ZNF226 Antibody

What is ZNF226 and why is it important in research settings?

ZNF226 (zinc finger protein 226) is a protein encoded by the ZNF226 gene in humans, located on chromosome 19q13.31. It belongs to the Krüppel C2H2-type zinc-finger protein family and contains a Kruppel associated box A (KRAB-A) domain, which functions as a transcriptional repressor. The protein contains 19 C2H2-type zinc finger structural motifs that can bind either zinc ions (Zn²⁺) or nucleic acids . This protein is believed to play a significant role in transcriptional regulation, making it an important subject for research in gene expression studies and developmental biology . Understanding ZNF226 function may provide insights into regulatory networks controlling gene expression in normal development and disease states.

What are the known isoforms of ZNF226 and how might this affect antibody selection?

ZNF226 is currently known to exist in three distinct isoforms in humans: ZNF226 isoform X1, ZNF226 isoform X2, and ZNF226 isoform X3. The longest variant, isoform X1, consists of 803 amino acids and contains the complete KRAB-A domain along with 18 C2H2 zinc finger structural motifs . When selecting antibodies for ZNF226 detection, researchers should carefully consider which epitopes are targeted by the antibody to ensure detection of the specific isoform(s) of interest. Antibodies targeting the common regions shared across all isoforms will detect all variants, while those targeting unique regions may be isoform-specific. This consideration is crucial for experimental design, especially when studying differential expression or function of specific ZNF226 isoforms in various cellular contexts.

What applications are commercially available ZNF226 antibodies validated for?

Based on available research materials, commercial ZNF226 antibodies have been validated for several experimental applications. These include Enzyme-Linked Immunosorbent Assay (ELISA) and Immunohistochemistry (IHC) . Some antibodies are conjugated with specific markers, such as Horseradish Peroxidase (HRP), which enhances their utility in certain detection systems . Researchers should verify the validation status of antibodies for specific applications before designing experiments. While the provided information indicates validation for ELISA and IHC, additional applications like Western blotting, immunofluorescence, or flow cytometry may also be possible but would require validation by individual researchers if not explicitly stated by manufacturers.

How should researchers optimize ZNF226 antibody concentrations for immunohistochemistry applications?

Optimizing antibody concentrations for IHC detection of ZNF226 requires a systematic titration approach. Begin with a dilution series based on manufacturer recommendations (typically in the range of 1:100 to 1:500 for polyclonal antibodies) . Perform parallel experiments on positive control tissues known to express ZNF226, such as placental tissue samples, which have been demonstrated to express related zinc finger proteins . Include negative controls (tissues known not to express ZNF226 or primary antibody omission) to assess background staining.

Optimal concentration is determined when specific staining is clearly visible in positive control sections with minimal background. For ZNF226 detection specifically, subcellular localization should be primarily nuclear, consistent with its role in transcriptional regulation . If using HRP-conjugated antibodies, optimize not only antibody concentration but also substrate development time to achieve optimal signal-to-noise ratio. Document all optimization parameters meticulously, including antigen retrieval methods, blocking conditions, and incubation times and temperatures to ensure reproducibility.

What are the recommended controls for validating ZNF226 antibody specificity?

Validating ZNF226 antibody specificity requires multiple complementary approaches:

• Positive and negative tissue controls: Use tissues with known ZNF226 expression patterns as positive controls and tissues where expression is absent as negative controls.

• Peptide competition assay: Pre-incubate the antibody with a synthetic peptide containing the target epitope before application to the sample. Specific binding should be blocked, resulting in signal reduction or elimination.

• Knockout/knockdown validation: Compare staining between wild-type samples and those where ZNF226 has been knocked down using siRNA . Commercial ZNF226 siRNA is available and can be used to generate appropriate controls. The signal should be significantly reduced in knockdown samples.

• Antibody validation using multiple detection methods: Confirm results using alternative detection methods such as Western blotting or immunofluorescence.

• Isotype controls: Use an irrelevant antibody of the same isotype (IgG for most ZNF226 antibodies) to distinguish between specific binding and Fc receptor-mediated or other non-specific interactions .

Thorough validation using these controls is essential for ensuring reliability of experimental results and avoiding artifactual findings.

What sample preparation methods are optimal for detecting ZNF226 in cellular and tissue samples?

The optimal sample preparation methods for ZNF226 detection depend on the application and sample type:

For tissue samples in IHC:

• Fixation: 10% neutral buffered formalin for 24-48 hours is standard for paraffin embedding. For immunofluorescence, 4% paraformaldehyde for 1-4 hours may preserve antigenicity better.

• Antigen retrieval: Since ZNF226 is a nuclear protein, heat-mediated antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is often necessary to expose epitopes masked during fixation.

• Sectioning: 4-6 μm sections are typically optimal for IHC detection of nuclear proteins.

• Blocking: Include thorough blocking steps (3-5% BSA or serum from the species of the secondary antibody) to minimize background staining.

For cellular samples in ELISA or Western blotting:

• Lysis: Use nuclear extraction buffers containing protease inhibitors since ZNF226 is primarily localized to the nucleus .

• Protein denaturation: For Western blotting, complete denaturation with SDS and reducing agents is typically required for antibody access to epitopes.

• Protein concentration: Standardize protein concentration across samples (typically 20-50 μg total protein per well for Western blotting).

In all cases, maintain sample integrity through proper handling, minimize freeze-thaw cycles, and process samples consistently across experimental conditions to ensure reproducible results.

How can researchers distinguish between ZNF226 and other highly homologous zinc finger proteins in experimental systems?

Distinguishing between ZNF226 and other zinc finger proteins presents a significant challenge due to structural similarities, particularly among the KRAB-domain containing C2H2 zinc finger proteins . Several strategies can address this challenge:

• Epitope mapping and antibody selection: Choose antibodies targeting unique regions of ZNF226 rather than conserved zinc finger domains or KRAB domains. The amino acid sequences between zinc finger structural motifs often contain greater variation between family members.

• Orthogonal validation approaches: Combine antibody-based detection with nucleic acid-based methods targeting the unique mRNA sequence of ZNF226. RT-qPCR with primers spanning unique exon junctions can confirm antibody specificity.

• Immunoprecipitation followed by mass spectrometry: This approach can definitively identify the protein being detected by determining its full amino acid sequence and distinguishing it from homologous proteins.

• Cross-reactivity testing: Test the antibody against recombinant proteins of closely related zinc finger family members to establish specificity boundaries.

• Bioinformatic analysis: Perform sequence alignment of ZNF226 with other zinc finger proteins to identify unique epitopes and predict potential cross-reactivity based on sequence homology.

For ZNF226 specifically, attention should be paid to distinguishing it from other chromosome 19 zinc finger cluster proteins, which emerged from gene duplication events and retain significant homology .

What methods can be used to study the transcriptional regulation function of ZNF226 using antibody-based approaches?

To investigate the transcriptional regulatory function of ZNF226 using antibody-based approaches, researchers can employ several sophisticated techniques:

• Chromatin Immunoprecipitation (ChIP): Use anti-ZNF226 antibodies to precipitate chromatin fragments bound by ZNF226, followed by sequencing (ChIP-seq) or qPCR to identify genomic binding sites. This provides direct evidence of the DNA sequences regulated by ZNF226.

• Co-Immunoprecipitation (Co-IP): Use ZNF226 antibodies to precipitate protein complexes containing ZNF226, followed by mass spectrometry to identify interacting partners in the transcriptional machinery.

• Proximity Ligation Assay (PLA): This technique can visualize and quantify interactions between ZNF226 and suspected protein partners involved in transcriptional regulation within intact cells.

• ChIP-MS (Chromatin Immunoprecipitation coupled with Mass Spectrometry): This advanced technique enables identification of both the DNA binding sites and protein interaction partners of ZNF226 in a single experiment.

• Immunofluorescence combined with RNA FISH: This approach can correlate the nuclear localization of ZNF226 protein with the expression of putative target genes.

• CUT&RUN or CUT&Tag: These newer alternatives to ChIP offer higher signal-to-noise ratios for mapping DNA-protein interactions and may provide clearer data on ZNF226 binding sites.

These methods should be combined with functional readouts, such as reporter gene assays or RNA-seq following ZNF226 knockdown or overexpression, to establish causal relationships between ZNF226 binding and transcriptional outcomes.

How do post-translational modifications affect ZNF226 antibody detection, and how can researchers account for this?

Post-translational modifications (PTMs) of ZNF226 can significantly impact antibody detection by altering epitope accessibility or recognition. Common PTMs that may affect zinc finger proteins include phosphorylation, SUMOylation, and ubiquitination, which can regulate protein function, localization, and stability.

To account for PTM effects on ZNF226 antibody detection:

• Use modification-specific antibodies: When studying a specific PTM of ZNF226, use antibodies specifically designed to detect the modified form of the protein.

• Employ multiple antibodies: Use several antibodies targeting different epitopes of ZNF226 to provide a more complete picture of protein expression and modification state.

• Pretreatment of samples: For phosphorylation studies, compare detection with and without phosphatase treatment. Similarly, for other modifications, use appropriate enzymes to remove specific PTMs before detection.

• 2D gel electrophoresis: This can separate different post-translationally modified forms of ZNF226 based on both molecular weight and isoelectric point before antibody detection . The isoelectric point of human ZNF226 ranges from 8.60 to 9.00, and modifications would alter this property .

• Mass spectrometry validation: Following immunoprecipitation with ZNF226 antibodies, perform mass spectrometry analysis to identify and quantify specific PTMs present on the protein.

• Consider cellular context: PTM patterns may vary with cellular conditions, differentiation state, or disease context. Document experimental conditions thoroughly and consider how they might affect the modification state of ZNF226.

By systematically addressing these considerations, researchers can develop more accurate and comprehensive understandings of ZNF226 biology.

What are common causes of false positive and false negative results when using ZNF226 antibodies, and how can they be mitigated?

Common causes of false results when using ZNF226 antibodies include:

False Positive Causes and Mitigations:

• Cross-reactivity with homologous zinc finger proteins: ZNF226 belongs to a large family of structurally similar proteins . Mitigate by using highly specific antibodies and validating with knockdown controls .

• Non-specific binding to Fc receptors: This is particularly problematic in immune cells. Mitigate by using appropriate blocking reagents and including isotype controls.

• Endogenous peroxidase activity: In IHC using HRP-conjugated antibodies , endogenous peroxidases can cause background. Mitigate with hydrogen peroxide quenching steps before antibody application.

• Overfixation: Excessive fixation can create artifactual epitopes. Mitigate by optimizing fixation protocols and including appropriate controls.

False Negative Causes and Mitigations:

• Epitope masking due to protein interactions or PTMs: ZNF226 functions in protein complexes that may mask antibody binding sites. Mitigate by testing multiple antibodies targeting different epitopes and optimizing antigen retrieval methods.

• Protein degradation: Nuclear proteins may degrade during sample processing. Mitigate by adding protease inhibitors promptly and maintaining cold temperatures during sample preparation.

• Insufficient sensitivity: Low expression levels of ZNF226 may fall below detection limits. Mitigate by using signal amplification methods or more sensitive detection systems.

• Suboptimal antibody concentration: Too dilute antibody solutions reduce detection probability. Perform careful titration experiments to determine optimal concentration for each application and batch of antibody.

In all cases, thorough validation using the controls described in question 2.2 is essential for distinguishing true from false results.

How can researchers interpret conflicting results from different detection methods when studying ZNF226?

When faced with conflicting results from different detection methods for ZNF226, researchers should follow a systematic approach to reconcile discrepancies:

• Evaluate method-specific limitations: Each detection technique has inherent strengths and weaknesses. For example, Western blotting provides molecular weight information but may denature conformational epitopes, while IHC preserves spatial context but may suffer from cross-reactivity issues.

• Consider epitope accessibility differences: The three-dimensional structure of ZNF226 may render certain epitopes inaccessible in native conditions but exposed in denatured states, explaining why an antibody might work in Western blotting but not in IHC or immunofluorescence.

• Examine isoform specificity: The three known isoforms of ZNF226 (X1, X2, and X3) may be differentially detected by various antibodies or methods. Map the epitope recognized by each antibody relative to the protein structure and isoform differences.

• Assess technical variables: Differences in sample preparation, fixation methods, antibody concentrations, and detection systems can significantly impact results. Standardize these variables as much as possible across methods.

• Implement orthogonal validation: Use nucleic acid-based methods (such as RT-qPCR or RNA-seq) alongside protein detection methods to corroborate findings and identify discrepancies.

• Conduct knockdown/knockout validation: siRNA knockdown experiments provide the most stringent validation. If signal persists after effective knockdown, it suggests non-specific detection.

• Consult literature for known caveats: Review published studies using the same antibodies and methods to identify previously reported limitations or considerations.

When reporting conflicting results, transparently document all method parameters and discuss possible explanations for discrepancies rather than selectively reporting only concordant results.

What strategies can be employed when commercially available ZNF226 antibodies fail to detect the protein in specific experimental contexts?

When commercial ZNF226 antibodies fail in specific experimental contexts, researchers can implement several advanced troubleshooting strategies:

• Epitope mapping and antibody selection: Different regions of ZNF226 may be accessible in different experimental contexts. Test antibodies targeting different epitopes, particularly those outside the zinc finger domains, which may be involved in protein-protein or protein-DNA interactions that could mask detection.

• Custom antibody development: Consider generating custom antibodies against unique peptide sequences in ZNF226, especially targeting regions predicted to be surface-exposed and unique to ZNF226 compared to other zinc finger proteins.

• Sample enrichment techniques: Use subcellular fractionation to enrich for nuclear proteins, where ZNF226 is expected to localize . This can increase signal-to-noise ratio in detection systems.

• Alternative detection strategies: Implement epitope tagging approaches by expressing tagged versions of ZNF226 (HA, FLAG, or GFP tags) that can be detected with highly specific and well-characterized tag antibodies.

• Proximity labeling approaches: Techniques like BioID or APEX2 can identify proteins in proximity to ZNF226 even when direct detection is challenging, providing indirect evidence of its presence and interactions.

• Mass spectrometry-based detection: Targeted mass spectrometry approaches can detect specific peptides from ZNF226 without relying on antibodies.

• Expression system optimization: If studying exogenously expressed ZNF226, optimize expression levels to ensure they fall within detectable ranges without causing aggregation or mislocalization.

• Consider biological context: ZNF226 expression or localization may be regulated in a cell-type or condition-specific manner. Verify that the experimental system is appropriate for studying this protein by first confirming mRNA expression.

Documentation of failed detection attempts is valuable to the scientific community and should be reported along with successful strategies.

How can ZNF226 antibodies be utilized in studying evolutionary conservation of zinc finger proteins across vertebrate lineages?

ZNF226 antibodies can serve as valuable tools for evolutionary studies of zinc finger proteins, providing insights into functional conservation and diversification across vertebrate lineages:

• Cross-species reactivity testing: Systematically test ZNF226 antibodies against tissue samples from diverse vertebrate species to establish cross-reactivity profiles. This can reveal conservation of epitopes across evolutionary distances and identify species boundaries where structural divergence occurs.

• Comparative expression mapping: Use validated cross-reactive antibodies to map expression patterns of ZNF226 orthologs across tissues in different vertebrate species. This approach can reveal conservation or divergence of regulatory mechanisms controlling zinc finger protein expression.

• Combined immunoprecipitation and phylogenetic analysis: Use antibodies to immunoprecipitate ZNF226-related proteins from different species, followed by mass spectrometry identification and phylogenetic analysis of the isolated proteins. This can provide direct evidence of structural conservation despite sequence divergence.

• Functional domain conservation assessment: Design experiments to test whether antibodies targeting specific functional domains (such as the KRAB-A domain or zinc finger motifs) show differential cross-reactivity compared to those targeting more variable regions. This approach can reveal which protein domains faced stronger evolutionary constraints.

• Co-evolution studies: Compare the conservation patterns of ZNF226 with its interacting partners across species. The evolution of KRAB-ZNF proteins has been shown to involve independent expansion and divergence in different vertebrate lineages , making this particularly relevant for understanding the evolution of transcriptional regulatory networks.

The data in search result showing the distribution of different types of zinc finger genes across vertebrate lineages provides context for such evolutionary studies, highlighting the dynamic nature of this gene family's evolution.

What are the implications of ZNF226's potential role in transcriptional regulation for disease research, and how can antibodies facilitate this investigation?

ZNF226's role as a potential transcriptional regulator has significant implications for disease research, particularly in conditions involving dysregulated gene expression. ZNF226 antibodies can facilitate these investigations in several ways:

• Disease-specific expression profiling: Use ZNF226 antibodies in tissue microarrays to compare expression levels and subcellular localization across healthy versus diseased tissues (particularly in cancers and developmental disorders where transcriptional regulation is altered).

• Chromatin landscape characterization: Combine ChIP-seq using ZNF226 antibodies with histone modification mapping to understand how ZNF226 binding correlates with specific chromatin states in normal versus pathological conditions.

• Target gene identification in disease contexts: Use ZNF226 antibodies for ChIP followed by sequencing to identify disease-specific alterations in the genomic binding profile of ZNF226, potentially revealing pathologically relevant target genes.

• Therapeutic target validation: In diseases where ZNF226 is implicated, antibodies can help validate it as a potential therapeutic target by confirming its expression in relevant tissues and cell types.

• Biomarker development: If ZNF226 expression or localization changes are consistently associated with specific disease states, antibody-based detection could form the basis for diagnostic or prognostic biomarkers.

• Functional studies in disease models: Use antibodies to track changes in ZNF226 expression, localization, or interactome in cellular or animal models of disease, particularly those involving the KRAB domain transcriptional repression function .

• Post-translational modification profiling: Develop modification-specific antibodies to determine whether disease states affect the PTM profile of ZNF226, potentially altering its function.

The KRAB domain's established role as a transcriptional repressor suggests that ZNF226 dysfunction could lead to inappropriate gene activation in disease contexts, making this an important area for investigation.

How might new antibody technologies advance our understanding of ZNF226 function in complex cellular systems?

Emerging antibody technologies offer promising approaches to better understand ZNF226 function in complex cellular contexts:

• Single-cell antibody-based proteomics: Technologies like mass cytometry (CyTOF) and CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) using ZNF226 antibodies can reveal cell-to-cell variation in ZNF226 expression and correlate it with other proteins and transcripts at single-cell resolution.

• Intrabodies and nanobodies: These engineered antibody formats can be expressed within living cells to track ZNF226 dynamics in real-time or to functionally perturb specific interactions, providing insights into dynamic regulation not possible with conventional antibodies.

• Antibody-based proximity labeling: Techniques like TurboID or APEX2 fused to anti-ZNF226 antibody fragments can identify proteins in the immediate vicinity of ZNF226 in living cells, revealing context-specific interaction networks.

• Super-resolution microscopy with advanced probes: Combining super-resolution imaging techniques with specially designed antibody probes can reveal the spatial organization of ZNF226 relative to chromatin structures and transcriptional machinery at nanometer resolution.

• Optogenetic antibody tools: Light-activatable antibody systems can allow temporal control over ZNF226 interactions or functions, enabling precise dissection of its role in transcriptional regulation.

• Combinatorial antibody barcoding: This approach can simultaneously detect ZNF226 along with dozens of other proteins in the same sample, revealing how ZNF226 expression correlates with broader cellular states.

• Degradation-inducing antibodies: Technologies like Trim-Away that use antibodies to target proteins for degradation could provide acute functional perturbation of ZNF226 without genetic manipulation.

These advanced applications extend far beyond the basic ELISA and IHC applications currently validated for commercial ZNF226 antibodies , but represent important future directions as the field advances toward understanding this protein's function in complex regulatory networks.