ZNF420 Antibody, HRP conjugated

What is ZNF420 and what are its key biological functions?

ZNF420 (Zinc Finger Protein 420) is a nuclear protein containing 19 C2H2-type zinc fingers and 1 KRAB domain that functions primarily as a transcriptional regulator . Also known as APAK (ATM and p53 associated KZNF protein), ZNF420 acts as a negative regulator of p53-mediated apoptosis . The protein's structure features multiple zinc finger domains that enable specific DNA binding, while the KRAB domain typically functions as a potent transcriptional repression module. Its subcellular localization in the nucleus is consistent with its role in transcriptional regulation . The combination of multiple zinc finger domains suggests ZNF420 likely recognizes specific DNA sequences with high specificity to regulate target gene expression.

What experimental validations should be performed when using ZNF420 antibody for the first time?

When using ZNF420 antibody, HRP conjugated for the first time, researchers should implement a systematic validation approach:

• Molecular weight verification: Confirm that detected bands in Western blot match the expected molecular weight of ZNF420.

• Positive and negative controls: Use cell lines or tissues with known expression levels of ZNF420, ideally including:

  • Cell lines with documented high ZNF420 expression

  • Negative controls through ZNF420 knockdown (siRNA/shRNA)

  • Recombinant ZNF420 as positive control for Western blots

• Antibody specificity testing: Pre-incubate the antibody with synthetic peptide immunogen (if available) to confirm binding specificity.

• Cross-species reactivity assessment: Verify antibody performance across species. According to the manufacturer, the antibody has confirmed reactivity in mouse samples with predicted reactivity to human, rat, cow, sheep, pig, and horse samples .

• Dilution optimization: Test multiple dilutions across the recommended ranges for each application:

  • ELISA: 1:500-1000

  • Western blot: 1:100-1000

  • IHC-P: 1:200-400

  • IHC-F: 1:100-500

• Nuclear localization confirmation: Verify proper nuclear staining pattern consistent with ZNF420's role as a transcription factor.

What are the optimal storage conditions to maintain ZNF420 antibody activity?

For optimal maintenance of ZNF420 antibody, HRP conjugated activity, implement the following storage protocol:

• Storage temperature: Store at -20°C as recommended by the manufacturer .

• Aliquoting strategy: Upon receipt, immediately prepare multiple small-volume aliquots (10-20 μL) to prevent repeated freeze-thaw cycles that degrade antibody function .

• Storage buffer composition: The antibody is supplied in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol . This formulation provides several stability advantages:

  • 50% Glycerol acts as a cryoprotectant during freezing

  • 1% BSA provides protein stability

  • TBS maintains physiological pH

  • Proclin300 prevents microbial growth

• Handling recommendations: Always thaw aliquots on ice, avoid vortexing (mix by gentle flicking), and return to -20°C immediately after use.

• Working stock preparation: For multi-day experiments, prepare a working dilution with additional 0.1-1% BSA as carrier protein, storable at 4°C for 1-2 weeks.

Proper storage significantly affects experimental reproducibility and prevents false negative results due to antibody degradation.

How should application-specific protocols be optimized for ZNF420 antibody, HRP conjugated?

Optimizing protocols for ZNF420 antibody requires application-specific adjustments:

Western Blotting (1:100-1000 dilution)

• Blocking optimization: Test both 5% non-fat milk and 3-5% BSA in TBST to determine optimal blocking conditions.

• Membrane selection: PVDF membranes generally require more stringent blocking than nitrocellulose.

• Detection system: Enhanced chemiluminescence (ECL) systems provide optimal sensitivity for HRP-conjugated antibodies.

• Antibody incubation: Start with overnight incubation at 4°C at 1:500 dilution, then optimize as needed.

Immunohistochemistry (IHC-P: 1:200-400; IHC-F: 1:100-500)

• Antigen retrieval: Since ZNF420 is a nuclear protein, heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be tested.

• Detection method: DAB (3,3'-diaminobenzidine) produces a brown precipitate compatible with nuclear counterstains.

• Counterstaining: Hematoxylin provides optimal nuclear contrast without obscuring nuclear ZNF420 staining.

• Controls: Include tissue sections known to express ZNF420 as positive controls.

ELISA (1:500-1000 dilution)

• Plate coating: Optimize antigen concentration and coating buffer.

• Blocking: 1-3% BSA in PBS is recommended to minimize background.

• Detection substrate: TMB (3,3',5,5'-tetramethylbenzidine) provides high sensitivity with HRP conjugates.

• Standard curve: Include recombinant ZNF420 protein at known concentrations (0-100 ng/mL).

For each application, perform systematic titration experiments to identify conditions providing optimal signal-to-noise ratio.

What strategies can minimize non-specific binding when using ZNF420 antibody?

To minimize non-specific binding with ZNF420 antibody, HRP conjugated, implement these research-validated strategies:

• Blocking optimization:

  • Test different blockers: 3-5% BSA, non-fat milk, normal serum, commercial blocking reagents

  • Include 0.1-0.3% Tween-20 in wash and blocking buffers

  • For tissue samples, include 10% normal serum from the host species of the secondary antibody (if using indirect detection)

• Antibody dilution optimization:

  • Begin with the manufacturer's recommended range (ELISA: 1:500-1000; WB: 1:100-1000; IHC-P: 1:200-400)

  • Perform titration experiments to identify minimal effective concentration

• Controls for background assessment:

  • Include isotype control antibodies

  • Perform primary antibody omission controls

  • For tissues, include known negative tissue types

• Pre-adsorption validation:

  • If available, pre-incubate antibody with immunizing peptide to verify signal specificity

• Tissue-specific treatments:

  • For IHC applications, block endogenous peroxidase (3% H₂O₂, 10 min)

  • Use avidin/biotin blocking for tissues with high endogenous biotin

• Sample preparation considerations:

  • Ensure complete antigen retrieval for fixed tissues

  • For cell lysates, include adequate detergent and reducing agents

  • Centrifuge antibody dilutions before use to remove aggregates

• Wash optimization:

  • Increase number of washes (minimum 3-5 washes)

  • Extend wash duration (5-10 minutes per wash)

  • Use gentle agitation during washing

These strategies systematically address the major sources of non-specific binding while maintaining specific signal detection.

How does the enhanced lyophilization method for HRP conjugation improve ZNF420 antibody performance?

The enhanced lyophilization method for HRP conjugation represents a significant methodological advancement for antibody preparation that can be applied to ZNF420 antibody with notable benefits:

The process involves significant modifications to the traditional periodate conjugation method:

• Initial HRP activation: Similar to classical method, using 0.15 M sodium metaperiodate to oxidize carbohydrate moieties on HRP, generating reactive aldehyde groups .

• Lyophilization step: After HRP activation and dialysis, the activated HRP undergoes freezing at -80°C for 5-6 hours followed by overnight lyophilization . This critical step significantly enhances conjugation efficiency.

• Conjugation reaction: The lyophilized activated HRP is mixed with antibody (1:4 molar ratio of antibody to HRP) and incubated at 37°C for 1 hour .

• Schiff's base stabilization: Sodium cyanoborohydride (1/10th volume) is added to stabilize the conjugate through reduction of Schiff's bases, followed by 2-hour incubation at 4°C .

The mechanism of enhancement derives from collision theory principles - reducing reaction volume without changing reactant quantities increases molecular collision probability and reaction efficiency . This allows more HRP molecules to conjugate to each antibody, creating a poly-HRP effect that significantly amplifies detection sensitivity while minimizing antibody consumption .

What is the molecular mechanism of ZNF420's regulation of p53-mediated apoptosis and its implications for cancer research?

ZNF420 (also known as APAK - ATM and p53 associated KZNF protein) functions as a negative regulator of p53-mediated apoptosis, with significant implications for cancer research :

Molecular Mechanism:
ZNF420 contains 19 C2H2-type zinc fingers that likely facilitate specific DNA binding, along with a KRAB domain that typically functions as a potent transcriptional repression module . As a negative regulator of p53-mediated apoptosis, ZNF420 likely acts through one or more of these mechanisms:

• Direct transcriptional repression of p53 target genes involved in apoptosis

• Direct interaction with p53 protein, inhibiting its transcriptional activity

• Recruitment of chromatin-modifying complexes to p53-responsive promoters

• Modulation of p53 post-translational modifications that regulate its activity

Research Implications:
Understanding ZNF420's role in p53 regulation presents several opportunities for cancer research:

• Therapeutic targeting potential: Inhibiting ZNF420 could potentially enhance p53-mediated apoptosis in cancers with wild-type p53, increasing sensitivity to DNA-damaging therapies.

• Biomarker development: ZNF420 expression patterns may serve as:

  • Predictive biomarkers for response to p53-activating therapies

  • Prognostic indicators in cancers with wild-type p53

  • Markers of functional p53 inactivation despite wild-type status

• Resistance mechanism identification: ZNF420 overexpression could represent a previously unrecognized mechanism of resistance to therapies relying on p53-mediated cell death.

• Synthetic lethality approaches: In p53-mutant cancers, targeting pathways that interact with ZNF420 might reveal synthetic lethal interactions.

Detection of ZNF420 using the HRP-conjugated antibody in tumor samples could help stratify patients for clinical trials targeting the p53 pathway and identify cancers where ZNF420-targeted approaches might prove beneficial.

How can ZNF420 antibody be incorporated into multiplexed detection systems for studying apoptotic pathways?

Incorporating ZNF420 antibody into multiplexed detection systems enables comprehensive analysis of p53-mediated apoptotic pathway regulation. Several methodological approaches are possible:

• Sequential multiplex immunohistochemistry/immunofluorescence:

  • Initial staining: ZNF420 antibody, HRP conjugated (1:200-400 dilution) with DAB detection

  • Stripping/blocking: Glycine-SDS buffer (pH 2.0) to remove primary antibody

  • Subsequent staining: Additional antibodies against p53, MDM2, BAX, PUMA with distinct chromogens

  • Analysis: Digital slide scanning with spectral unmixing algorithms

• Fluorescent multiplex immunoassays:

  • Tyramide signal amplification: Utilize HRP activity of the ZNF420 conjugate to deposit fluorescent tyramide

  • Compatible fluorophores: FITC, Cy3, Cy5, DAPI for nuclear counterstaining

  • Antibody panels: Combine with antibodies against apoptotic markers (cleaved caspase-3, PARP)

  • Analysis: Confocal microscopy or high-content imaging systems

• Flow cytometry applications:

  • Intracellular staining: Fixation/permeabilization followed by ZNF420 antibody (1:100-500)

  • HRP substrate: Use fluorogenic substrates compatible with flow cytometry

  • Complementary markers: Co-stain for apoptotic status (Annexin V, propidium iodide)

  • Analysis: Multiparameter flow cytometry with compensation controls

• Multiplex bead-based assays:

  • Antibody coupling: Conjugate capture antibodies to distinct bead populations

  • Detection system: Use ZNF420 antibody, HRP conjugated as detection reagent

  • Signal development: TMB substrate for colorimetric readout

  • Analysis: Bead-specific signal quantification

• Optimal experimental design:

  • Include appropriate single-stain controls for signal separation

  • Perform antibody titration to prevent signal spillover

  • Include biological controls representing activated and inactivated pathways

  • Apply bioinformatic analysis to interpret pathway interactions

This multiplex approach enables simultaneous visualization of multiple components of the p53-mediated apoptotic pathway, providing spatial and temporal understanding of ZNF420's regulatory role.

What are the critical factors affecting reproducibility in ZNF420 detection across different experimental systems?

Ensuring reproducible ZNF420 detection across experimental systems requires systematic attention to several critical factors:

• Antibody-specific variables:

  • Lot-to-lot variation: Different antibody lots may show varying affinities and specificities

  • Storage conditions: Improper storage (-20°C, aliquoted to prevent freeze-thaw cycles) can degrade antibody performance

  • HRP conjugation stability: The enhanced lyophilization method provides more stable conjugates than classical methods

  • Working concentration optimization: Titration should be performed for each new experimental system

• Sample preparation considerations:

  • Fixation impact: Different fixatives alter protein epitope accessibility

  • Antigen retrieval methods: Heat-induced vs. enzymatic retrieval yield different results

  • Protein extraction protocols: Varied lysis buffers extract different subcellular fractions

  • Post-translational modifications: Sample handling may affect ZNF420 phosphorylation status

• Detection system variables:

  Detection Method	Critical Parameters	Optimization Approach
  Western Blot	Transfer efficiency, blocking conditions	Standardize protein loading (20-40 μg/lane), optimize transfer time
  IHC/ICC	Background, antigen masking	Optimize blocking (3-5% BSA), standardize antigen retrieval
  ELISA	Coating efficiency, detection limit	Establish standard curves (1.5-100 ng range), use consistent plate type

• Data analysis standardization:

  • Signal quantification: Consistent methods for densitometry or fluorescence quantification

  • Background subtraction: Apply identical algorithms across experiments

  • Normalization strategy: Use appropriate housekeeping genes or total protein normalization

  • Statistical approach: Apply consistent statistical tests with appropriate sample sizes

• Protocol documentation:

  • Detailed methods sections including antibody catalog number (bs-7092R-HRP), lot number, and dilution

  • Explicit description of optimization steps

  • Sharing of raw data and analysis workflows

  • Inclusion of all negative and positive controls

By systematically addressing these factors, researchers can significantly improve reproducibility of ZNF420 detection across different experimental platforms and laboratories.

What are common causes of signal variability with ZNF420 antibody and how can they be addressed?

Signal variability with ZNF420 antibody, HRP conjugated can be systematically diagnosed and addressed using this troubleshooting framework:

Specific recommendations for enhanced lyophilization conjugates:
The enhanced lyophilization method for HRP conjugation significantly improves sensitivity (1:5000 vs 1:25 dilution for classical methods) , but this higher sensitivity makes these conjugates more susceptible to certain sources of variability:

• For highly sensitive detection, use freshly prepared substrates

• Extend washing steps to eliminate trace unbound antibody

• Include more dilute standard curves (detection threshold ~1.5 ng)

• Use automated methods for enhanced reproducibility of timed reaction steps

Advanced quantitative analysis of signal variability can help distinguish between technical and biological sources of variation, improving experimental design and result interpretation.

How can non-specific signals be distinguished from specific ZNF420 detection?

Distinguishing specific ZNF420 signals from non-specific background requires implementing a comprehensive validation strategy:

• Control-based validation:

  • Peptide competition assay: Pre-incubate ZNF420 antibody with excess immunizing peptide (derived from human ZNF420, range 301-688) . Specific signals should disappear or significantly diminish.

  • Knockout/knockdown controls: Compare staining between wild-type samples and those with ZNF420 knockdown by siRNA/shRNA. Specific signals should be absent or reduced in knockdown samples.

  • Isotype controls: Use rabbit IgG-HRP conjugate at matching concentration to identify non-specific binding.

• Signal pattern analysis:

  • Subcellular localization: ZNF420 is a nuclear protein ; specific staining should be predominantly nuclear.

  • Molecular weight verification: In Western blots, specific bands should match predicted molecular weight.

  • Expression pattern consistency: Specific staining should show consistent patterns across different samples of the same tissue type.

• Methodological approaches:

  • Signal intensity gradient: Perform antibody titration (1:100, 1:200, 1:500, 1:1000); specific signals typically show dose-dependence while background often remains constant.

  • Multiple detection methods: Compare results across different platforms (Western blot, IHC, ELISA); specific signals should be consistent across methods.

  • Orthogonal validation: Use alternative detection methods like mRNA analysis (RT-PCR) to corroborate protein detection patterns.

• Advanced validation methods:

  • Immunoprecipitation-Mass Spectrometry: Confirm ZNF420 as the predominant protein detected by the antibody.

  • Multiple antibody validation: Compare results with different antibodies targeting distinct ZNF420 epitopes.

  • Cross-species confirmation: Test antibody in species with predicted reactivity (human, mouse, rat) ; staining patterns should correlate with evolutionary conservation.

• Statistical analysis of signal distribution:

  • Calculate signal-to-noise ratios across different experimental conditions

  • Apply thresholding based on negative control distribution

  • Consider machine learning approaches for pattern recognition in complex tissues

Rigorous validation across these dimensions provides strong evidence for specific ZNF420 detection and enables confident interpretation of experimental results.

How can ZNF420 antibody be utilized in studying the relationship between apoptosis regulation and cancer therapeutics?

ZNF420 antibody can be strategically employed to investigate the relationship between apoptosis regulation and cancer therapeutics through several methodological approaches:

• Patient-derived xenograft (PDX) studies:

  • Profile ZNF420 expression levels in patient tumors before implantation

  • Monitor expression changes during treatment with p53-activating therapies

  • Correlate ZNF420 levels with treatment response and resistance development

  • Applications: IHC-P (1:200-400 dilution) on FFPE tumor sections

• Predictive biomarker development:

  • Screen cancer cell line panels for ZNF420 expression using Western blot (1:100-1000)

  • Correlate expression with sensitivity to apoptosis-inducing drugs

  • Develop threshold values for high vs. low expression that predict therapeutic response

  • Validate in retrospective patient cohorts using tissue microarrays

• Mechanistic studies of drug resistance:

  • Monitor ZNF420 expression changes during acquired resistance development

  • Perform ChIP-seq to identify ZNF420 binding sites in resistant vs. sensitive cells

  • Analyze p53 pathway activity using multiplexed approaches that include ZNF420

  • Test whether ZNF420 knockdown resensitizes resistant cells to therapy

• Combination therapy rational design:

  • Screen for compounds that modulate ZNF420 expression or activity

  • Test synergy between these compounds and conventional p53-activating therapies

  • Use the antibody to confirm on-target effects in combination studies

  • Applications: ELISA (1:500-1000) for high-throughput screening

• Post-translational modification analysis:

  • Investigate how cancer therapeutics affect ZNF420 phosphorylation or other modifications

  • Combine ZNF420 antibody with phospho-specific antibodies in multiplex detection

  • Correlate modification status with protein function and therapeutic response

This systematic approach leveraging ZNF420 antibody can reveal new insights into how this negative regulator of p53-mediated apoptosis influences treatment outcomes and may identify novel therapeutic vulnerabilities in cancers.

What considerations are important when incorporating ZNF420 antibody into single-cell analysis techniques?

Incorporating ZNF420 antibody into single-cell analysis techniques requires specific methodological considerations to ensure robust and interpretable results:

• Single-cell immunofluorescence optimization:

  • Signal amplification: Convert HRP activity to fluorescent signal using tyramide signal amplification

  • Antibody titration: Traditional dilutions (1:100-500) may require adjustment for single-cell sensitivity

  • Signal-to-noise optimization: Include additional blocking steps (Fc receptor blocking, avidin/biotin blocking)

  • Nuclear marker co-staining: Include DAPI to confirm nuclear localization of ZNF420 signals

• Flow cytometry/mass cytometry applications:

  • Cell fixation/permeabilization: Optimize for nuclear protein detection (methanol-based protocols)

  • Compensation controls: Include single-stained controls to account for spectral overlap

  • Antibody validation: Confirm specificity using positive/negative controls and titration series

  • Panel design: Combine with apoptosis markers (cleaved PARP, caspase-3) and cell cycle markers

• Single-cell Western techniques:

  • Protein capture: Optimize lysis conditions to extract nuclear proteins

  • Detection sensitivity: Enhanced HRP conjugates provide better detection limits (~1.5 ng)

  • Size verification: Confirm signal corresponds to ZNF420's expected molecular weight

  • Quantitative analysis: Establish detection thresholds based on negative controls

• Imaging mass cytometry/CODEX multiplexed imaging:

  • Metal conjugation: Consider converting HRP-conjugated antibody to metal-tagged format

  • Epitope accessibility: Optimize antigen retrieval specifically for multiplexed protocols

  • Antibody compatibility: Test for interference with other antibodies in high-parameter panels

  • Batch effects: Implement batch correction algorithms for multi-sample studies

• Single-cell sequencing integration:

  • Protein verification: Use antibody-based sorting to validate transcriptional signatures

  • Multi-omics approaches: Combine with scRNA-seq through CITE-seq or similar technologies

  • Tissue context preservation: Consider spatial transcriptomics with antibody validation

• Data analysis considerations:

  • Binary vs. continuous expression: Determine appropriate thresholds for positive/negative classification

  • Heterogeneity quantification: Apply clustering algorithms to identify ZNF420 expression patterns

  • Trajectory analysis: Incorporate ZNF420 expression in pseudotime analyses of differentiation/response

  • Spatial context: For imaging-based techniques, analyze subcellular localization and neighbor relationships

These methodological considerations enable robust incorporation of ZNF420 antibody into the expanding toolkit of single-cell analysis techniques, providing insights into heterogeneous expression patterns within complex tissues and cell populations.