ZNF335 Antibody, Biotin conjugated

What is ZNF335 and why is it significant for research?

ZNF335 (Zinc finger protein 335), also known as NIF-1 (NRC-interacting factor 1), functions as a component of histone methyltransferase complexes and regulates transcription through recruitment of these complexes to gene promoters . This protein enhances ligand-dependent transcriptional activation by nuclear hormone receptors and plays important roles in neural progenitor cell proliferation and self-renewal through regulation of genes involved in brain development, including REST . ZNF335 also controls the expression of genes involved in somatic development and regulates lymphoblast proliferation . Studies in mouse models have demonstrated that Zfp335 (the mouse ortholog) is essential for T cell maturation and functions as a transcription factor that binds to promoter regions via a specific consensus motif .

What are the key characteristics of ZNF335 Antibody, Biotin conjugated?

ZNF335 Antibody, Biotin conjugated is a high-quality polyclonal antibody raised in rabbits against a recombinant fragment of human ZNF335 protein (amino acids 672-909) . The antibody is conjugated to biotin, which facilitates detection in various immunological assays, particularly ELISA applications . It demonstrates reactivity against human samples and is purified via Protein G with >95% purity . The antibody is provided in liquid form in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative .

What is the molecular mechanism of ZNF335 function in cellular processes?

ZNF335 functions through a C2H2 zinc finger domain, which is the largest class of transcription factors in mammalian genomes . As a transcription factor, ZNF335 binds to promoter regions of target genes through a specific consensus motif . Genome-wide studies have shown that ZNF335 binding sites are strongly enriched in gene promoters and located upstream of transcriptional start sites (TSS) . These binding regions are associated with high levels of H3K4me3 (a hallmark of active gene promoters) and low levels of the enhancer-associated modification H3K27ac and the repressive chromatin mark H3K27me3 . This pattern is consistent with ZNF335 functioning primarily as a regulator of promoter-dependent gene transcription. Target genes regulated by ZNF335 are enriched in functional categories related to protein synthesis and metabolism, mitochondrial function, cell cycle regulation, RNA processing, and transcriptional regulation .

How should I design an ELISA experiment using ZNF335 Antibody, Biotin conjugated?

When designing an ELISA using ZNF335 Antibody, Biotin conjugated, follow this sandwich ELISA methodology:

• Plate Preparation: Pre-coat a 96-well plate with a capturing anti-ZNF335 antibody (not biotinylated) .

• Sample Addition: Add standards and test samples to the wells and incubate to allow ZNF335 protein binding to the capturing antibody .

• Detection Antibody: Add the biotinylated anti-ZNF335 antibody (ZNF335 Antibody, Biotin conjugated) as the detection antibody and incubate .

• Washing: Thoroughly wash to remove unbound conjugates using an appropriate wash buffer .

• Enzyme Conjugate Addition: Add HRP-Streptavidin, which binds to the biotin on the detection antibody .

• Final Washing: Perform another washing step to remove unbound HRP-Streptavidin .

• Substrate Addition: Add TMB substrates to visualize the HRP enzymatic reaction, producing a blue color that turns yellow after adding the stop solution .

• Measurement: Read the optical density (OD) at 450nm using a microplate reader .

• Data Analysis: Calculate the concentration of ZNF335 in samples by comparing to a standard curve. The concentration is proportional to the OD450 value .

What are the optimal storage conditions for maintaining ZNF335 Antibody activity?

For optimal preservation of ZNF335 Antibody, Biotin conjugated activity, follow these storage guidelines:

• Upon receipt, store the sealed antibody at -20°C or -80°C .

• Avoid repeated freeze-thaw cycles as this can denature the antibody and reduce its efficacy .

• The antibody is supplied in a buffer containing 50% glycerol, which helps maintain stability during freezing .

• When working with the antibody, keep it on ice or at 4°C and return to -20°C as soon as possible after use.

• If aliquoting the antibody for multiple uses, prepare small volumes to minimize freeze-thaw cycles.

• Monitor the expiration date, as antibody reactivity may decrease over time even with proper storage.

How can I validate the specificity of ZNF335 Antibody in my experimental system?

To validate the specificity of ZNF335 Antibody, Biotin conjugated in your experimental system, implement these approaches:

• Positive and Negative Controls:

  • Use samples with known ZNF335 expression as positive controls

  • Include samples lacking ZNF335 expression as negative controls

  • Consider using cell lines with ZNF335 knockdown or knockout for comparison

• Western Blot Validation:

  • Perform western blotting to confirm the antibody recognizes a protein of the expected molecular weight (approximately 140 kDa for ZNF335)

  • Look for a single, clean band that corresponds to ZNF335

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess recombinant ZNF335 protein (preferably the immunogen fragment, amino acids 672-909)

  • Run parallel assays with and without peptide competition

  • Specific binding should be blocked in the peptide-treated sample

• Cross-Reactivity Testing:

  • Test the antibody against samples from different species if cross-reactivity is a concern

  • The antibody is generated against human ZNF335, so confirmation of reactivity in other species is necessary

• Immunoprecipitation Followed by Mass Spectrometry:

  • For advanced validation, perform immunoprecipitation using the ZNF335 antibody

  • Analyze the precipitated proteins by mass spectrometry to confirm ZNF335 identity

What are common issues when using ZNF335 Antibody in ELISA and how can they be resolved?

When performing ELISA with ZNF335 Antibody, Biotin conjugated, researchers may encounter several challenges. Here are common issues and their solutions:

For optimal results, titrate the antibody concentration to determine the ideal working dilution for your specific experimental conditions .

How can I minimize non-specific binding when using ZNF335 Antibody, Biotin conjugated?

To minimize non-specific binding when using ZNF335 Antibody, Biotin conjugated, implement these strategies:

• Optimal Blocking: Use high-quality blocking reagents (2-5% BSA or casein) in both washing and dilution buffers to occupy non-specific binding sites .

• Antibody Dilution Optimization: Perform titration experiments to determine the minimum effective concentration of antibody that provides specific signal with minimal background .

• Buffer Optimization:

  • Add 0.05-0.1% Tween-20 to washing buffers to reduce hydrophobic interactions

  • Consider adding 0.1-0.5% Triton X-100 for more stringent washes if background persists

  • Add 0.1-0.5M NaCl to reduce ionic interactions if they contribute to non-specific binding

• Pre-adsorption: Pre-incubate the antibody solution with tissues or cells lacking ZNF335 to remove antibodies that might bind non-specifically.

• Cross-Adsorption: If possible, use cross-adsorbed secondary reagents (like streptavidin-HRP) that have been purified to minimize cross-reactivity.

• Wash Protocol Enhancement:

  • Increase the number of washing steps (6-8 washes rather than 3-4)

  • Extend washing time to allow better removal of unbound antibodies

  • Use fresh wash buffer for each washing step

• Streptavidin-HRP Dilution: Optimize the concentration of streptavidin-HRP conjugate to minimize background while maintaining sensitivity.

How can ZNF335 Antibody, Biotin conjugated be used to study transcriptional regulation in neural development?

ZNF335 Antibody, Biotin conjugated can be utilized in several advanced approaches to study transcriptional regulation in neural development:

• Chromatin Immunoprecipitation (ChIP) Analysis:

  • Use the antibody to pull down ZNF335-bound chromatin

  • Sequence the immunoprecipitated DNA (ChIP-seq) to identify genome-wide binding sites

  • Focus on regions enriched in gene promoters and upstream of transcriptional start sites where ZNF335 typically binds

  • Analyze the binding sites for the ZNF335 consensus motif

• Co-Immunoprecipitation (Co-IP) to Identify Protein Complexes:

  • Utilize the antibody to precipitate ZNF335 along with its interacting partners

  • Mass spectrometry analysis can identify components of histone methyltransferase complexes that associate with ZNF335

  • Investigate interactions with other neural developmental regulators, particularly REST which is regulated by ZNF335

• Target Gene Expression Analysis:

  • Combine ChIP with RNA-seq to correlate ZNF335 binding with gene expression changes

  • Focus on genes involved in neural progenitor cell proliferation and self-renewal

  • Utilize RT-qPCR to validate expression changes in specific target genes like those identified in the mouse ortholog studies (e.g., Ankle2)

• Developmental Time-Course Studies:

  • Apply the antibody in ELISA assays to quantify ZNF335 expression across different stages of neural development

  • Compare ZNF335 levels with expression patterns of target genes to establish temporal relationships

• Immunofluorescence Co-Localization:

  • Use biotinylated antibody with fluorescent streptavidin for detection

  • Perform co-localization studies with markers of active chromatin (H3K4me3) and repressive marks (H3K27me3)

  • Analyze nuclear distribution patterns in developing neural tissue

What methodologies can be used to investigate ZNF335-mediated gene regulation in T cell maturation?

Based on research with the mouse ortholog Zfp335, several methodologies can be employed to investigate ZNF335-mediated gene regulation in T cell maturation:

How can I design experiments to explore ZNF335's role in epigenetic regulation using the biotinylated antibody?

To investigate ZNF335's role in epigenetic regulation using ZNF335 Antibody, Biotin conjugated, consider these experimental designs:

• Sequential ChIP (ChIP-reChIP) Analysis:

  • First ChIP: Use anti-ZNF335 antibody to precipitate ZNF335-bound chromatin

  • Second ChIP: Re-precipitate with antibodies against histone modifiers (e.g., methyltransferases) or histone marks (H3K4me3, H3K27me3)

  • This approach identifies genomic regions where ZNF335 co-localizes with specific epigenetic modifications

• CUT&RUN or CUT&Tag with Biotinylated Antibody:

  • Leverage the biotin conjugation for highly sensitive chromatin profiling

  • These techniques provide higher resolution than traditional ChIP

  • Map ZNF335 binding sites in relation to chromatin accessibility and histone modifications

• Proximity Ligation Assay (PLA):

  • Detect in situ interactions between ZNF335 and components of histone methyltransferase complexes

  • Visualize the spatial distribution of these interactions within the nucleus

  • Quantify changes in interaction frequency under different cellular conditions

• CRISPR-dCas9 Epigenetic Editing:

  • Target dCas9 fused to epigenetic modifiers to ZNF335 binding sites

  • Assess how altering histone modifications at these sites affects ZNF335 binding and target gene expression

  • Create a functional map of the relationship between specific epigenetic marks and ZNF335 activity

• 3D Chromatin Organization Analysis:

  • Combine ZNF335 ChIP with Hi-C or other chromosome conformation capture techniques

  • Determine how ZNF335 binding influences higher-order chromatin structure

  • Investigate whether ZNF335 mediates long-range chromatin interactions between its target promoters

How should I interpret contradictory results between ZNF335 binding and target gene expression?

When encountering contradictory results between ZNF335 binding and target gene expression, consider these analytical approaches:

• Contextual Analysis:

  • Examine the cellular context – ZNF335 may function differently in various cell types or developmental stages

  • The mouse Zfp335 study showed differential binding effects across target genes, with some sites more affected by the R1092W mutation than others

  • Consider that ZNF335 may function as both an activator and a repressor depending on context

• Cooperative Factor Analysis:

  • Investigate co-binding factors that might influence ZNF335's regulatory outcome

  • ZNF335 enhances ligand-dependent transcriptional activation by nuclear hormone receptors, suggesting context-dependent activity

  • The absence of essential co-factors might explain why binding doesn't always correlate with expression changes

• Epigenetic Landscape Assessment:

  • Analyze the chromatin environment at binding sites with contradictory outcomes

  • Examine histone modification patterns (H3K4me3, H3K27ac, H3K27me3) at these loci

  • Consider the influence of chromatin accessibility on the functional outcome of ZNF335 binding

• Binding Affinity and Occupancy Quantification:

  • Quantitatively compare ZNF335 occupancy levels between sites with different expression outcomes

  • Low-affinity binding might be detected by ChIP but may not be functionally significant

  • The specific binding motif elements contribute differentially to binding strength, with mutations in the first element having stronger effects than the second element

• Temporal Resolution Analysis:

  • Conduct time-course experiments to detect delayed effects on gene expression following ZNF335 binding

  • Some regulatory events may require multiple cell cycles or developmental transitions to manifest

What integrative bioinformatic approaches are recommended for analyzing ZNF335 ChIP-seq data?

For comprehensive analysis of ZNF335 ChIP-seq data, implement these integrative bioinformatic approaches:

• Motif Discovery and Enrichment Analysis:

  • Apply de novo motif discovery tools (MEME, Homer) to identify the ZNF335 binding consensus

  • Compare with the bipartite motif identified in mouse studies

  • Perform motif enrichment analysis to determine if the consensus motif is significantly enriched in peaks

• Genomic Feature Association:

  • Analyze the distribution of binding sites relative to genomic features (promoters, enhancers, gene bodies)

  • ZNF335 peaks are expected to be strongly enriched in gene promoters and located upstream of transcriptional start sites

  • Create metaplots showing the average binding profile around TSSs

• Multi-omics Data Integration:

  • Integrate ChIP-seq with RNA-seq data to correlate binding with expression changes

  • Incorporate ATAC-seq or DNase-seq data to assess chromatin accessibility at binding sites

  • Include histone modification ChIP-seq data (H3K4me3, H3K27ac, H3K27me3) to characterize the epigenetic environment

• Network Analysis:

  • Construct gene regulatory networks centered on ZNF335 target genes

  • Identify hub genes and regulatory circuits

  • Perform pathway enrichment analysis on target genes (expected categories: protein metabolism, mitochondrial function, transcriptional regulation)

• Differential Binding Analysis:

  • Compare binding profiles between experimental conditions or cell types

  • Identify sites with differential occupancy and correlate with expression changes

  • The Zfp335 R1092W mutation study showed selective effects on a subset of target sites rather than global binding reduction

• Comparative Genomics:

  • Analyze conservation of binding sites across species

  • Examine evolutionary conservation of the binding motif

  • Compare binding profiles with data from orthologous proteins (e.g., mouse Zfp335)

How can I design a comprehensive study to investigate the role of ZNF335 in lymphocyte development beyond T cells?

To investigate ZNF335's role in broader lymphocyte development, design a comprehensive study with these components:

• Comparative Expression Profiling:

  • Quantify ZNF335 expression across multiple lymphocyte lineages (T cells, B cells, NK cells)

  • Use ELISA with ZNF335 Antibody, Biotin conjugated to measure protein levels

  • Perform RT-qPCR to assess transcript levels

  • Conduct single-cell RNA-seq to identify cell subsets with distinctive ZNF335 expression patterns

• Multi-lineage ChIP-seq Analysis:

  • Perform ChIP-seq using ZNF335 Antibody, Biotin conjugated across different lymphocyte populations

  • Compare binding profiles to identify:
    a) Core binding sites common to all lymphocytes
    b) Lineage-specific binding patterns
    c) Developmental stage-specific occupancy changes

  • Correlate binding with expression changes in each lineage

• Conditional Knockout Model System:

  • Generate lymphocyte-specific conditional knockout models for ZNF335

  • Create separate models for T cells, B cells, and NK cells

  • Analyze developmental progression using flow cytometry

  • Perform functional assays to assess maturation and activation potential

• Rescue Experiments with Target Gene Panels:

  • Based on the findings that Ankle2 partially rescues T cell maturation defects in Zfp335 mutant mice

  • Test multiple ZNF335 target genes individually and in combinations

  • Compare rescue efficiency across different lymphocyte lineages

  • Identify lineage-specific essential target genes

• Protein Interaction Network Mapping:

  • Perform co-immunoprecipitation followed by mass spectrometry

  • Identify ZNF335 interaction partners in different lymphocyte lineages

  • Look for lineage-specific interactions that might explain differential functions

  • Focus on interactions with known developmental regulators of each lineage

• Epigenetic Landscape Comparison:

  • Generate chromatin accessibility maps (ATAC-seq) for each lineage

  • Profile histone modifications associated with active (H3K4me3, H3K27ac) and repressive (H3K27me3) chromatin

  • Correlate ZNF335 binding with epigenetic changes during development

  • Identify pioneering factor activity that might be lineage-specific

What emerging technologies can enhance the utility of ZNF335 Antibody, Biotin conjugated in single-cell studies?

Several cutting-edge technologies can significantly enhance the application of ZNF335 Antibody, Biotin conjugated in single-cell research:

• Single-Cell CUT&Tag/CUT&RUN:

  • These techniques require minimal cell input and provide high resolution

  • The biotin conjugation makes this antibody particularly suitable for these methods

  • Can map ZNF335 binding sites in rare cell populations or developmental intermediates

  • Enables correlation of binding profiles with cell state at single-cell resolution

• Spatial Transcriptomics Integration:

  • Combine immunofluorescence detection of ZNF335 using the biotinylated antibody with spatial transcriptomics

  • Map the spatial relationship between ZNF335 protein localization and target gene expression

  • Particularly valuable for studying developmental contexts where spatial organization is critical

• Microfluidic Antibody-based Chromatin Profiling:

  • Implement microfluidic platforms for parallel processing of single cells

  • Use the biotinylated antibody for automated chromatin profiling

  • Increase throughput while maintaining sensitivity

• CyTOF (Mass Cytometry) Extension:

  • Adapt the biotinylated antibody for use in CyTOF by conjugating metal isotopes to streptavidin

  • Include ZNF335 in multiparameter panels to correlate its expression with cell surface markers

  • Enable high-dimensional phenotyping of cells based on ZNF335 expression and other markers

• Live-Cell Imaging of ZNF335 Dynamics:

  • Develop cell-permeable streptavidin conjugates with fluorescent dyes

  • Use cell-penetrating peptides to deliver the biotinylated antibody into living cells

  • Monitor real-time changes in ZNF335 localization during cell division or differentiation

• Droplet-based Single-Cell ChIP-seq:

  • Leverage the biotin-streptavidin interaction for efficient chromatin capture in droplet-based platforms

  • Enable high-throughput analysis of ZNF335 binding across thousands of individual cells

  • Identify rare cell populations with distinct ZNF335 regulatory programs

How can the ZNF335 consensus binding motif be utilized to predict novel regulatory targets?

The identification of a ZNF335 consensus binding motif provides powerful opportunities for predicting novel regulatory targets:

• Genome-Wide Motif Scanning:

  • Use the bipartite motif identified in mouse studies to scan the human genome

  • Apply position weight matrices (PWMs) derived from ChIP-seq data

  • Prioritize sites based on conservation, accessibility, and proximity to genes

  • Focus on promoter regions, where ZNF335 primarily binds

• Integrated Prediction Models:

  • Combine motif information with epigenetic data (H3K4me3, H3K27ac, H3K27me3)

  • Develop machine learning models that integrate motif strength, chromatin state, and gene expression

  • Train models using validated ZNF335 targets to predict new targets

  • The model should account for the observation that both elements of the bipartite motif contribute to binding, with the first element having a stronger effect

• Cross-Species Conservation Analysis:

  • Identify evolutionarily conserved instances of the ZNF335 binding motif

  • Focus on motifs conserved between human and mouse, given the functional data available for mouse Zfp335

  • Prioritize motifs in promoters of genes with conserved expression patterns

• Experimental Validation Pipeline:

  • Design a systematic approach to validate predicted targets:
    a) In vitro: EMSA assays with oligonucleotides containing predicted motifs
    b) In cellulo: Reporter assays using promoters with wild-type or mutated motifs
    c) In vivo: CRISPR-based mutation of motifs followed by expression analysis

  • The validation should test both elements of the bipartite motif, as mutations in both parts affected binding in gel shift assays

• Network-Based Prediction Enhancement:

  • Utilize known ZNF335 targets to identify common regulatory networks

  • Predict additional members of these networks as potential ZNF335 targets

  • Integrate protein-protein interaction data to improve predictions

  • Focus on pathways related to protein metabolism, mitochondrial function, and transcriptional regulation

What are the implications of ZNF335 research for understanding neurodevelopmental and immunological disorders?

Research on ZNF335 has significant implications for understanding both neurodevelopmental and immunological disorders:

• Neurodevelopmental Disorders:

  • ZNF335 plays crucial roles in neural progenitor cell proliferation and brain development

  • It regulates REST, a key factor in neuronal development

  • Mutations in ZNF335 could contribute to microcephaly and other neurodevelopmental disorders

  • Research using ZNF335 Antibody, Biotin conjugated could help identify dysregulated target genes in patient-derived neural cells

  • Therapeutic strategies might target downstream pathways affected by ZNF335 dysfunction

• Primary Immunodeficiencies:

  • The mouse ortholog Zfp335 is essential for T cell maturation

  • Mutations in human ZNF335 might contribute to T cell development disorders

  • Antibody-based studies could identify patient populations with altered ZNF335 expression or function

  • The partial rescue of T cell maturation by Ankle2 suggests potential therapeutic targets

  • Combined immunodeficiency syndromes might involve ZNF335 pathway dysregulation

• Autoimmune Disorders:

  • Proper T cell development is critical for maintaining self-tolerance

  • ZNF335's role in T cell maturation suggests potential involvement in autoimmunity

  • Abnormal expression of ZNF335 target genes might contribute to loss of tolerance

  • The protein's function in transcriptional regulation makes it a candidate for epigenetic dysregulation in autoimmune conditions

• Cancer Immunology:

  • ZNF335 regulates lymphoblast proliferation

  • Altered ZNF335 function could contribute to lymphoid malignancies

  • Its dual roles in neural development and immune function suggest potential involvement in neuroimmunological conditions

  • ZNF335 target genes related to cell cycle regulation and metabolism are frequently dysregulated in cancer

• Therapeutic Target Development:

  • Understanding the ZNF335 regulatory network could identify novel therapeutic targets

  • Small molecules targeting ZNF335 binding or protein-protein interactions might modulate its function

  • Gene therapy approaches could be developed to correct ZNF335 mutations

  • The finding that Ankle2 partially rescues T cell maturation defects suggests functional redundancy that could be therapeutically exploited