ZNF423 Antibody, Biotin conjugated

What is ZNF423 and why is it an important research target?

ZNF423 (Zinc finger protein 423) is a multifunctional transcription factor that can act as both an activator and repressor depending on cellular context. It plays critical roles in BMP signaling pathways, olfactory neurogenesis, and B cell differentiation. This protein contains multiple zinc finger domains that mediate its interactions with various partners including SMADs (in response to BMP2) and EBF1 (Early B-cell Factor 1) .

ZNF423 has gained significant research interest because:

• It regulates critical developmental pathways in neural and B cell differentiation

• Its aberrant expression has been linked to acute lymphoblastic leukemia (ALL), particularly in ETV6-RUNX1 negative cases

• It participates in a developmental switch regulating the transition from differentiation to maturation in olfactory receptor neurons

• It controls the proliferation and differentiation of neural precursors in cerebellar vermis formation

How do biotin-conjugated ZNF423 antibodies differ from unconjugated versions in experimental applications?

Biotin-conjugated ZNF423 antibodies offer several methodological advantages over unconjugated versions:

• Enhanced signal amplification: The biotin-avidin/streptavidin system provides significant signal enhancement through multiple binding sites, improving detection sensitivity in techniques like ELISA, immunohistochemistry, and flow cytometry .

• Flexible detection systems: Biotin conjugation allows researchers to use various secondary detection systems (streptavidin-HRP, streptavidin-fluorophores) without changing the primary antibody .

• Reduced background: In multi-step staining protocols, biotin conjugation can reduce non-specific binding compared to secondary antibody approaches .

• Application compatibility: Biotin-conjugated antibodies are particularly advantageous for ELISA, flow cytometry, and immunohistochemistry applications where signal amplification is desired .

What are the optimal storage conditions for maintaining ZNF423 antibody, Biotin conjugated activity?

To maintain optimal activity of biotin-conjugated ZNF423 antibodies, researchers should follow these evidence-based storage protocols:

• Temperature: Store at -20°C for long-term storage. For antibodies in frequent use, small aliquots can be maintained at 2-8°C for up to 2 weeks .

• Light protection: All biotin-conjugated antibodies should be stored in light-protected vials or covered with a light-protecting material (e.g., aluminum foil) to prevent photobleaching of the biotin moiety .

• Glycerol stabilization: For extended storage (>12 months), conjugates may be diluted with up to 50% glycerol and stored at -20°C to -80°C .

• Aliquoting: To prevent protein degradation from repeated freeze-thaw cycles, divide the antibody into small working aliquots before freezing .

• Buffer conditions: Optimal buffer typically contains preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) .

Laboratory data indicates that properly stored biotin-conjugated antibodies maintain stability for at least 12 months at 4°C, while glycerol-stabilized preparations at -20°C can maintain reactivity for up to 24 months .

What are the validated applications for ZNF423 antibody, Biotin conjugated in current research protocols?

Based on comprehensive validation studies, biotin-conjugated ZNF423 antibodies have been successfully employed in the following applications with specific methodological considerations:

For investigating ZNF423's nuclear localization, researchers should note that optimization of permeabilization protocols is critical due to the protein's nuclear compartmentalization. The calculated molecular weight of human ZNF423 is approximately 144.6 kDa, which should be considered when validating western blot results .

How can researchers optimize immunoprecipitation protocols using ZNF423 antibody, Biotin conjugated for protein interaction studies?

Optimizing immunoprecipitation (IP) protocols with biotin-conjugated ZNF423 antibodies for protein interaction studies requires careful consideration of several methodological factors:

• Capture system selection:

  • Streptavidin-agarose beads provide high affinity binding (Kd~10^-15 M) and are recommended for complex samples

  • NeutrAvidin-coated magnetic beads offer reduced non-specific binding and are optimal for nuclear protein complexes

• Nuclear protein extraction optimization:

  • Since ZNF423 is a nuclear protein, use high-salt extraction buffers (300-400 mM NaCl) with gentle detergents

  • Include DNase I treatment (10-50 units/mL) to release chromatin-bound protein complexes

  • Add phosphatase inhibitors to preserve protein-protein interactions mediated through phosphorylated residues

• Cross-linking considerations:

  • For transient interactions (especially with EBF1 and SMAD proteins), implement formaldehyde cross-linking (0.1-1% for 10 minutes)

  • For detection of the ZNF423-NuRD repressor complex, consider using DSP (dithiobis[succinimidyl propionate]) cross-linker

• Washing stringency balance:

  • For ZNF423-SMAD interactions: use moderate stringency (150-200 mM NaCl)

  • For ZNF423-EBF1 complexes: use lower stringency with 0.1% NP-40 to preserve zinc finger-mediated interactions

Research has demonstrated that using biotin-conjugated antibodies directed against the central domain of ZNF423 (amino acids 864-897) provides superior results in co-immunoprecipitation of EBF interaction partners compared to C-terminal antibodies .

What controls should be included when using ZNF423 antibody, Biotin conjugated for chromatin immunoprecipitation (ChIP) experiments?

For robust ChIP experiments using biotin-conjugated ZNF423 antibodies, the following comprehensive control system should be implemented:

• Antibody validation controls:

  • Peptide competition assay using the immunizing peptide (amino acids 39-57 or 864-897 depending on antibody)

  • IP-Western blot verification of antibody specificity prior to ChIP

  • Parallel ChIP with non-biotin conjugated ZNF423 antibody to verify equivalent enrichment

• Technical controls:

  • Input control (1-5% of starting chromatin)

  • IgG control matched to antibody host species (rabbit polyclonal IgG)

  • Mock IP control (beads only, no antibody)

  • Biotin blocking control (pre-incubation with free biotin to assess endogenous biotin interference)

• Positive genomic controls:

  • Known ZNF423 target regions containing the EBF binding motif (5'-CCCNNGGG-3')

  • Validated ZNF423-binding sites in B lymphocyte gene promoters (CD79a, CD79b, IGLL1)

  • BMP2-responsive genomic regions containing SMAD binding elements

• Negative genomic controls:

  • Transcriptionally silent regions (gene deserts)

  • Housekeeping gene promoters lacking ZNF423/EBF binding motifs

• Cell type controls:

  • ZNF423 high-expressing cells (B-lineage ALL cells)

  • ZNF423 low/non-expressing cells (mature B cells or liver cells)

Recent studies have demonstrated that optimized ChIP protocols using biotin-conjugated ZNF423 antibodies showed significant enrichment at sites containing the EBF response element, confirming the specificity of these reagents for genomic studies .

How can researchers use ZNF423 antibody, Biotin conjugated to investigate the differential roles of ZNF423 in BMP signaling versus EBF1-mediated transcriptional repression?

Investigating the dual roles of ZNF423 requires sophisticated experimental designs that can distinguish between its BMP-SMAD interaction function and its EBF1-mediated repression activity:

• Domain-specific functional analysis:

  • Target biotin-conjugated antibodies to different ZNF423 domains in sequential ChIP (re-ChIP) experiments:

    • ZFs 9-13: DNA binding domain for ZNF423-SMAD complexes

    • ZFs 14-19: Binding domain for phosphorylated SMAD1-SMAD4 complex

    • ZFs 28-30: EBF1 interaction domain

• Differential protein complex isolation:

  • Utilize biotin-conjugated ZNF423 antibodies in proximity-dependent biotin identification (BioID) assays

  • Compare protein interaction networks under BMP2 stimulation versus B-cell differentiation conditions

  • Verify specific interactions with co-immunoprecipitation followed by mass spectrometry

• Chromatin occupancy dynamics:

  • Implement dual-ChIP-seq protocols using biotin-conjugated ZNF423 antibodies with:

    • Anti-SMAD1/4 antibodies to identify BMP-responsive elements

    • Anti-EBF1 antibodies to identify sites of transcriptional repression

  • Motif analysis can distinguish between ZNF423 binding to EBF sites versus SMAD sites

• Functional validation through targeted mutations:

  • Combine antibody studies with expression of ZNF423 variants containing specific zinc finger mutations:

    • ZF 28 (H1224N): Maintains EBF1 interaction

    • ZF 29 (H1255N): Partially disrupts EBF1 interaction

    • ZF 30 (H1285N): Completely disrupts EBF1 interaction without affecting SMAD functions

Recent studies have revealed that ZNF423 can recruit the NuRD corepressor complex to EBF1 target genes, while simultaneously promoting BMP2-dependent gene activation through different domains, highlighting the importance of domain-specific analysis in understanding this protein's dual functionality .

What methodological approaches can distinguish between different ZNF423 isoforms using biotin-conjugated antibodies?

Distinguishing between ZNF423 isoforms requires carefully designed antibody-based experimental approaches:

• Isoform-specific epitope targeting:

  • Select biotin-conjugated antibodies raised against unique epitopes in specific isoforms

  • Human ZNF423 has at least 2 confirmed isoforms with distinct functional properties

  • Antibodies targeting amino acids 39-57 can detect both isoforms, while those targeting the central region (864-897) may show isoform selectivity

• Differential immunoprecipitation protocols:

  • Implement size-based separation following immunoprecipitation with biotin-conjugated antibodies

  • Use gradient gels (4-15%) for optimal resolution of high molecular weight isoforms

  • Verify isoform identity through mass spectrometry following streptavidin pull-down

• Sequential epitope accessibility analysis:

  • Utilize a panel of biotin-conjugated antibodies targeting different ZNF423 domains

  • Compare binding patterns under native versus denaturing conditions

  • Differential epitope accessibility can identify conformational variants of ZNF423

• Functional isoform discrimination:

  • Combine biotin-conjugated antibody immunoprecipitation with activity-based assays

  • ZNF423α has been shown to repress CD79b promoter activity in concentration-dependent manner

  • Differential repression capacities can distinguish functional isoforms

• Subcellular fractionation approach:

  • Different ZNF423 isoforms may exhibit distinct subcellular localization patterns

  • Combine nuclear/cytoplasmic fractionation with immunodetection using biotin-conjugated antibodies

  • Compare against known localization markers for verification

Research has demonstrated that ZNF423α isoform specifically inhibits EBF-1 target genes and leads to B cell maturation arrest in vivo, highlighting the importance of isoform-specific detection methods in functional studies .

How can researchers use ZNF423 antibody, Biotin conjugated to investigate the epigenetic mechanisms controlling ZNF423 expression in different leukemia subtypes?

Investigating epigenetic regulation of ZNF423 in leukemia requires sophisticated experimental approaches combining biotin-conjugated antibodies with epigenetic profiling techniques:

• Chromatin landscape analysis at the ZNF423 locus:

  • Use ChIP-seq with biotin-conjugated anti-ZNF423 antibodies alongside histone modification markers

  • Correlate ZNF423 occupancy with active (H3K4me3, H3K27ac) versus repressive (H3K27me3, H3K9me3) histone marks

  • Compare patterns between ETV6-RUNX1 positive versus negative ALL samples

• DNA methylation profiling correlated with expression:

  • Implement methyl-DNA immunoprecipitation (MeDIP) at the ZNF423 promoter

  • Correlate methylation status with ZNF423 protein levels detected by biotin-conjugated antibodies

  • Research has shown that 5'-aza-2-deoxycytidine treatment can activate ZNF423 in CD34+ cells that normally don't express it

• Chromatin accessibility and BMP2 responsiveness:

  • Combine ATAC-seq with biotin-conjugated ZNF423 ChIP-seq

  • Assess chromatin remodeling at the ZNF423 locus following BMP2 stimulation

  • Determine the relationship between chromatin state and ZNF423 expression in different leukemia subtypes

• NuRD complex recruitment and activity:

  • Use biotin-conjugated ZNF423 antibodies to pull down associated chromatin modifiers

  • Specifically examine recruitment of the NuRD corepressor complex components (CHD4, MTA1/2, RBAP46/48, MBD3, HDAC1/2)

  • Compare NuRD recruitment patterns in different leukemia contexts

• Therapeutic implications:

  • Evaluate changes in ZNF423 expression and localization following treatment with epigenetic modifying agents

  • Correlate ZNF423 levels with treatment resistance metrics

  • ZNF423 has been linked to DNA damage response pathways, suggesting a potential association with chemotherapy resistance

Research has demonstrated that epigenetic deregulation and BMP2-dependent transactivation of ZNF423 represent novel synergistic mechanisms in ALL. High ZNF423 expression levels were associated with adverse outcomes in ETV6-RUNX1 negative ALL patients, highlighting the clinical relevance of understanding its epigenetic regulation .

What are common challenges when using ZNF423 antibody, Biotin conjugated in flow cytometry, and how can they be overcome?

Flow cytometry with biotin-conjugated ZNF423 antibodies presents several technical challenges due to the nuclear localization of the target protein:

• Insufficient permeabilization:

  • Challenge: ZNF423 is a nuclear transcription factor requiring complete nuclear membrane permeabilization

  • Solution: Implement two-step permeabilization protocol:

    • Primary membrane permeabilization with 0.1% Triton X-100

    • Secondary nuclear permeabilization with 0.5% saponin or 90% methanol (10 minutes at -20°C)

    • Verify permeabilization efficiency with control nuclear antigens

• High background signal:

  • Challenge: Endogenous biotin in cells can create false positive signals

  • Solution: Pre-block with avidin/biotin blocking kit before adding biotin-conjugated antibodies

  • Include unstained and isotype controls with matched biotin conjugation

• Signal intensity limitations:

  • Challenge: Variable ZNF423 expression levels in different cell populations

  • Solution: Implement signal amplification using sequential streptavidin-PE or streptavidin-APC

  • Titrate antibody concentration (typically 1:50 - 1:200) to optimize signal-to-noise ratio

• Fixation-induced epitope masking:

  • Challenge: Some fixation methods can mask the ZNF423 epitope

  • Solution: Compare mild fixation protocols (1-2% paraformaldehyde for 10 minutes) versus alcohol fixation

  • For some epitopes, perform antibody staining before fixation

• Multi-parameter analysis challenges:

  • Challenge: Combining ZNF423 detection with cell surface markers

  • Solution: Implement sequential staining protocol:

    • First stain surface markers with directly conjugated antibodies

    • Then fix, permeabilize, and stain for ZNF423 using biotin-conjugated antibody

    • Complete with streptavidin-fluorophore detection

Validation studies have confirmed that when properly optimized, biotin-conjugated ZNF423 antibodies can achieve detection sensitivity suitable for distinguishing ZNF423-high versus ZNF423-low expressing cell populations in leukemia samples .

How can researchers verify the specificity of ZNF423 antibody, Biotin conjugated when working with complex tissue samples?

Verifying antibody specificity in complex tissue environments requires a multi-layered validation approach:

• Peptide competition assay:

  • Pre-incubate biotin-conjugated ZNF423 antibody with excess immunizing peptide (5-10 μg/mL)

  • Perform parallel staining with blocked and unblocked antibody

  • Specific staining should be eliminated in the peptide-blocked condition

• Genetic validation controls:

  • Utilize tissues from ZNF423 knockout/knockdown models (when available)

  • Compare staining patterns in tissues with confirmed differential ZNF423 expression

  • For human samples, include tissues known to express ZNF423 (brain, lung, skeletal muscle) versus negative controls (liver, placenta)

• Multi-antibody concordance testing:

  • Compare biotin-conjugated ZNF423 antibody staining patterns with antibodies targeting different epitopes

  • Concordant staining patterns across multiple antibodies increases confidence in specificity

  • Evaluate different conjugates (HRP, fluorophores) to rule out conjugation-specific artifacts

• Subcellular localization assessment:

  • Confirm nuclear localization pattern consistent with transcription factor function

  • Co-stain with nuclear markers (DAPI, Hoechst) to verify compartmentalization

  • Unexpected cytoplasmic staining may indicate non-specific binding

• Western blot correlation:

  • Perform parallel Western blot analysis on tissue lysates to confirm detection of correctly sized protein

  • ZNF423 should appear as a band at approximately 144.6 kDa

  • Multiple bands may indicate detection of isoforms, proteolytic fragments, or non-specific binding

• RNA-protein correlation:

  • Correlate antibody signal intensity with ZNF423 mRNA expression in the same tissues

  • Techniques like BaseScope in situ hybridization can provide spatial mRNA expression data for comparison

Studies have demonstrated that properly validated biotin-conjugated ZNF423 antibodies show nuclear localization in ZNF423-expressing tissues and cell types, with signal intensities that correlate with known expression patterns across different tissue types .

What are the critical considerations when selecting between different biotin-conjugated ZNF423 antibodies for specific research applications?

When selecting between different commercially available biotin-conjugated ZNF423 antibodies, researchers should evaluate several critical parameters:

• Epitope specificity and location:

  • Antibodies targeting different domains exhibit distinct functional properties:

    • N-terminal antibodies (aa 39-57): Suitable for total ZNF423 detection across isoforms

    • Central region antibodies (aa 191-290, 864-897): Optimal for functional studies involving EBF1 interaction

    • C-terminal antibodies (aa 1235-1284): Best for SMAD interaction studies

• Validation rigor and documentation:

  • Assess validation data comprehensiveness:

    • Western blot showing single band at expected MW (144.6 kDa)

    • Immunohistochemistry demonstrating expected nuclear localization

    • Peptide competition controls confirming specificity

    • Knockout/knockdown validation

• Application-specific performance:

  • ELISA applications: Consider antibodies validated at 1:1000-1:2000 dilutions

  • IHC applications: Select antibodies validated on FFPE tissues at 1:200-1:400

  • Flow cytometry: Choose antibodies with demonstrated permeabilized cell performance

• Species reactivity and cross-reactivity:

  • Verify species reactivity claims with experimental validation data

  • Consider epitope conservation across species:

    • Human and mouse ZNF423 share high homology but contain some divergent regions

    • Some antibodies exhibit cross-reactivity with rat, bovine, and zebrafish orthologs

• Manufacturing consistency and lot validation:

  • Polyclonal antibodies may show lot-to-lot variation

  • Assess supplier quality control documentation

  • Consider monoclonal options for highly standardized applications

• Biotin conjugation quality:

  • Optimal biotin:antibody ratio (typically 3-5 biotin molecules per antibody)

  • Conjugation chemistry that preserves epitope binding

  • Demonstrated stability under recommended storage conditions

Research requirements should drive antibody selection decisions. For functional studies examining ZNF423's role in transcriptional repression of B cell differentiation genes, antibodies targeting the EBF1-interaction domain (ZFs 28-30) have demonstrated superior performance .

How might biotin-conjugated ZNF423 antibodies facilitate the development of prognostic markers for acute lymphoblastic leukemia (ALL)?

Biotin-conjugated ZNF423 antibodies present promising opportunities for developing clinically relevant prognostic tools for ALL through several innovative approaches:

• Multi-parameter flow cytometry prognostic panels:

  • Integration of biotin-conjugated ZNF423 antibodies into leukemia immunophenotyping panels

  • Quantitative assessment of ZNF423 expression levels correlated with patient outcomes

  • Research has established that high ZNF423 expression is associated with adverse outcomes in ETV6-RUNX1 negative ALL patients

• Tissue microarray-based prognostic stratification:

  • Development of standardized immunohistochemistry protocols using biotin-conjugated antibodies

  • Quantitative image analysis of ZNF423 nuclear expression intensity

  • Correlation with minimal residual disease (MRD) measurements at day 28 of treatment

• Combined biomarker approach:

  • Integration of ZNF423 and SMAD1 expression analysis

  • Multivariate analysis incorporating established prognostic factors (age, white blood cell count, genetic alterations)

  • Development of risk prediction algorithms for treatment stratification

• Monitoring treatment response:

  • Sequential measurement of ZNF423 expression levels during therapy

  • Association with chemotherapy resistance mechanisms

  • ZNF423 has been linked to DNA damage response pathways, suggesting potential involvement in treatment resistance

• Companion diagnostics for targeted therapies:

  • Identification of patients with aberrant ZNF423 expression who might benefit from BMP pathway modulation

  • Screening for epigenetic therapies targeting ZNF423 regulation

  • Studies have shown that demethylating agents can activate ZNF423 in CD34+ cells

Research has demonstrated that ZNF423 expression predicts ALL outcome independently of established clinical and genetic risk features, highlighting its potential value as a prognostic biomarker worthy of further clinical development .

What novel research applications might emerge from combining biotin-conjugated ZNF423 antibodies with emerging single-cell technologies?

The integration of biotin-conjugated ZNF423 antibodies with cutting-edge single-cell technologies opens exciting new research frontiers:

• Single-cell multiomics integration:

  • Combining CITE-seq (antibody-based detection) with scRNA-seq to correlate ZNF423 protein levels with transcriptional profiles

  • Implementing index sorting with biotin-conjugated ZNF423 antibodies followed by single-cell genomics

  • Identifying rare cell populations with specific ZNF423 expression patterns in heterogeneous samples

• Spatial transcriptomics applications:

  • Utilizing biotin-conjugated ZNF423 antibodies in spatial proteomics platforms

  • Mapping ZNF423 protein expression in tissue microenvironments in relation to cellular niches

  • Correlating spatial ZNF423 expression with disease progression in lymphoid tissues

• Live-cell imaging of ZNF423 dynamics:

  • Developing membrane-permeable biotin-conjugated antibody fragments (scFv)

  • Real-time visualization of ZNF423 nuclear translocation and chromatin association

  • Tracking ZNF423 dynamics during B cell differentiation or in response to BMP signaling

• Proximity-based interaction mapping:

  • Implementing proximity-dependent biotin identification (BioID) using ZNF423 fusion proteins

  • Characterizing context-specific protein interaction networks in different cell states

  • Mapping the recruitment dynamics of NuRD complex components to ZNF423

• Single-cell epigenomics integration:

  • Combining CUT&Tag approaches with biotin-conjugated ZNF423 antibodies

  • Single-cell resolution mapping of ZNF423 chromatin binding sites

  • Correlating ZNF423 binding with chromatin accessibility and histone modification states