ZNF221 Antibody

What is ZNF221 and what is its primary cellular function?

ZNF221 (Zinc Finger Protein 221) is a transcription factor that belongs to the C2H2-type zinc finger protein family. It plays a crucial role in gene regulation through DNA binding and transcriptional control activities. ZNF221 is primarily involved in controlling gene expression and cell growth, serving as a key player in various biological processes including development, differentiation, and potentially tumorigenesis . The protein contains multiple zinc finger domains that facilitate its interaction with specific DNA sequences to regulate target gene expression. As a transcriptional regulator, ZNF221 may contribute to both activation and repression of gene expression depending on cellular context and interacting partners .

How is ZNF221 protein distributed within cellular compartments?

Immunofluorescence studies using ZNF221 antibodies have demonstrated that ZNF221 localizes predominantly to nuclear bodies within cells . This nuclear localization is consistent with its function as a transcription factor. Specifically, immunofluorescent staining in the human cell line RH-30 shows clear nuclear body localization pattern . This subcellular distribution supports ZNF221's role in transcriptional regulation, as nuclear bodies are often sites of transcriptional activity and RNA processing. The protein appears to concentrate in discrete nuclear domains rather than showing diffuse nuclear distribution, suggesting it may participate in specific transcriptional complexes at defined genomic loci.

What is currently known about ZNF221's involvement in pathological conditions?

Current research suggests ZNF221 may have implications in cancer biology and other disease contexts . As a transcription factor that regulates cell growth and differentiation, dysregulation of ZNF221 could potentially contribute to tumorigenesis through aberrant control of cell proliferation pathways. Researchers are investigating its expression patterns across various cancers and normal tissues to better understand its potential role in disease progression. Immunohistochemical studies using ZNF221 antibodies have been conducted on both normal human tissues and common cancer types to map its distribution and expression levels . These investigations aim to elucidate whether ZNF221 might serve as a biomarker or therapeutic target in specific pathological conditions.

What types of ZNF221 antibodies are available for research applications?

Several types of ZNF221 antibodies are available for research applications, including:

• Polyclonal antibodies: Rabbit-derived polyclonal antibodies are the most common type available for ZNF221 research . These recognize multiple epitopes on the ZNF221 protein.

• Conjugated antibodies: Various conjugated forms including:

  • FITC-conjugated ZNF221 antibodies for fluorescence applications

  • HRP-conjugated antibodies for enzymatic detection methods

  • Biotin-conjugated antibodies for amplification strategies

• Unconjugated primary antibodies: These are versatile options that can be paired with different secondary detection systems based on experimental needs .

The choice between these antibody types depends on the specific experimental application, with polyclonal antibodies offering broader epitope recognition while conjugated antibodies provide direct detection capabilities without secondary antibodies.

What epitopes do commercially available ZNF221 antibodies target?

Commercial ZNF221 antibodies target different regions of the protein:

• N-terminal region antibodies: Some antibodies specifically target the immunogen sequence "MISPSLELLHSGLCKFPEVEGKMTTFKEA" located in the N-terminal portion of ZNF221 . This region is distinct from the zinc finger domains and may be more accessible for antibody binding.

• Amino acids 1-300 antibodies: Several polyclonal antibodies are raised against recombinant human ZNF221 protein spanning amino acids 1-300 . This larger region encompasses multiple functional domains of the protein.

• Specific peptide region (AA 108-157): Some antibodies target this narrower middle region of the protein for more specific detection .

The epitope selection impacts specificity, with N-terminal antibodies potentially offering different detection characteristics compared to those targeting other regions of the protein. Researchers should select antibodies targeting epitopes relevant to their specific experimental questions.

What is the species reactivity profile of available ZNF221 antibodies?

The currently available ZNF221 antibodies demonstrate the following reactivity profile:

Most commercial ZNF221 antibodies have been specifically validated for human samples . The sequence homology between human ZNF221 and its orthologs in mouse (41%) and rat (34%) suggests potential cross-reactivity, though this would need experimental validation . The antibodies have been extensively tested on human tissues and cell lines, including normal human tissues (spleen, kidney) and various cancer tissues . Researchers working with non-human models should conduct preliminary validation tests to confirm cross-reactivity before proceeding with full experiments.

What applications have ZNF221 antibodies been validated for?

ZNF221 antibodies have been validated for several experimental applications:

• Immunohistochemistry (IHC): Extensively validated for paraffin-embedded tissue sections, with documented results in human spleen and kidney tissues . This application allows visualization of ZNF221 distribution within tissue architecture.

• Enzyme-Linked Immunosorbent Assay (ELISA): Several antibodies have been validated for ELISA applications, allowing quantitative measurement of ZNF221 levels in biological samples .

• Immunofluorescence (IF): Validated for cellular localization studies, particularly demonstrating nuclear body localization in human cell lines like RH-30 . This technique provides high-resolution information about subcellular distribution.

• Western Blotting (WB): Some antibody variants have been validated for protein detection via western blot, allowing size verification and semi-quantitative analysis .

The extensive validation across multiple techniques makes these antibodies versatile tools for both localization and quantitative studies of ZNF221 expression.

What are the optimal working dilutions for different ZNF221 antibody applications?

The recommended working dilutions for ZNF221 antibodies vary by application:

These recommended dilutions should serve as starting points for optimization. Researchers should perform titration experiments to determine the optimal concentration for their specific experimental conditions, sample types, and detection methods. Factors that may influence optimal dilution include tissue fixation method, antigen retrieval protocol, detection system sensitivity, and endogenous expression levels of ZNF221 in the target sample.

What are the proper storage and handling requirements for maintaining ZNF221 antibody activity?

To maintain optimal activity of ZNF221 antibodies, the following storage and handling guidelines should be followed:

• Storage temperature: Store at -20°C to -80°C for long-term preservation . Some products may be stored at 4°C for short periods if frequently used.

• Storage buffer: Most ZNF221 antibodies are supplied in a stabilizing buffer containing:

  • 50% Glycerol

  • 0.01M PBS, pH 7.4

  • 0.03% Proclin 300 as preservative

• Aliquoting: For antibodies that will be used multiple times, prepare small aliquots to minimize freeze-thaw cycles.

• Freeze-thaw considerations: Avoid repeated freeze-thaw cycles as they can decrease antibody activity and specificity . Upon receipt, prepare working aliquots if needed.

• Safety precautions: Note that some preservatives like Proclin 300 are hazardous substances that should be handled by trained personnel with appropriate safety measures .

• Transport considerations: Antibodies are typically shipped on wet ice and should be stored immediately upon receipt at the recommended temperature .

Proper storage and handling are essential for maintaining antibody performance and extending shelf life. Documentation of lot numbers, receipt dates, and any observed performance changes is recommended as part of good laboratory practice.

How can ZNF221 antibodies be optimized for challenging tissue types or detection methods?

Optimizing ZNF221 antibody performance for challenging samples requires systematic approach:

• Antigen retrieval optimization:

  • For formalin-fixed tissues, test multiple retrieval methods (heat-induced vs. enzymatic)

  • Optimize pH conditions (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Adjust retrieval duration based on fixation time of samples

• Signal amplification strategies:

  • For low-expressing samples, implement tyramide signal amplification (TSA)

  • Consider biotin-conjugated secondary antibodies with streptavidin-HRP systems

  • Use polymer-based detection systems for increased sensitivity

• Background reduction:

  • Implement prolonged blocking (3-5% BSA or serum from secondary antibody host species)

  • Include detergents (0.1-0.3% Triton X-100) for membrane permeabilization

  • Consider tissue-specific autofluorescence quenching protocols for IF applications

• Validation approaches:

  • Always run parallel negative controls (isotype control and secondary-only)

  • Include positive control tissues known to express ZNF221

  • Consider dual-labeling with markers of nuclear bodies to confirm localization pattern

For particularly challenging applications, researchers might need to test multiple antibody clones targeting different epitopes of ZNF221 to identify the optimal reagent for their specific sample type and detection method.

What experimental controls are essential when working with ZNF221 antibodies?

A robust control strategy is crucial for reliable ZNF221 antibody experiments:

• Negative controls:

  • Isotype control: Use non-specific IgG from the same species and at the same concentration as the ZNF221 antibody

  • Secondary-only control: Omit primary antibody but include all other reagents

  • Peptide competition: Pre-incubate antibody with immunizing peptide to demonstrate specificity

• Positive controls:

  • Known positive tissues: Human spleen and kidney tissues have been validated for ZNF221 expression

  • Cell lines with confirmed ZNF221 expression: RH-30 has demonstrated nuclear body staining

  • Recombinant ZNF221 protein: Can be used as a western blot or ELISA standard

• Expression validation controls:

  • siRNA/shRNA knockdown of ZNF221 to confirm signal specificity

  • Parallel detection with alternative methods (e.g., RT-PCR for mRNA expression)

  • Use of multiple antibodies targeting different ZNF221 epitopes

• Technical controls:

  • Antibody titration to determine optimal concentration

  • Inclusion of internal staining controls (e.g., nuclear counterstain)

  • Processing control samples alongside experimental samples in each batch

Implementing these controls helps distinguish specific ZNF221 signal from background or non-specific binding, increasing confidence in experimental results and interpretations.

How does post-translational modification status affect ZNF221 antibody detection?

The impact of post-translational modifications (PTMs) on ZNF221 antibody detection is an important consideration:

• Potential ZNF221 modifications:

  • Phosphorylation: Like many transcription factors, ZNF221 may undergo phosphorylation that could alter antibody epitope accessibility

  • SUMOylation: Common in zinc finger proteins and may affect protein localization and antibody binding

  • Ubiquitination: May influence protein stability and detection in certain applications

• Epitope considerations:

  • Antibodies targeting amino acids 1-300 or the N-terminal region may have different sensitivities to PTMs

  • Modifications near the epitope may block antibody binding or alter recognition efficiency

  • Modification-specific antibodies might be required to study specific ZNF221 isoforms

• Experimental approaches:

  • Phosphatase treatment: Pre-treating samples to remove phosphorylation if this modification affects detection

  • Denaturing conditions: May expose epitopes hidden by conformational changes induced by PTMs

  • Nuclear extraction protocols: May need optimization to retain differently modified forms of ZNF221

• Validation strategies:

  • Compare detection in different sample preparation methods

  • Use mass spectrometry to identify modification sites on ZNF221

  • Correlate antibody detection with specific cellular states known to modify zinc finger proteins

Researchers should be aware that most commercial ZNF221 antibodies detect the unmodified form of the protein , and special considerations may be needed to study specific modified variants.

How should researchers interpret varying ZNF221 expression patterns across different tissue types?

When analyzing ZNF221 expression across different tissues, consider these interpretation guidelines:

• Expression pattern evaluation:

  • Nuclear localization: ZNF221 normally shows nuclear localization, particularly in nuclear bodies . Aberrant cytoplasmic localization may indicate pathological conditions or technical artifacts.

  • Expression intensity: Quantify staining intensity using standardized scoring methods (H-score, Allred score) for comparison across tissues.

  • Distribution within tissues: Note cell type-specific expression patterns, as ZNF221 may show differential expression across cell types within the same tissue.

• Comparative analysis framework:

  • Use validated positive controls like spleen and kidney tissues as reference points for expression comparison.

  • Consider tissue-specific nuclear morphology differences when evaluating nuclear body localization.

  • Account for tissue-specific background levels when interpreting signal-to-noise ratios.

• Technical considerations for cross-tissue comparison:

  • Standardize fixation times and processing methods across tissue types.

  • Use identical antibody concentrations and detection protocols for valid comparisons.

  • Consider tissue-specific antigen retrieval optimization if necessary.

• Biological interpretation:

  • Correlate ZNF221 expression with tissue-specific transcriptional programs.

  • Consider developmental stage and physiological state when interpreting expression differences.

  • Analyze co-expression with known interacting partners or downstream targets.

Systematic documentation of staining patterns with standardized protocols enables reliable cross-tissue comparisons and identification of biologically significant expression patterns versus technical variations.

What are common causes of false positive and false negative results when using ZNF221 antibodies?

Understanding potential sources of error is critical for accurate interpretation:

To distinguish true results from artifacts, researchers should implement comprehensive control strategies as outlined in section 4.2 and validate findings with complementary detection methods when possible.

How can researchers address contradictory results when using different ZNF221 antibody clones?

When faced with contradictory results from different ZNF221 antibodies, implement this systematic approach:

By systematically addressing discrepancies, researchers can often reconcile contradictory results or identify important biological variables affecting ZNF221 detection.

How can ZNF221 antibodies be utilized in ChIP experiments to study transcriptional targets?

Chromatin Immunoprecipitation (ChIP) using ZNF221 antibodies allows identification of direct transcriptional targets:

• Antibody selection considerations:

  • Choose antibodies specifically validated for immunoprecipitation applications

  • Ensure the targeted epitope is accessible in chromatin-bound ZNF221

  • Consider antibodies targeting regions outside the DNA-binding zinc finger domains to avoid interference with DNA binding

• Protocol optimization:

  • Cross-linking conditions: Typically 1% formaldehyde for 10 minutes at room temperature

  • Sonication parameters: Optimize to yield 200-500bp DNA fragments

  • Antibody concentration: Typically 2-5μg per ChIP reaction, requiring titration

  • Washing stringency: Balance between reducing background and maintaining specific interactions

• Controls framework:

  • Input chromatin: Essential reference for enrichment calculation

  • IgG control: Same species as ZNF221 antibody to establish background

  • Positive control regions: Known or predicted ZNF221 binding sites

  • Negative control regions: Genomic regions unlikely to bind ZNF221

• Data analysis approach:

  • Peak calling algorithms appropriate for transcription factor ChIP

  • Motif analysis to identify ZNF221 binding sequences

  • Integration with expression data to correlate binding with transcriptional effects

• Validation strategies:

  • Confirm selected targets by ChIP-qPCR

  • Perform reporter assays to validate functional significance

  • Manipulate ZNF221 levels to observe effects on target gene expression

While specific ChIP protocols for ZNF221 are not detailed in the provided sources, these guidelines align with standard approaches for transcription factor ChIP experiments.

What methodologies combine ZNF221 antibodies with other detection systems for multi-parameter analysis?

Advanced multi-parameter approaches enable contextual understanding of ZNF221 function:

• Multiplexed immunofluorescence techniques:

  • Sequential immunostaining: Apply, image, and strip/quench ZNF221 antibody before applying next antibody

  • Spectral unmixing: Use spectrally distinct fluorophores for simultaneous detection of ZNF221 and other proteins

  • Antibody conjugates: Utilize available FITC-conjugated ZNF221 antibodies in combination with other distinctly labeled antibodies

• Flow cytometry applications:

  • Intracellular staining protocols: Required for nuclear ZNF221 detection

  • Multicolor panels: Combine ZNF221 staining with cell type markers and functional readouts

  • Phospho-flow: Correlate ZNF221 expression with signaling pathway activation

• Proximity-based detection systems:

  • Proximity Ligation Assay (PLA): Detect protein-protein interactions involving ZNF221

  • FRET/BRET: Study dynamic interactions between ZNF221 and binding partners

  • Co-immunoprecipitation: Identify protein complexes containing ZNF221

• Spatial transcriptomics integration:

  • Combine ZNF221 immunodetection with in situ hybridization for target genes

  • Correlate ZNF221 protein localization with local transcriptional activity

  • Implement digital spatial profiling technologies for comprehensive analysis

• Live-cell imaging approaches:

  • Fluorescently tagged ZNF221 constructs to complement antibody staining

  • Photobleaching techniques (FRAP/FLIP) to study ZNF221 dynamics

  • Single-molecule tracking to analyze ZNF221 binding kinetics

These advanced methodologies extend beyond basic detection to provide mechanistic insights into ZNF221 function in complex biological contexts.

How can researchers quantitatively assess ZNF221 expression levels across experimental conditions?

Robust quantitative analysis of ZNF221 requires standardized approaches:

• Western blot quantification:

  • Densitometric analysis normalized to loading controls

  • Standard curves using recombinant ZNF221 protein

  • Digital imaging systems with extended linear dynamic range

• ELISA-based quantification:

  • Sandwich ELISA development using available antibodies

  • Standard curve generation with recombinant protein

  • Validation across sample types (cell lysates, tissue extracts)

• Immunohistochemistry quantification:

  • Digital pathology approaches:

    • H-score calculation (staining intensity × percentage positive cells)

    • Automated image analysis with AI-assisted recognition

  • Controls for staining batch effects (reference samples)

  • Statistical methods for analyzing distribution patterns

• Flow cytometry analysis:

  • Mean/median fluorescence intensity measurements

  • Population frequency determination (% ZNF221-positive cells)

  • Standardization with calibration beads

• qRT-PCR correlation:

  • Parallel analysis of ZNF221 mRNA and protein

  • Calculation of protein/mRNA ratios to assess post-transcriptional regulation

  • Time-course analysis to determine expression dynamics

• Normalization considerations:

  • Selection of appropriate housekeeping proteins

  • Tissue-specific reference standards

  • Absolute quantification using recombinant protein standards