ZNF335 Antibody, FITC conjugated

What is the molecular target of ZNF335 Antibody, FITC conjugated?

ZNF335 Antibody, FITC conjugated specifically targets the Zinc Finger Protein 335, also known as NRC-interacting factor 1 (NIF-1). The antibody targets amino acids 672-909 of the human ZNF335 protein and is generated using recombinant human Zinc finger protein 335 as the immunogen . ZNF335 is a transcription factor that functions as a component or associated component of histone methyltransferase complexes and plays a crucial role in regulating transcription through recruitment of these complexes to gene promoters . The protein enhances ligand-dependent transcriptional activation by nuclear hormone receptors and is involved in neural progenitor cell proliferation and self-renewal through regulating specific genes involved in brain development .

What are the optimal storage conditions for ZNF335 Antibody, FITC conjugated?

ZNF335 Antibody, FITC conjugated should be stored at -20°C or -80°C immediately upon receipt. The antibody is shipped at 4°C, but long-term storage requires freezing to maintain activity . It's crucial to avoid repeated freeze-thaw cycles as these can significantly reduce antibody performance and lead to protein degradation . The antibody is provided in a storage buffer consisting of 0.03% Proclin 300 (as a preservative), 50% Glycerol, and 0.01M PBS at pH 7.4, which helps maintain stability during storage . For researchers planning extended studies, it is advisable to aliquot the antibody into smaller volumes upon receipt to minimize freeze-thaw cycles.

What validation methods confirm the specificity of ZNF335 Antibody, FITC conjugated?

The ZNF335 Antibody, FITC conjugated has undergone rigorous validation through several methods to confirm its specificity. The antibody is purified using Protein G affinity purification methods, achieving >95% purity . Specificity for human ZNF335 has been validated through ELISA testing, which is the primary recommended application for this antibody . The antibody's reactivity is specific to human samples, with no confirmed cross-reactivity to other species reported in the available data . Researchers should note that additional validation might be necessary when applying this antibody to experimental systems or techniques beyond those tested by the manufacturer.

What are the validated applications for ZNF335 Antibody, FITC conjugated?

ZNF335 Antibody, FITC conjugated has been primarily validated for Enzyme-Linked Immunosorbent Assay (ELISA) applications . The FITC conjugation makes this antibody particularly suitable for direct detection methods without requiring secondary antibodies. While the primary validated application is ELISA, researchers might consider testing this antibody for other fluorescence-based applications such as flow cytometry, immunofluorescence microscopy, or immunohistochemistry with fluorescence detection, though these would require additional optimization and validation . When designing experiments with this antibody, researchers should consider the excitation (approximately 495 nm) and emission (approximately 519 nm) wavelengths of FITC to ensure compatibility with their detection systems.

How should researchers design immunofluorescence experiments using ZNF335 Antibody, FITC conjugated?

When designing immunofluorescence experiments with ZNF335 Antibody, FITC conjugated, researchers should consider the following methodological approach:

• Sample preparation: Fix cells with 4% paraformaldehyde (10-15 minutes) or cold methanol (10 minutes), then permeabilize with 0.1-0.5% Triton X-100 in PBS if using paraformaldehyde fixation.

• Blocking: Block non-specific binding sites with 1-5% BSA or normal serum from the same species as the secondary antibody (if used) for 30-60 minutes.

• Antibody dilution: Prepare appropriate dilutions of the ZNF335 Antibody, FITC conjugated in blocking buffer. Since the optimal concentration is not specified in the search results, start with a 1:100 to 1:500 dilution range for optimization.

• Incubation conditions: Apply the diluted antibody to the sample and incubate in a humidified chamber (2 hours at room temperature or overnight at 4°C).

• Nuclear counterstaining: Use DAPI or Hoechst dyes for nuclear visualization, being careful to select compatible fluorophores to avoid spectral overlap with FITC.

• Controls: Include appropriate negative controls (samples without primary antibody) and positive controls (samples known to express ZNF335) .

• Subcellular localization: Based on ZNF335's function as a transcription factor, expect predominantly nuclear localization, with potential enrichment in regions of active transcription .

What are the recommended protocols for using ZNF335 Antibody, FITC conjugated in ELISA?

For ELISA applications using ZNF335 Antibody, FITC conjugated, researchers should follow these methodological guidelines:

• Plate preparation: Coat a high-binding 96-well plate with the target antigen (recombinant ZNF335 protein or cell/tissue lysates containing ZNF335) in carbonate/bicarbonate buffer (pH 9.6) overnight at 4°C.

• Blocking: Block non-specific binding sites with 1-5% BSA or appropriate blocking buffer for 1-2 hours at room temperature.

• Sample addition: Add samples containing ZNF335 protein to the wells and incubate for 2 hours at room temperature.

• Antibody dilution: Prepare the ZNF335 Antibody, FITC conjugated at an appropriate dilution (starting with 1:1000 is recommended, with optimization as needed).

• Antibody incubation: Add the diluted antibody to the wells and incubate for 1-2 hours at room temperature, protected from light to prevent photobleaching of the FITC.

• Detection: For FITC detection, use a fluorescence plate reader with appropriate excitation (495 nm) and emission (519 nm) filters.

• Data analysis: Generate a standard curve using known concentrations of recombinant ZNF335 protein to quantify the ZNF335 in experimental samples .

How does ZNF335's role in neural development inform research applications?

ZNF335 plays a critical role in neural progenitor cell proliferation and self-renewal through regulating specific genes involved in brain development, including REST (RE1-Silencing Transcription factor) . Research has shown that mutations in ZNF335 are associated with severe neurodevelopmental disorders, including Microcephaly 10, Primary, Autosomal Recessive . This makes the ZNF335 Antibody, FITC conjugated particularly valuable for neurodevelopmental research applications, including:

• Neural progenitor studies: Investigating ZNF335 expression patterns during different stages of neural development and in various neural progenitor populations.

• Gene regulation analysis: Examining how ZNF335 regulates downstream target genes involved in neurogenesis and neural differentiation.

• Disease modeling: Studying ZNF335's role in cellular and animal models of microcephaly and other neurodevelopmental disorders.

• Protein complex identification: Identifying and characterizing ZNF335's interactions with histone methyltransferase complexes in neural cells .

When designing experiments targeting ZNF335's neural functions, researchers should consider developmental timing, regional specificity within the nervous system, and potential differences between in vitro and in vivo models.

What is known about ZNF335's involvement in T cell development and how can this antibody contribute to immunological research?

ZNF335 has been identified as a critical factor in T cell development, particularly in the formation of naïve T lymphocytes. Research using a mouse model with a hypomorphic mutation in Zfp335 (the mouse ortholog of human ZNF335) revealed an intrinsic developmental defect in T cells beginning in the thymus and continuing into peripheral T cells that have recently undergone thymic egress . This developmental defect cannot be attributed to altered thymic selection, proliferation, or Bcl2-dependent survival mechanisms .

Researchers investigating T cell development can utilize ZNF335 Antibody, FITC conjugated to:

• Track ZNF335 expression: Monitor ZNF335 expression levels during different stages of T cell development, particularly during late-stage intrathymic and post-thymic maturation.

• Analyze gene regulation: Investigate how ZNF335 regulates its target genes, which are enriched in categories related to protein metabolism, mitochondrial function, and transcriptional regulation in T cells.

• Study developmental defects: Examine the relationship between ZNF335 expression and T cell maturation defects in various immunological disorders.

• Investigate Ankle2 pathway: Explore the relationship between ZNF335 and Ankle2, as restoring Ankle2 expression has been shown to partially rescue T cell maturation in Zfp335-deficient models .

When designing immunological experiments using this antibody, researchers should note that while the current antibody has been validated for human samples, the available functional data on ZNF335 in T cell development comes primarily from mouse models. Cross-species validation would be necessary for comparative studies.

How can ZNF335 Antibody, FITC conjugated be used to study histone methyltransferase complex recruitment to gene promoters?

ZNF335 functions as a component or associated component of histone methyltransferase complexes and regulates transcription through recruitment of these complexes to gene promoters . Researchers investigating this process can use ZNF335 Antibody, FITC conjugated in the following advanced applications:

• Chromatin Immunoprecipitation followed by Fluorescence Imaging (ChIP-FISH): While not yet validated for this specific application, researchers could potentially adapt this antibody for ChIP-FISH protocols to visualize ZNF335's association with specific genomic loci and histone methyltransferase complexes simultaneously.

• Co-localization studies: Using multi-channel fluorescence microscopy to examine the co-localization of ZNF335 (detected with this FITC-conjugated antibody) with other components of histone methyltransferase complexes labeled with spectrally distinct fluorophores.

• Live-cell imaging: For researchers developing cell-permeable variants of this antibody or using cell delivery techniques, the FITC conjugation could allow real-time visualization of ZNF335's dynamic association with chromatin during transcriptional activation.

• Sequential ChIP (re-ChIP): While requiring protocol optimization, the antibody could potentially be used in sequential ChIP experiments to identify genomic regions where ZNF335 co-localizes with specific histone modifications or other transcriptional regulators .

These approaches would require significant optimization beyond the antibody's validated ELISA application, but they represent important avenues for understanding ZNF335's role in epigenetic regulation.

What experimental strategies can address the multi-functional nature of ZNF335 in both neural and immune system development?

ZNF335 has demonstrated important roles in both neural development and immune system function, particularly T cell maturation . Researchers investigating this multi-functional nature can employ the following experimental strategies using ZNF335 Antibody, FITC conjugated:

• Comparative expression analysis: Quantify ZNF335 expression levels across neural progenitors, mature neurons, thymic T cells, and peripheral T cell populations to identify tissue-specific expression patterns.

• ChIP-seq comparative analysis: Combine the antibody (after ChIP protocol optimization) with sequencing approaches to compare ZNF335 binding sites in neural versus immune cells, potentially identifying tissue-specific and shared target genes.

• Protein complex precipitation: Adapt the antibody for immunoprecipitation to identify tissue-specific binding partners of ZNF335 in neural versus immune contexts.

• Cell-type specific knockdown/knockout studies: Use the antibody to validate ZNF335 depletion efficiency in knockdown/knockout models of neural and immune cells, then analyze differential phenotypic effects.

• Rescue experiments: In ZNF335-deficient models, perform tissue-specific rescue experiments and use the antibody to validate restoration of expression, analyzing whether neural and immune functions require different domains or expression levels of ZNF335 .

The challenge in these approaches lies in distinguishing direct versus indirect effects of ZNF335 given its role as a transcription factor that potentially regulates hundreds of downstream genes.

What are common technical challenges when using FITC-conjugated antibodies and how can they be addressed?

When working with ZNF335 Antibody, FITC conjugated, researchers should be aware of these common technical challenges and their solutions:

• Photobleaching: FITC is relatively susceptible to photobleaching compared to other fluorophores.

  • Solution: Minimize exposure to light during all experimental steps; use anti-fade mounting media; consider image acquisition settings that minimize exposure while maintaining adequate signal.

• Autofluorescence: Cellular components (especially in fixed tissues) can exhibit autofluorescence in the FITC emission range.

  • Solution: Include unstained controls; use autofluorescence quenchers like Sudan Black B (0.1-0.3%); consider spectral unmixing during image analysis.

• pH sensitivity: FITC fluorescence is optimal at alkaline pH (8-9) and decreases significantly at acidic pH.

  • Solution: Ensure buffers are at appropriate pH; avoid acidic conditions during fixation and staining; consider this sensitivity when staining compartments with low pH (e.g., lysosomes).

• Signal intensity limitations: Direct conjugation provides one fluorophore per antibody molecule, potentially limiting detection of low-abundance targets.

  • Solution: For low-abundance targets, consider signal amplification methods or alternative detection systems.

• Spectral overlap: FITC's emission spectrum may overlap with other commonly used fluorophores.

  • Solution: Carefully design multi-color experiments with appropriate filter sets and compensation controls; consider sequential imaging rather than simultaneous acquisition .

Researchers should perform appropriate controls, including negative controls (samples known not to express ZNF335) and compensation controls when multiplexing with other fluorophores.

How can researchers optimize detection of nuclear ZNF335 in fixed tissue samples?

Detecting nuclear ZNF335 in fixed tissue samples using the FITC-conjugated antibody requires special considerations:

• Fixation optimization:

  • Overfixation can mask nuclear antigens. Test different fixation durations with 4% paraformaldehyde (12-24 hours is often optimal for tissue sections).

  • Consider antigen retrieval methods such as heat-induced epitope retrieval in citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0).

• Permeabilization protocol:

  • Nuclear transcription factors often require enhanced permeabilization. Consider 0.5% Triton X-100 (higher than typically used for cytoplasmic proteins) or 0.1-0.5% SDS to improve nuclear access.

• Signal-to-noise optimization:

  • Increase blocking duration (2-3 hours) with 3-5% BSA or normal serum plus 0.1-0.3% Triton X-100.

  • Include 0.1-0.3% Sudan Black B treatment post-staining to reduce tissue autofluorescence.

• Nuclear context visualization:

  • Counterstain with DAPI or Hoechst (far enough spectrally from FITC to avoid bleed-through).

  • Consider co-staining with nuclear structure markers (lamin, nuclear pore complex proteins) to provide context for ZNF335 localization.

• Image acquisition settings:

  • Use confocal microscopy with appropriate optical sectioning to accurately visualize nuclear localization.

  • Adjust detector gain to capture the typically punctate pattern of transcription factors while avoiding saturation .

These optimizations are particularly important given ZNF335's role as a transcription factor that would be expected to show nuclear localization, often in a punctate pattern corresponding to sites of active transcription.

How should researchers quantify and interpret ZNF335 expression levels in different cell populations?

When analyzing ZNF335 expression using the FITC-conjugated antibody across different cell populations, researchers should:

• Establish quantification parameters:

  • For flow cytometry: Report median fluorescence intensity (MFI) rather than mean, as transcription factor expression often shows non-normal distribution.

  • For microscopy: Measure nuclear intensity specifically, using nuclear masks defined by DAPI counterstaining.

  • For ELISA: Generate standard curves using recombinant ZNF335 protein at known concentrations.

• Apply appropriate normalization:

  • Normalize to appropriate housekeeping proteins or total protein content.

  • For microscopy, normalize to nuclear area or DAPI intensity.

  • Account for cell-cycle variations, as transcription factor levels may fluctuate during different cell cycle phases.

• Statistical analysis considerations:

  • Use non-parametric tests when appropriate, as transcription factor expression may not follow normal distribution.

  • Consider hierarchical analysis for tissue samples with multiple cell types.

  • Report both biological and technical replicates separately.

• Biological interpretation framework:

  • Interpret ZNF335 expression in the context of its known roles in neural development and T cell maturation .

  • Consider expression levels in conjunction with downstream target genes, particularly those involved in neural progenitor cell proliferation and T cell development.

  • Compare expression patterns across developmental timepoints, as ZNF335 functions may be highly stage-specific .

The interpretation should consider that ZNF335 acts as a transcriptional regulator, so even modest changes in expression could have significant biological impact through amplification at the level of target gene expression.

What analytical approaches should be used when studying ZNF335's association with histone methyltransferase complexes?

When investigating ZNF335's association with histone methyltransferase complexes using the FITC-conjugated antibody, researchers should employ these analytical approaches:

• Co-localization analysis:

  • Calculate Pearson's or Mander's correlation coefficients between ZNF335-FITC signal and markers of histone methyltransferase complexes.

  • Perform object-based co-localization analysis to identify discrete nuclear foci where ZNF335 and methyltransferase components overlap.

  • Use super-resolution microscopy techniques when possible to overcome diffraction limits when examining nuclear protein complexes.

• Chromatin association analysis:

  • If adapting the antibody for ChIP applications, analyze enrichment at promoter regions containing the known ZNF335 binding motifs.

  • Compare ZNF335 binding sites with maps of specific histone modifications (H3K4me3, H3K27me3) to establish functional correlations.

  • Integrate with RNA-seq data to correlate ZNF335 binding with transcriptional outcomes.

• Quantitative interaction assessment:

  • If using the antibody for immunoprecipitation followed by protein identification, employ stable isotope labeling approaches for quantitative interactome analysis.

  • Apply computational network analysis to identify core and peripheral components of ZNF335-associated complexes.

• Temporal dynamics analysis:

  • Consider stimulation-dependent changes in ZNF335 association with histone methyltransferase complexes.

  • Analyze time-course data to identify sequential recruitment events at target loci.

• Data integration framework:

  • Integrate findings with published datasets on histone modifications and chromatin accessibility.

  • Compare ZNF335 binding patterns with those of other transcription factors involved in neural development or T cell maturation .

These approaches should be guided by ZNF335's known role as a component or associated component of histone methyltransferase complexes that regulates transcription through recruitment of these complexes to gene promoters.