ZNF555 Antibody

What is the molecular basis for Anti-ZNF555 antibody specificity?

The Anti-ZNF555 antibody produced by Sigma-Aldrich demonstrates high specificity through its development against the unique immunogen sequence KQYKCNVGHPPANEFMCSASEKSHQERDLIKVVNMVLP, which represents a distinctive epitope region of the human ZNF555 protein . This specificity is achieved through affinity isolation techniques that select antibodies with optimal binding kinetics to the target sequence. The polyclonal nature of this rabbit-derived antibody provides recognition of multiple epitopes within the target protein, enhancing detection sensitivity while maintaining specificity across various experimental platforms . The extensive validation through the Human Protein Atlas project further confirms the antibody's selective binding to ZNF555 protein targets across diverse human tissue samples.

How does the ZNF555 antibody compare to antibodies targeting other zinc finger proteins?

The Anti-ZNF555 antibody is specifically engineered to recognize unique epitope sequences not shared with other zinc finger family members, distinguishing it from antibodies targeting related proteins such as ZNF155 . While both ZNF555 and other zinc finger proteins contain characteristic C2H2-type zinc finger motifs, the Anti-ZNF555 antibody's immunogen sequence targets regions outside these conserved domains to minimize cross-reactivity. Comparative analysis between Anti-ZNF555 and other zinc finger antibodies (such as PCRP-ZNF155-1A5) reveals distinct differences in host species generation (rabbit versus mouse), clonality (polyclonal versus monoclonal), and recommended applications (immunohistochemistry versus immunoprecipitation and microarray) . These differences significantly impact experimental design considerations when studying specific zinc finger proteins in complex biological systems.

What post-translational modifications of ZNF555 can be detected with current antibodies?

The existing Anti-ZNF555 antibody from Sigma-Aldrich is specifically designed to target the unmodified form of the protein, as indicated in its technical specifications . This antibody does not have confirmed reactivity against phosphorylated, acetylated, methylated, or otherwise modified variants of ZNF555. Researchers investigating potential post-translational regulation of ZNF555 should note this limitation when designing experiments focused on protein modifications. The detection of modified ZNF555 would require specialized antibodies specifically raised against the modified epitopes of interest. Given the importance of post-translational modifications in regulating zinc finger protein function, including nuclear localization, DNA binding affinity, and protein-protein interactions, this represents a significant gap in current research tools available for comprehensive ZNF555 characterization.

What are the optimal immunohistochemistry protocols for ZNF555 antibody in formalin-fixed, paraffin-embedded tissues?

For optimal immunohistochemical detection of ZNF555 in FFPE tissues, researchers should implement a heat-induced epitope retrieval (HIER) protocol using citrate buffer (pH 6.0) followed by antibody incubation at dilutions of 1:200-1:500 as recommended by the manufacturer . The protocol should include:

• Deparaffinization and rehydration through xylene and graded alcohols

• Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

• Peroxidase blocking with 3% H₂O₂ for 10 minutes

• Protein blocking with 5% normal serum for 30 minutes

• Primary antibody incubation with Anti-ZNF555 (1:200-1:500) overnight at 4°C

• Secondary antibody incubation with HRP-conjugate for 30 minutes

• DAB chromogen development with 3-5 minute monitoring

• Counterstaining with hematoxylin

This protocol has been validated through the Human Protein Atlas project across multiple tissue types, with attention to both nuclear and cytoplasmic staining patterns that reflect ZNF555's functional localization . Modifications may be required for specific tissue types with high autofluorescence or endogenous peroxidase activity.

How can researchers optimize ZNF555 antibody conditions for co-immunoprecipitation experiments?

While the Anti-ZNF555 antibody from Sigma-Aldrich is primarily validated for immunohistochemistry applications, researchers can adapt it for co-immunoprecipitation studies with the following optimizations :

• Lysis Buffer Selection: Use a mild non-denaturing lysis buffer (150mM NaCl, 50mM Tris-HCl pH 7.5, 1% NP-40, 0.5% sodium deoxycholate) supplemented with protease inhibitors to preserve protein-protein interactions.

• Antibody Concentration: Increase antibody concentration to 5-10μg per immunoprecipitation reaction.

• Pre-clearing Step: Pre-clear lysates with Protein A/G beads for 1 hour at 4°C to reduce non-specific binding.

• Extended Incubation: Extend antibody-lysate incubation to overnight at 4°C with gentle rotation.

• Cross-linking Consideration: For transient interactions, consider using a membrane-permeable crosslinker prior to lysis.

These modifications address the polyclonal nature of the antibody while maximizing the capture of ZNF555 and its interacting partners. Researchers should validate each step through appropriate controls, including IgG control immunoprecipitations and input sample comparisons to evaluate pull-down efficiency and specificity.

What are the considerations for multiplexing ZNF555 antibody with other markers in fluorescence microscopy?

When designing multiplexed immunofluorescence experiments incorporating ZNF555 detection, researchers should consider several technical factors:

Additionally, researchers should perform careful titration experiments to determine optimal antibody concentrations for each marker in the multiplexed panel, as antibody performance can be affected by the presence of other antibodies and detection reagents in complex immunofluorescence protocols.

How can ZNF555 antibody be utilized in ChIP-seq experiments to identify genome-wide binding sites?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) with ZNF555 antibody requires careful optimization beyond standard protocols due to the specific characteristics of zinc finger transcription factors. While the Anti-ZNF555 antibody is not explicitly validated for ChIP applications, researchers can develop an effective protocol with these modifications :

• Cross-linking Protocol: Implement dual cross-linking with 1.5mM EGS (ethylene glycol bis(succinimidyl succinate)) for 30 minutes followed by 1% formaldehyde for 10 minutes to better preserve protein-DNA interactions typical of zinc finger proteins.

• Sonication Parameters: Optimize sonication conditions to achieve chromatin fragments between 200-300bp, smaller than typical ChIP-seq protocols, to improve resolution of binding sites.

• Antibody Quantity: Increase antibody amount to 10μg per reaction to compensate for potentially lower affinity in chromatin context.

• Negative Controls: Include both input DNA and IgG immunoprecipitation controls, along with chromatin from cells with ZNF555 knockdown when possible.

• Bioinformatic Analysis: Employ motif discovery algorithms specific for C2H2 zinc finger binding patterns when analyzing peak data.

This approach accommodates the technical challenges of ChIP-seq with transcription factors that may have transient DNA interactions or function within large multi-protein complexes. Researchers should validate ChIP-seq findings with orthogonal methods such as EMSA or reporter assays to confirm the functional relevance of identified binding sites.

What approaches can resolve contradictory results when using ZNF555 antibody across different experimental systems?

When researchers encounter discrepancies in ZNF555 detection across different experimental platforms or biological systems, a systematic troubleshooting approach is essential:

• Epitope Accessibility Analysis: Different fixation or preparation methods may differentially affect the exposure of the immunogen sequence (KQYKCNVGHPPANEFMCSASEKSHQERDLIKVVNMVLP), particularly in techniques that involve protein denaturation versus native conditions .

• Isoform-Specific Expression: Verify whether different experimental systems express varying ZNF555 isoforms that might lack or modify the epitope region targeted by the antibody.

• Cross-Validation Strategy: Implement orthogonal detection methods including:

  • RT-qPCR for mRNA expression correlation

  • Tagged recombinant expression for antibody validation

  • Alternative antibodies targeting different epitopes when available

• Cellular Context Considerations: Evaluate whether post-translational modifications, protein-protein interactions, or subcellular compartmentalization might mask epitopes in specific experimental systems.

• Quantitative Calibration: Develop standard curves using recombinant ZNF555 protein to establish detection thresholds across different platforms.

This comprehensive approach acknowledges that antibody performance can vary substantially based on experimental conditions, and that biological differences in ZNF555 presentation may underlie seemingly contradictory results.

How can researchers distinguish between ZNF555 and other closely related zinc finger proteins in complex samples?

Discriminating ZNF555 from other zinc finger family members presents significant challenges due to sequence homology. Researchers can implement a multi-faceted approach to ensure specificity:

• Peptide Competition Assays: Pre-incubate Anti-ZNF555 antibody with excess immunizing peptide (KQYKCNVGHPPANEFMCSASEKSHQERDLIKVVNMVLP) to confirm signal specificity .

• Knockout Validation: Utilize CRISPR/Cas9-mediated ZNF555 knockout samples as negative controls.

• Mass Spectrometry Verification: Follow immunoprecipitation with tryptic digestion and mass spectrometry to confirm the identity of captured proteins through unique peptide signatures.

• Parallel Detection Strategy: Design experiments incorporating both:

  • The polyclonal Anti-ZNF555 antibody for higher sensitivity

  • Monoclonal antibodies against unique ZNF555 regions for higher specificity when available

• Bioinformatic Sequence Analysis: Prior to experimental design, conduct in silico analysis of potential cross-reactivity with other zinc finger proteins based on epitope sequence alignment.

This integrated approach provides multiple layers of validation, particularly important when studying zinc finger proteins that share conserved zinc finger domains but differ in their specific biological functions and interaction partners.

What is the significance of nuclear versus cytoplasmic ZNF555 localization in experimental interpretation?

The subcellular localization pattern of ZNF555 has profound implications for functional interpretation in research studies. While ZNF555, like most zinc finger proteins, primarily functions as a nuclear transcription factor, cytoplasmic detection may indicate several biologically relevant scenarios :

• Regulatory Dynamics: Cytoplasmic retention may represent a regulatory mechanism controlling ZNF555 activity, similar to other transcription factors regulated by nuclear-cytoplasmic shuttling.

• Post-Translational Regulation: Modifications like phosphorylation can alter nuclear localization signal (NLS) functionality, potentially redirecting ZNF555 localization.

• Alternative Functions: Cytoplasmic ZNF555 may participate in non-transcriptional roles, including RNA binding or cytoplasmic protein complex formation.

• Pathological Significance: Aberrant cytoplasmic accumulation could indicate disrupted nuclear transport machinery or protein misfolding in disease states.

When interpreting immunohistochemistry or immunofluorescence results, researchers should quantify nuclear-to-cytoplasmic ratios across experimental conditions rather than simply noting presence or absence in either compartment. This quantitative approach allows for more nuanced understanding of potential ZNF555 regulatory mechanisms and functional states in different cellular contexts.

How should researchers approach the integration of ZNF555 antibody data with genomic and transcriptomic datasets?

Multi-omics integration involving ZNF555 antibody-derived data requires sophisticated analytical frameworks:

• Correlation Analysis Framework:

  • Map ZNF555 protein levels detected via antibody-based methods to corresponding mRNA expression from RNA-seq data

  • Identify discordant patterns indicating post-transcriptional regulation

  • Calculate protein-mRNA correlation coefficients across different tissue or cell types

• Functional Network Construction:

  • Combine ChIP-seq data using Anti-ZNF555 antibody with RNA-seq differential expression after ZNF555 perturbation

  • Distinguish direct from indirect regulatory targets through motif analysis at binding sites

  • Construct directed regulatory networks incorporating protein interaction data

• Temporal Resolution Considerations:

  • Account for time delays between transcriptional and translational events when integrating datasets

  • Implement time-series analyses when data from multiple time points is available

• Statistical Integration Methods:

  • Apply Bayesian integration methods to combine evidence from protein, genomic, and transcriptomic data

  • Utilize dimensionality reduction techniques like MOFA (Multi-Omics Factor Analysis) to identify principal sources of variation across dataset types

This integrated analytical approach maximizes the value of antibody-generated data by placing it within broader molecular contexts, enabling more comprehensive understanding of ZNF555's biological roles and regulatory mechanisms.

What emerging technologies might enhance or replace current ZNF555 antibody applications in future research?

Several cutting-edge technologies are poised to complement or potentially supersede traditional antibody-based detection of ZNF555:

• CRISPR-Based Tagging Systems:

  • CRISPR knock-in of fluorescent or affinity tags at the endogenous ZNF555 locus

  • Advantages: Elimination of antibody specificity concerns; monitoring of dynamic expression in live cells

  • Current limitations: Technical challenges in efficient knock-in; potential disruption of protein function

• Aptamer Development:

  • Selection of DNA/RNA aptamers specific to ZNF555 protein structure

  • Advantages: Greater consistency between batches; potential for in vivo applications; reduced cross-reactivity

  • Current limitations: Lower affinity than high-quality antibodies; less established validation protocols

• Mass Cytometry and Imaging Mass Cytometry:

  • Metal-tagged antibodies for high-dimensional protein detection

  • Advantages: Dramatically increased multiplexing capacity; reduced autofluorescence issues

  • Current limitations: Requires specialized equipment; lower spatial resolution than advanced microscopy

• Proximity Ligation Assays and Proximity Extension Assays:

  • DNA-coupled antibody pairs for ultrasensitive protein detection

  • Advantages: Femtomolar sensitivity; reduced background; quantitative readout

  • Current limitations: Requires two distinct antibodies or one antibody against ZNF555 and another against an interacting protein

These emerging technologies will likely transform how researchers detect and study ZNF555, particularly in contexts requiring higher sensitivity, specificity, or multiplexing capabilities than current antibody-based methods can provide.

What modifications to standard protocols are required when using ZNF555 antibody in primary human tissue samples?

Working with primary human tissues introduces several variables that necessitate protocol modifications for optimal ZNF555 detection:

• Fixation Optimization: Reduce standard formalin fixation time to 8-12 hours for surgical specimens to prevent epitope masking, as zinc finger protein epitopes can be particularly sensitive to overfixation .

• Antigen Retrieval Enhancement: Implement extended antigen retrieval (25-30 minutes) with EDTA buffer (pH 9.0) as an alternative to standard citrate buffer to improve epitope accessibility in dense tissues.

• Signal Amplification Integration: Incorporate tyramide signal amplification to enhance detection sensitivity, particularly in tissues with naturally low ZNF555 expression.

• Background Reduction Strategy: Add a 30-minute blocking step with human serum (5%) prior to primary antibody incubation to minimize non-specific binding in human tissues.

• Tissue-Specific Controls: Include tissue-specific positive and negative controls based on The Human Protein Atlas data for ZNF555 expression patterns across human tissues .

These modifications address the inherent challenges of working with human biospecimens, including variable pre-analytical factors (ischemia time, fixation consistency) and tissue-specific autofluorescence or endogenous peroxidase activity that can interfere with antibody-based detection systems.

How can ZNF555 antibody be validated for use in non-human experimental models?

Cross-species application of Anti-ZNF555 antibody requires rigorous validation due to potential sequence variations in the epitope region:

This comprehensive validation strategy ensures that results obtained in non-human models can be confidently attributed to the intended orthologous protein target, enabling valid cross-species comparisons in evolutionary and functional studies of zinc finger protein biology.