ZNF264 Antibody

What is ZNF264 and what cellular functions does it perform?

ZNF264 (zinc finger protein 264) is a member of the krueppel C2H2-type zinc-finger protein family. These proteins typically localize to the nucleus, bind nucleic acids, and regulate transcription . ZNF264 has a predicted protein size of approximately 70.4 kDa and is believed to be involved in transcriptional regulation mechanisms . As a zinc finger protein, it contains multiple zinc finger domains that enable sequence-specific DNA binding, allowing it to function as a transcriptional regulator by controlling the expression of target genes. The gene encoding ZNF264 is also sometimes referred to as KIAA0412 in literature .

What types of ZNF264 antibodies are currently available for research applications?

Researchers have multiple options when selecting ZNF264 antibodies, which vary by host species, clonality, and target epitope:

The choice between these antibodies depends on experimental needs, with monoclonal antibodies offering higher specificity and polyclonal antibodies providing stronger signal amplification .

How should ZNF264 antibodies be properly stored to maintain reactivity?

Proper storage is crucial for maintaining antibody functionality. For most ZNF264 antibodies, the recommended storage conditions are:

• Long-term storage: -20°C (some manufacturers suggest -80°C as an alternative)

• Some antibodies are supplied in lyophilized form and require reconstitution before use

• Liquid antibodies typically contain preservatives like 0.02-0.09% sodium azide

• Many formulations include glycerol (often 50%) as a cryoprotectant to prevent freeze-thaw damage

• For reconstituted or liquid antibodies, avoid repeated freeze-thaw cycles by preparing small aliquots

Upon receipt, antibodies should be immediately stored at the recommended temperature. For working solutions, storage at 4°C for short periods (typically 1-2 weeks) is acceptable .

What are the recommended applications for ZNF264 antibodies?

ZNF264 antibodies have been validated for several experimental applications, with variations based on the specific antibody:

• Western Blotting (WB): All examined ZNF264 antibodies have been validated for WB applications, making this the most universal application .

• Immunohistochemistry (IHC): Several polyclonal antibodies are suitable for IHC, including paraffin-embedded tissues .

• ELISA: Some antibodies are specifically recommended for ELISA applications .

• Immunofluorescence (IF): Select antibodies may be suitable for immunofluorescence studies, though this application appears less common .

When selecting an antibody, researchers should consider which applications are critical for their experimental design and choose accordingly. For comprehensive studies involving multiple techniques, selecting an antibody validated across all required applications is advisable .

How can Western blot protocols be optimized when using ZNF264 antibodies?

Optimizing Western blot protocols for ZNF264 detection requires attention to several factors:

• Antibody dilution: Recommended dilutions vary by manufacturer:

  • Mouse monoclonal antibodies: 1:500-2000

  • Rabbit polyclonal antibodies: 1:1000-5000 for WB , 0.2-1 μg/mL

• Sample preparation:

  • Human cell lysates, particularly B cell lysates (e.g., 721_B), have been successfully used as positive controls

  • Denaturation conditions should be optimized as protein conformation affects epitope accessibility

• Detection system selection:

  • For enhanced sensitivity, consider HRP-conjugated antibodies like the OTI2A6 clone

  • Unconjugated antibodies require appropriate secondary antibodies matched to the host species (mouse or rabbit)

• Blocking conditions:

  • Standard blocking agents (5% non-fat milk or BSA) are typically sufficient

  • For phospho-specific applications, BSA is preferred over milk proteins

• Troubleshooting considerations:

  • Expected band size is approximately 70.4 kDa

  • Non-specific binding can be reduced through optimization of antibody concentration and washing steps

For consistent results, researchers should perform preliminary dilution series experiments to determine optimal antibody concentration for their specific sample type and detection system .

What tissue types and experimental models are recommended for studying ZNF264 expression?

Based on available data, researchers investigating ZNF264 should consider the following experimental models:

• Human cell lines:

  • B cell lines (721_B) have been used successfully as positive controls in Western blot applications

  • Human lung tissue, particularly alveolar cells, has shown detectable expression in IHC applications

• Species considerations:

  • Most antibodies show primary reactivity with human ZNF264

  • Select antibodies demonstrate cross-reactivity with rodent models (mouse, rat) and other species (cow, dog, horse, monkey)

  • When working with non-human models, antibodies with confirmed cross-reactivity should be selected

• Expression systems:

  • Recombinant ZNF264 expressed in E. coli has been used as an immunogen for antibody development

  • Synthetic peptide fragments (e.g., AA 1-220) have also been used to generate specific antibodies

For comparative studies across species, researchers should validate antibody cross-reactivity in their specific experimental system, as sequence homology varies (85-100% identity has been reported across different species) .

How do epitope-specific ZNF264 antibodies differ in research applications?

ZNF264 antibodies targeting different epitopes provide complementary research capabilities:

• N-Terminal antibodies (AA 1-220):

  • Useful for detecting full-length protein and N-terminal fragments

  • May recognize isoforms sharing the N-terminal domain

  • Show cross-reactivity with multiple species (human, rat, cow, mouse, dog)

  • Recommended for WB and IHC applications

• C-Terminal antibodies:

  • Specific for detecting full-length protein and C-terminal fragments

  • May recognize different isoforms than N-terminal antibodies

  • Show cross-reactivity with human, horse, rabbit, and monkey samples

  • Primarily validated for WB applications

• Full-length protein antibodies:

  • Typically offer broader epitope recognition

  • The OTI2A6 monoclonal clone was raised against full-length human recombinant ZNF264

  • May provide more specific detection of the intact protein

For comprehensive studies, using antibodies targeting different regions can provide validation of results and potentially reveal the presence of protein isoforms or processing products. This approach is particularly valuable when investigating novel aspects of ZNF264 biology or when working with experimental systems where protein processing might occur .

What are the critical differences between monoclonal and polyclonal ZNF264 antibodies?

The choice between monoclonal and polyclonal ZNF264 antibodies involves important research considerations:

The mouse monoclonal OTI2A6 clone offers high specificity and consistency, making it ideal for quantitative experiments and longitudinal studies requiring consistent antibody performance . In contrast, rabbit polyclonal antibodies provide signal amplification through recognition of multiple epitopes, potentially increasing detection sensitivity, especially in samples with low ZNF264 expression .

For critical experiments, researchers should consider validating findings with both antibody types to ensure robust results. Additionally, the availability of conjugated versions (HRP, biotin) of the monoclonal antibody provides flexibility for direct detection methods .

How can researchers distinguish between ZNF264 and other related zinc finger proteins?

Distinguishing ZNF264 from related zinc finger proteins requires careful experimental design:

• Antibody selection considerations:

  • Choose antibodies raised against unique regions of ZNF264 that lack homology with other zinc finger proteins

  • C-terminal regions often show greater sequence divergence than the conserved zinc finger domains

  • Monoclonal antibodies like OTI2A6 may offer enhanced specificity

• Validation approaches:

  • Western blot analysis should show a band at the predicted molecular weight of 70.4 kDa

  • Preabsorption with the immunizing peptide should eliminate specific signals

  • If available, knockout or knockdown models provide definitive controls

  • For polyclonal antibodies, affinity purification against the target peptide improves specificity

• Experimental controls:

  • Include positive controls from tissues/cells known to express ZNF264

  • Consider including samples expressing related zinc finger proteins to assess cross-reactivity

  • Use blocking peptides (when available) to confirm antibody specificity

• Complementary techniques:

  • Combine antibody-based detection with mRNA analysis (qPCR, RNA-seq)

  • Mass spectrometry can provide definitive protein identification

  • DNA binding assays may help distinguish functionally similar zinc finger proteins

Using antibodies targeting different epitopes of ZNF264 in parallel experiments can provide additional validation by confirming consistent detection patterns .

Why might Western blots with ZNF264 antibodies show unexpected bands or patterns?

Unexpected bands or patterns in Western blots using ZNF264 antibodies can result from several factors:

• Multiple protein isoforms:

  • Alternative splicing may generate multiple ZNF264 isoforms

  • Different epitope-targeting antibodies may detect distinct subsets of isoforms

• Post-translational modifications:

  • Phosphorylation, ubiquitination, or SUMOylation can alter protein mobility

  • Modified forms may appear as higher molecular weight bands

• Proteolytic processing:

  • Sample preparation conditions may affect protein integrity

  • Endogenous proteases can generate fragments with altered mobility

• Technical considerations:

  • Insufficient blocking or antibody concentration issues may cause non-specific binding

  • Secondary antibody cross-reactivity can generate unexpected signals

  • Sample overloading can lead to distorted band patterns

• Experimental approaches to resolve issues:

  • Titrate antibody concentration (recommended ranges: 1:500-2000 for monoclonal , 1:1000-5000 for polyclonal )

  • Include positive control samples (e.g., 721_B cell lysate)

  • Optimize blocking and washing conditions

  • Compare results using antibodies targeting different epitopes

  • Consider denaturing vs. non-denaturing conditions

The expected molecular weight of ZNF264 is 70.4 kDa , but modifications or processing may generate additional bands that require careful validation to interpret correctly.

What are the recommended reconstitution and dilution protocols for lyophilized ZNF264 antibodies?

Proper reconstitution of lyophilized ZNF264 antibodies is essential for optimal performance:

• Initial reconstitution:

  • For the OTI2A6 monoclonal antibody, add 100 μL distilled water to achieve approximately 1 mg/mL concentration

  • Allow complete dissolution at room temperature with gentle agitation (avoid vortexing)

  • For conjugation experiments, an additional desalting step is recommended (e.g., using Zeba Spin Desalting Columns, 7KMWCO)

• Working dilution preparation:

  • For Western blotting:

    • Monoclonal antibodies: 1:500-2000 dilution

    • Polyclonal antibodies: 1:1000-5000 or 0.2-1 μg/mL

  • For immunohistochemistry:

    • Polyclonal antibodies: 1:20-200 dilution is recommended

• Buffer considerations:

  • Original buffer composition for lyophilized antibodies typically includes 1X PBS, pH 7.3, with 8% trehalose

  • For long-term storage, some manufacturers recommend adding glycerol to 50% final concentration

• Storage after reconstitution:

  • Prepare small aliquots to avoid repeated freeze-thaw cycles

  • Store at -20°C for long-term storage

  • Working dilutions can be kept at 4°C for short periods (typically 1-2 weeks)

Following these protocols ensures optimal antibody performance and extends the usable lifetime of the reagent .

How can researchers validate the specificity of ZNF264 antibodies in their experimental system?

Validating ZNF264 antibody specificity is a critical step for ensuring reliable experimental results:

• Positive and negative controls:

  • Positive controls: 721_B cell lysate has been validated for Western blot ; human lung (alveolar cells) for IHC

  • Negative controls: Cell lines with low/no ZNF264 expression or tissues where expression is not expected

  • Genetic controls: If available, ZNF264 knockout or knockdown models provide definitive validation

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide

  • Specific signals should be significantly reduced or eliminated

  • Non-specific binding will persist even with peptide competition

• Multi-antibody validation:

  • Compare results using antibodies targeting different epitopes (N-terminal vs. C-terminal)

  • Consistent detection patterns across different antibodies increase confidence in specificity

• Orthogonal techniques:

  • Correlate protein detection with mRNA expression (qPCR, RNA-seq)

  • Mass spectrometry identification of immunoprecipitated proteins

  • Immunofluorescence localization should be consistent with expected nuclear localization

• Titration experiments:

  • Perform antibody dilution series to identify optimal concentration

  • Specific signals should decrease proportionally with dilution

  • Non-specific background may not show the same proportional reduction

These validation approaches should be documented as part of quality control measures for publications and ensure that observed results genuinely reflect ZNF264 biology .

How can ZNF264 antibodies be used to investigate transcriptional regulatory mechanisms?

ZNF264 antibodies can facilitate multiple approaches to study transcriptional regulation:

• Chromatin Immunoprecipitation (ChIP):

  • ZNF264 antibodies can precipitate DNA-protein complexes to identify genomic binding sites

  • Both monoclonal and polyclonal antibodies may be suitable, though validation for ChIP applications is essential

  • Results can be analyzed by qPCR, sequencing (ChIP-seq), or microarray (ChIP-chip)

• Co-Immunoprecipitation (Co-IP):

  • Identify protein interaction partners within transcriptional complexes

  • May reveal associations with co-activators, co-repressors, or chromatin modifiers

  • Both N-terminal and C-terminal antibodies can provide complementary information

• Immunofluorescence microscopy:

  • Examine nuclear localization and potential co-localization with transcription factories

  • Assess changes in localization during different cell cycle phases or in response to stimuli

  • Polyclonal antibodies validated for immunocytochemistry applications are preferred

• Functional assays:

  • Combine with reporter gene assays to assess transcriptional activity

  • Use in combination with gene knockdown/knockout approaches to validate targets

  • Employ to confirm ZNF264 recruitment to specific promoters

As a member of the krueppel C2H2-type zinc-finger protein family that regulates transcription , ZNF264 likely binds specific DNA sequences through its zinc finger domains. Research using these antibodies can help elucidate its role in normal cellular processes and potential dysregulation in disease states.

What methodological considerations are important when using ZNF264 antibodies for immunohistochemistry?

For optimal immunohistochemistry results with ZNF264 antibodies, researchers should consider:

• Tissue preparation and fixation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues are compatible with validated antibodies

  • Fixation time should be optimized to preserve epitope accessibility

  • Antigen retrieval methods may be necessary to expose epitopes masked by fixation

• Antibody selection and dilution:

  • Polyclonal antibodies are generally preferred for IHC applications

  • Recommended dilution ranges from 1:20-1:200 for optimal staining

  • Preliminary titration experiments should determine optimal concentration for specific tissues

• Detection systems:

  • DAB (3,3'-diaminobenzidine) or AEC (3-amino-9-ethylcarbazole) chromogens are commonly used

  • Amplification systems may enhance sensitivity for low-abundance expression

  • Fluorescent secondary antibodies enable co-localization studies with other markers

• Controls and validation:

  • Positive control: Human lung tissue (alveolar cells) has shown detectable expression

  • Negative controls: Primary antibody omission and tissues not expressing ZNF264

  • Compare staining patterns between antibodies targeting different epitopes

• Result interpretation:

  • ZNF264 is expected to show predominantly nuclear localization

  • Evaluate staining intensity, pattern, and percentage of positive cells

  • Consider quantitative image analysis for comparative studies

Careful optimization of these parameters will ensure reliable detection of ZNF264 in tissue sections, providing valuable insights into its expression patterns in normal and pathological contexts .

What emerging research applications might benefit from ZNF264 antibodies?

ZNF264 antibodies could support several emerging research directions:

• Single-cell analysis:

  • Imaging mass cytometry or CyTOF with ZNF264 antibodies could reveal expression heterogeneity at single-cell resolution

  • Combined with other markers, this approach could identify specific cell populations expressing ZNF264

• Spatial transcriptomics integration:

  • Correlating ZNF264 protein localization with spatial gene expression patterns

  • May reveal regional specialization of transcriptional regulation in tissues

• 3D organoid and tissue models:

  • Investigating ZNF264 expression and function in more physiologically relevant models

  • Comparing expression patterns between 2D cultures and 3D organoids

• Super-resolution microscopy:

  • Nanoscale localization within nuclear subcompartments

  • Co-localization with chromatin marks or transcriptional machinery

• Therapeutic target validation:

  • Evaluating ZNF264 as a potential biomarker in various conditions

  • Monitoring changes in expression or localization in response to therapeutic interventions

• Developmental biology:

  • Tracking expression during differentiation and development

  • Understanding temporal regulation of ZNF264 activity

As transcription factors like ZNF264 often serve as master regulators of gene expression networks , these emerging approaches could provide deeper insights into its biological functions and potential relevance to human disease.