for 10 minutes, followed by protein blocking with 5% normal goat serum.

• Primary antibody incubation: Dilute ZNF365 antibody to 1:150 and incubate overnight at 4°C .

• Detection system:

  • For unconjugated antibodies: Use HRP-conjugated secondary antibodies and DAB chromogen

  • For biotin-conjugated antibodies: Use streptavidin-HRP and DAB chromogen

• Counterstaining: Counterstain with hematoxylin for 1 minute at room temperature .

• Special considerations: When using mouse antibodies on mouse tissues, a Mouse-On-Mouse blocking reagent may be necessary to reduce background signal .

• Analysis guidelines: ZNF365 may be observed in both cytoplasmic and nuclear compartments, and scoring systems should account for both localization patterns .

How can flow cytometry protocols be optimized for ZNF365 detection?

For optimal detection of ZNF365 using flow cytometry:

• Cell preparation: Harvest cells during logarithmic growth phase. Fix with 2-4% paraformaldehyde for 10-15 minutes if detecting total ZNF365, or use 70-90% methanol for detection of intracellular epitopes.

• Permeabilization: Use 0.1% Triton X-100 or 0.1% saponin in PBS for 10-15 minutes to allow antibody access to intracellular ZNF365.

• Blocking: Block with 2-5% serum from the same species as the secondary antibody for 30 minutes to reduce non-specific binding.

• Antibody dilution: For biotin-conjugated ZNF365 antibodies (OTI5B9 and OTI8G7), use at 1:100 dilution as recommended by manufacturers .

• Detection reagent: Use fluorophore-conjugated streptavidin (e.g., streptavidin-PE, streptavidin-APC) as the detection reagent.

• Controls: Include:

  • Unstained cells

  • Isotype control (mouse IgG-biotin)

  • Single-color controls if performing multicolor analysis

  • Positive control cells known to express ZNF365

• Analysis considerations: As ZNF365 can be expressed in both cytoplasm and nucleus, ensure your permeabilization protocol allows access to both compartments.

What strategies are recommended when ZNF365 antibody produces high background in immunostaining?

High background with ZNF365 antibodies can be addressed through several strategic approaches:

• Mouse-on-Mouse interference: When using mouse-derived ZNF365 antibodies on mouse tissues, significant background can occur. Implement Mouse-on-Mouse blocking reagents (e.g., products available under catalog numbers PK-2200-NB and MP-2400-NB) .

• Endogenous biotin interference: For biotin-conjugated antibodies, endogenous biotin in tissues can cause high background. Implement an avidin/biotin blocking step before antibody incubation.

• Dilution optimization: Titrate antibody concentration beyond manufacturer recommendations. For OTI1B7, if 1:150 produces high background, test 1:200 or 1:300 dilutions .

• Alternative blocking agents: Instead of standard BSA or serum blockers, try 5% milk protein or commercial blockers specifically designed for immunohistochemistry.

• Extended washing: Implement additional wash steps (at least 5 washes with PBS) after primary antibody incubation .

• Secondary detection optimization: If using streptavidin-based detection systems, dilute streptavidin conjugates further or reduce incubation time.

• Alternative fixation: Overfixation can increase background staining. Consider optimizing fixation time or using alternative fixatives.

How can researchers validate the specificity of ZNF365 antibody signals?

Validating ZNF365 antibody specificity requires implementing multiple controls and verification approaches:

• Positive control samples: Use HEK293T cells transfected with pCMV6-ENTRY ZNF365 as demonstrated in validation studies. This provides a clear positive control with overexpressed protein .

• Negative control samples:

  • Untransfected cells or cells transfected with empty vector

  • Secondary antibody-only controls

  • Isotype controls matching the primary antibody

• Knockdown validation: Implement siRNA or shRNA knockdown of ZNF365 to confirm signal reduction correlates with reduced expression. Evidence from research indicates that lentivirus encoding ZNF365 hairpin effectively reduces expression .

• Peptide competition: Pre-incubate the antibody with immunizing peptide (amino acids 108-407 of human ZNF365) to block specific binding sites .

• Cross-validation with multiple antibodies: Compare staining patterns using different clones targeting distinct epitopes (OTI1B7, OTI5B9, OTI8G7).

• Expected localization and molecular weight verification: Confirm that the detected protein shows the expected subcellular localization (cytoplasmic and nuclear) and molecular weight (approximately 46.4 kDa) .

How can ZNF365 antibodies be utilized to investigate DNA damage response pathways?

ZNF365 antibodies can be instrumental in elucidating DNA damage response pathways through several advanced experimental approaches:

• Colocalization with DNA damage markers: Perform dual immunofluorescence staining with ZNF365 antibodies and established DNA damage markers (γH2AX, 53BP1, RAD51) to investigate recruitment to DNA damage sites. Research has shown that ZNF365 depletion leads to increased 53BP1-positive foci, particularly after treatment with PARP inhibitors like ABT-888 .

• Chromatin immunoprecipitation (ChIP): Use ZNF365 antibodies for ChIP to identify genomic regions where ZNF365 binds, particularly after DNA damage induction with agents like ionizing radiation or chemical genotoxins.

• Proximity ligation assay (PLA): Combine ZNF365 antibodies with antibodies against potential interacting partners (BRCA1, BRCA2, etc.) to visualize and quantify protein-protein interactions in situ.

• Recovery dynamics after replication stress: Implement stalled replication fork recovery assays using DNA fiber analysis combined with ZNF365 immunostaining to investigate its role in replication stress response .

• Immunoprecipitation-mass spectrometry: Use ZNF365 antibodies to pull down protein complexes, followed by mass spectrometry to identify novel interacting partners in the DNA damage response pathway.

• FRAP (Fluorescence Recovery After Photobleaching): Combine ZNF365 antibodies with live-cell imaging to study dynamic recruitment to DNA damage sites.

These approaches should be designed with appropriate controls, as ZNF365 has been implicated in the BRCA-dependent DNA repair pathway, which is crucial for genome stability maintenance .

What experimental designs are recommended to investigate the role of ZNF365 in cancer progression?

To investigate ZNF365's role in cancer progression, researchers should consider these experimental approaches:

• Expression analysis in clinical samples:

  • Perform immunohistochemistry with ZNF365 antibodies on tissue microarrays of cancer specimens

  • Score expression levels using standardized immunoreactive scoring systems (IRS)

  • Correlate expression with clinicopathological parameters and survival outcomes

  Research has shown that high ZNF365 expression correlates with improved survival in colorectal cancer patients (P=0.009) .

• Epigenetic regulation studies:

  • Analyze ZNF365 promoter methylation status using bisulfite sequencing

  • Treat cells with demethylating agents (5-aza-2-deoxycytidine) and histone deacetylase inhibitors (trichostatin A) to assess re-expression

  • Correlate methylation with expression levels

  Evidence indicates that ZNF365 is frequently inactivated by promoter methylation in colorectal cancer .

• Functional assays:

  • Overexpression studies: Transfect cancer cell lines with ZNF365 expression constructs

  • Knockdown studies: Use siRNA or shRNA to deplete ZNF365

  • Assess effects on:

    • Proliferation (MTT/CCK-8 assays)

    • Apoptosis (Annexin V/PI staining)

    • Migration and invasion (Transwell assays)

    • Colony formation

• Mechanistic studies:

  • Investigate correlation between ZNF365 and phosphorylated p53 (Ser15), as research has shown a positive correlation (r=0.18; P=0.038)

  • Perform western blot analysis to assess proteins involved in DNA damage response pathways

  • Utilize protein-protein interaction networks (e.g., GeneMANIA database) to identify functional relationships

• Animal models:

  • Generate xenograft models using ZNF365-overexpressing or ZNF365-knockdown cancer cells

  • Assess tumor growth, metastasis, and response to therapy

How can ZNF365 antibodies contribute to understanding the protein's role in specific disease contexts?

ZNF365 antibodies can provide valuable insights into disease-specific roles through targeted investigative approaches:

• Breast cancer susceptibility:

  • Perform immunohistochemistry on breast tissues from individuals with BRCA1/2 mutations versus non-carriers

  • Analyze ZNF365 expression patterns in different breast cancer subtypes

  • Correlate expression with genetic variants at the ZNF365 locus that confer risk

  • Investigate colocalization with BRCA1/2 proteins in tissue sections

  This is particularly relevant as the ZNF365 locus is associated with breast cancer risk in carriers of mutated BRCA1 and BRCA2 .

• Colorectal cancer progression:

  • Implement multiplexed immunohistochemistry to simultaneously detect ZNF365 and P-p53 (Ser15) in colorectal tissues

  • Analyze expression patterns across different stages of colorectal carcinogenesis

  • Correlate expression with patient survival data

  • Investigate the relationship between ZNF365 methylation status and protein expression

  Research has shown that ZNF365 expression is an independent prognostic factor for survival in colorectal cancer (P=0.046) .

• Uric acid nephrolithiasis (UAN):

  • Examine ZNF365 expression in kidney tissues from UAN patients

  • Correlate expression with specific mutations associated with UAN

  • Analyze ZNF365 isoform expression patterns in renal tissues

  This approach is supported by evidence that mutation in the ZNF365 gene is associated with uric acid nephrolithiasis .

• Genomic instability syndromes:

  • Quantify 53BP1 foci formation in patient-derived cells with and without ZNF365 knockdown

  • Assess DNA damage accumulation in response to replication stress

  • Evaluate chromosomal aberrations in cells with altered ZNF365 expression

  Research has demonstrated that ZNF365 depletion leads to increased 53BP1-positive foci, indicating its role in genome stability .

What experimental approaches can elucidate the interaction between ZNF365 and the p53 pathway?

To investigate the functional relationship between ZNF365 and the p53 pathway, researchers should consider these experimental approaches:

• Co-immunoprecipitation (Co-IP):

  • Use ZNF365 antibodies to pull down protein complexes, followed by western blot analysis for p53 and phosphorylated p53 (Ser15)

  • Perform reciprocal Co-IP using p53 antibodies to confirm interaction

  • Conduct Co-IP under different conditions (normal vs. DNA damage)

• Proximity Ligation Assay (PLA):

  • Utilize ZNF365 and p53 antibodies in PLA to visualize and quantify their interaction in situ

  • Compare PLA signals in normal cells versus cells exposed to DNA damaging agents

• Expression correlation studies:

  • Expand on existing findings showing positive correlation between ZNF365 expression and phosphorylated p53 (Ser15) protein expression (r=0.18; P=0.038)

  • Implement GeneMANIA analysis to assess the association between ZNF365 and p53 expression networks

• Functional rescue experiments:

  • Deplete ZNF365 using siRNA/shRNA and assess the impact on p53 phosphorylation

  • Reintroduce wild-type or mutant ZNF365 to determine which domains are essential for p53 pathway interaction

  • Employ p53 inhibitors (e.g., pifithrin-α) to determine if the effects of ZNF365 depletion are p53-dependent

• ChIP-seq analysis:

  • Perform ChIP-seq with ZNF365 and p53 antibodies to identify shared genomic binding sites

  • Analyze whether ZNF365 affects p53 binding to target genes

• Transcriptome analysis:

  • Compare gene expression profiles in cells with normal versus altered ZNF365 expression

  • Focus on p53 target genes to determine if ZNF365 modulates p53 transcriptional activity

How should researchers design experiments to investigate ZNF365's role in maintaining genome integrity?

To investigate ZNF365's role in genome integrity, researchers should implement these methodological approaches:

• DNA damage response assays:

  • Quantify 53BP1 foci formation in ZNF365-depleted cells before and after treatment with DNA damaging agents

  • Research has demonstrated that lentivirus encoding ZNF365 hairpin-infected U2OS cells exhibited increased 53BP1-positive foci, particularly after treatment with the PARP inhibitor ABT-888

  • Assess γH2AX foci dynamics to measure DNA damage accumulation and resolution

• Replication fork studies:

  • Implement DNA fiber analysis to measure replication fork progression and restart after stalling

  • Use DNA combing to visualize individual DNA molecules and replication dynamics

  • Assess the impact of ZNF365 depletion on fork stability and recovery after hydroxyurea-induced stalling

• Homologous recombination assays:

  • Utilize DR-GFP reporter systems to quantify homologous recombination efficiency

  • Measure RAD51 foci formation as a marker of HR initiation

  • Analyze sister chromatid exchange frequencies in cells with altered ZNF365 expression

• Cell cycle checkpoints:

  • Analyze cell cycle distribution using flow cytometry in ZNF365-depleted cells

  • Assess checkpoint activation by measuring CHK1 and CHK2 phosphorylation

  • Determine if ZNF365 influences cell cycle arrest in response to DNA damage

• Chromosomal stability:

  • Perform metaphase spread analysis to quantify chromosomal aberrations

  • Use fluorescence in situ hybridization (FISH) to detect specific structural abnormalities

  • Implement micronucleus assays to measure genomic instability

• Genetic interaction studies:

  • Conduct synthetic lethality screens with ZNF365 depletion and known DNA repair factors

  • Investigate epistatic relationships with BRCA1/2 and other HR factors

  • Design double-knockdown experiments to uncover functional redundancies

• High-throughput microscopy:

  • Develop high-content screening approaches to simultaneously measure multiple DNA damage response parameters

  • Implement live-cell imaging to track DNA damage foci dynamics in real-time

These methodologies should be implemented with appropriate controls and in multiple cell lines to ensure reproducibility and biological relevance.

How should researchers interpret contradictory results in ZNF365 expression studies across different cancers?

When encountering contradictory results regarding ZNF365 expression across different cancer types or studies, researchers should implement the following analytical framework:

• Context-dependent functions:

  • Consider that ZNF365 has multiple isoforms with potentially distinct functions. Different antibodies may recognize different isoforms, leading to apparently contradictory results .

  • Analyze which specific isoform was detected in each study and whether the antibodies used target different epitopes.

• Methodological differences:

  • Compare detection methods (IHC vs. Western blot vs. qPCR) and their sensitivities.

  • Evaluate scoring systems used for immunohistochemistry (e.g., immunoreactive scores from 0-9) .

  • Assess differences in sample preparation, fixation methods, and antigen retrieval protocols.

• Subcellular localization:

  • ZNF365 is expressed in both cytoplasm and nucleus . Different studies may focus on different compartments.

  • Implement detailed subcellular fractionation studies to clarify localization-specific functions.

• Epigenetic regulation:

  • Determine if differences in ZNF365 promoter methylation status explain expression variations.

  • Treatment with demethylating agents (5-aza) and histone deacetylase inhibitors (TSA) can reveal epigenetic silencing mechanisms .

• Genetic background:

  • Analyze whether genetic variants in the ZNF365 locus contribute to expression differences.

  • Consider population-specific genetic factors, particularly in breast cancer studies where ZNF365 variants confer risk .

• Meta-analysis approach:

  • Implement formal meta-analysis incorporating all available studies.

  • Weight studies by sample size, methodological rigor, and consistency of antibody validation.

• Functional validation:

  • Design experiments to test whether ZNF365 has context-dependent functions in different cancer types.

  • Implement consistent methodologies across multiple cancer models to allow direct comparison.

What standards should be applied when evaluating ZNF365 expression in patient tissue samples?

When evaluating ZNF365 expression in patient tissues, researchers should implement these standardized approaches:

• Antibody validation standards:

  • Verify antibody specificity through positive controls (e.g., HEK293T cells transfected with ZNF365) .

  • Implement negative controls, including isotype controls and secondary-only controls.

  • Use multiple antibody clones (e.g., OTI1B7, OTI5B9, OTI8G7) to confirm expression patterns .

• Scoring methodology:

  • Implement standardized immunoreactive scoring (IRS) systems combining staining intensity and percentage of positive cells.

  • Use a scale of 0-9 as demonstrated in published research, with high expression defined as IRS 6-9 and low expression as IRS 0-5 .

  • Have multiple experienced pathologists score samples independently and blind to clinical outcomes .

• Tissue processing standardization:

  • Standardize fixation protocols (e.g., 10% formalin for 24 hours at room temperature) .

  • Implement consistent antigen retrieval methods.

  • Process normal control tissues alongside tumor samples.

• Subcellular localization assessment:

  • Evaluate both cytoplasmic and nuclear expression of ZNF365 .

  • Consider separate scoring for different cellular compartments.

  • Implement digital image analysis for objective quantification.

• Clinical correlation standards:

  • Collect comprehensive clinicopathological data.

  • Use multivariate analysis to determine if ZNF365 is an independent prognostic factor.

  • Apply Cox proportional hazards models for survival analysis .

• Reporting standards:

  • Report antibody clone, dilution, incubation conditions, and detection methods.

  • Provide representative images showing different expression levels.

  • Include details of statistical methods used for correlation analyses.

• Reproducibility measures:

  • Calculate inter-observer agreement statistics (e.g., kappa coefficients).

  • Implement tissue microarrays where possible to reduce batch effects.

  • Consider multicenter validation of findings.