ZNF268 Antibody, FITC conjugated

What is ZNF268 and what are its key structural features?

ZNF268 (Zinc finger protein 268, also known as HZF3) is a typical KRAB-containing zinc finger protein (KRAB-ZFP). The protein exists in multiple isoforms, with ZNF268a being the most extensively studied. ZNF268a contains a KRAB domain and 24 zinc fingers, functioning primarily as a transcriptional repressor. This isoform has been implicated in inhibiting erythroid differentiation and tumor cell proliferation. In contrast, ZNF268b2 lacks the KRAB domain and has different biological functions, including potential involvement in carcinogenesis through NF-κB signaling pathway activation .

What is the optimal storage condition for ZNF268 Antibody, FITC conjugated?

For maximum stability and activity retention, ZNF268 Antibody, FITC conjugated should be stored at -20°C or -80°C immediately upon receipt. It is critical to avoid repeated freeze-thaw cycles as this can significantly degrade the antibody and reduce FITC fluorescence intensity. The antibody is typically supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative, which helps maintain stability during storage .

What applications can ZNF268 Antibody, FITC conjugated be used for?

The FITC-conjugated ZNF268 antibody is primarily designed for applications requiring direct fluorescence detection. These include flow cytometry for quantitative analysis of ZNF268 expression in cell populations, fluorescence microscopy for examining subcellular localization patterns, and immunohistochemistry on frozen sections. The antibody's polyclonal nature makes it suitable for detecting multiple epitopes of the target protein, potentially increasing sensitivity but requiring additional validation steps compared to monoclonal antibodies .

How can researchers experimentally validate the nuclear localization function of ZNF268?

To validate ZNF268 nuclear localization function, researchers should employ a multi-method approach. First, construct expression vectors containing full-length ZNF268, KRAB-domain-only, and zinc-finger-only variants fused to a reporter such as GFP. After transfection into appropriate cell lines, observe subcellular localization using confocal microscopy. Complementary approaches include subcellular fractionation followed by Western blotting and co-immunoprecipitation with known nuclear proteins. For more detailed analysis, mutagenesis studies targeting specific zinc fingers can help determine which of the 24 zinc fingers possess nuclear localization activity .

What are critical considerations for studying the cooperation between KRAB domain and zinc fingers in ZNF268?

When studying the cooperative function of KRAB domain and zinc fingers in ZNF268, researchers must consider that these domains function both independently and synergistically. Design experiments with deletion constructs that systematically remove specific zinc fingers or the KRAB domain. Utilize fluorescence recovery after photobleaching (FRAP) to assess protein mobility differences between full-length and truncated variants. Also consider that the KRAB domain interacts with KAP1, targeting proteins specifically to the nucleoplasm (but not nucleolus), while zinc fingers target proteins throughout the nucleus. Co-immunoprecipitation experiments with KAP1 can confirm this interaction in your experimental system .

How should researchers design experiments to investigate ZNF268's role in tumor suppression?

To investigate ZNF268's tumor suppression role, implement a comprehensive experimental design incorporating both in vitro and in vivo approaches. Begin with establishing stable cell lines with ZNF268 overexpression and knockdown in relevant cancer models (e.g., Caki-1 for renal cell carcinoma studies). Assess proliferation using EdU incorporation assays and colony formation experiments. Evaluate migration and invasion capabilities through wound-healing and transwell assays. Measure apoptosis via flow cytometry and Western blot analysis of Bcl-2/Bax expression. For in vivo validation, use xenograft tumor models in immunodeficient mice with tumor volume measurements and immunohistochemical analysis of harvested tumors .

What protocol optimizations are recommended when using ZNF268 Antibody, FITC conjugated for flow cytometry?

When optimizing flow cytometry protocols for ZNF268 Antibody, FITC conjugated, start with titration experiments to determine the optimal antibody concentration (typically beginning with 1-5 μg/ml). For intracellular staining, use a gentle fixation method (0.5-2% paraformaldehyde) followed by permeabilization with 0.1% saponin or 0.1% Triton X-100. Include appropriate compensation controls to account for spectral overlap with other fluorophores. Importantly, protect samples from light throughout the procedure to prevent photobleaching of the FITC conjugate. Use freshly prepared cells when possible and include both positive and negative controls to validate staining specificity .

How can researchers troubleshoot weak or non-specific signals when using ZNF268 Antibody, FITC conjugated?

For troubleshooting weak signals, first verify antibody integrity by testing fluorescence on a known positive control. Increase antibody concentration incrementally and extend incubation times. If background fluorescence is high, optimize blocking conditions using 2-5% BSA or serum from the same species as the secondary antibody. Reduce autofluorescence by treating samples with 0.1% sodium borohydride or 100mM glycine. For verification of signal specificity, perform competitive blocking with the immunizing peptide (residues 416-603 of ZNF268). Additionally, perform parallel experiments with alternative antibodies against ZNF268 to confirm staining patterns .

What are the recommended fixation and permeabilization methods compatible with ZNF268 Antibody, FITC conjugated?

For optimal results with ZNF268 Antibody, FITC conjugated, choose fixation and permeabilization methods that preserve protein epitopes while allowing antibody access to nuclear proteins. Paraformaldehyde (2-4%) fixation for 10-15 minutes at room temperature followed by permeabilization with 0.1-0.5% Triton X-100 works well for most nuclear proteins. For flow cytometry, methanol fixation (-20°C for 10 minutes) can simultaneously fix and permeabilize cells. When working with tissue sections, optimize antigen retrieval methods (such as citrate buffer pH 6.0 with heat treatment) to expose epitopes that may be masked during fixation. Always validate your chosen method with positive control samples expressing known quantities of ZNF268 .

How does ZNF268 function in nuclear localization and what experimental evidence supports this?

ZNF268 achieves precise nuclear localization through a cooperative mechanism involving both its KRAB domain and zinc fingers. Experimental evidence from mutagenesis studies demonstrates that while both domains can independently target proteins to the nucleus, their combined effect is necessary for proper localization. The KRAB domain specifically directs proteins to the nucleoplasm but excludes them from the nucleolus, mediated through interaction with KAP1 (KRAB-associated protein 1). In contrast, zinc fingers distribute proteins uniformly throughout the nucleus. This cooperative mechanism ensures ZNF268 is precisely localized to nucleoplasmic regions where it can perform its transcriptional regulatory functions .

How is ZNF268 expression regulated at the molecular level?

ZNF268 expression appears to be regulated by a complex network of non-coding RNAs (ncRNAs). In ccRCC, a regulatory axis involving the long non-coding RNA AC093157.1 and microRNA miR-27a-3p has been identified. This regulation occurs through a competing endogenous RNA (ceRNA) mechanism, where AC093157.1 acts as a sponge for miR-27a-3p, preventing it from binding to and downregulating ZNF268. This regulation has been experimentally validated through luciferase reporter assays showing direct binding between miR-27a-3p and ZNF268. Additional regulatory mechanisms may include transcriptional control through its promoter region and post-translational modifications, though these aspects require further investigation to fully elucidate ZNF268's regulatory network .

How can ZNF268 Antibody, FITC conjugated be used to study immune cell interactions in tumor microenvironments?

To study immune cell interactions involving ZNF268 in tumor microenvironments, implement multiparameter flow cytometry with ZNF268 Antibody, FITC conjugated alongside markers for T helper cells, central memory T cells, and regulatory T cells. This approach allows correlation of ZNF268 expression with specific immune cell populations. For spatial analysis, perform multiplex immunofluorescence on tissue sections using ZNF268 Antibody, FITC conjugated with antibodies against CD4, CD8, FOXP3, and other relevant immune markers. Complementary approaches include single-cell RNA sequencing to identify gene expression patterns in ZNF268-expressing cells versus surrounding immune populations. These methodologies will help determine how ZNF268 expression influences immune cell recruitment and activation in the tumor microenvironment .

What experimental approaches can differentiate the functions of ZNF268a and ZNF268b2 isoforms?

To differentiate functions between ZNF268a and ZNF268b2 isoforms, design isoform-specific knockdown and overexpression systems using CRISPR-Cas9 or siRNA targeting unique exons. Generate stable cell lines expressing either isoform tagged with different fluorescent proteins to track localization differences. Perform RNA-seq and ChIP-seq analyses to identify differentially regulated genes and binding sites for each isoform. For protein interactions, conduct co-immunoprecipitation followed by mass spectrometry to identify isoform-specific binding partners. Functionally, compare the effects of each isoform on NF-κB pathway activation, erythroid differentiation, and proliferation using reporter assays and cell-based functional tests. These approaches will comprehensively map the distinct roles of each isoform in normal physiology and disease states .

How can researchers validate the AC093157.1/miR-27a-3p/ZNF268 regulatory axis in different experimental systems?

To validate the AC093157.1/miR-27a-3p/ZNF268 regulatory axis across experimental systems, employ a multi-step validation approach. First, quantify expression correlations between all three components in relevant cell lines and tissue samples using qRT-PCR. Perform luciferase reporter assays with wild-type and mutated binding sites to confirm direct interactions between miR-27a-3p and both ZNF268 and AC093157.1. Use RNA immunoprecipitation (RIP) assays to verify physical interactions between miRNAs and target RNAs. Functionally validate the axis by systematically overexpressing or knocking down each component and measuring effects on the others. Additionally, utilize rescue experiments where phenotypes caused by modulating one component are reversed by appropriate modulation of downstream components. These comprehensive approaches will establish the biological relevance of this regulatory axis across different experimental systems and potentially identify tissue-specific variations in regulatory mechanisms .

What quantitative methods are recommended for analyzing ZNF268 expression levels in experimental samples?

For robust quantitative analysis of ZNF268 expression, employ multiple complementary methods. For protein level quantification, use Western blotting with densitometric analysis normalized to appropriate loading controls (β-actin, GAPDH). In flow cytometry experiments, calculate mean fluorescence intensity (MFI) values and compare against isotype controls. For immunohistochemistry or immunofluorescence, utilize digital image analysis software (ImageJ, QuPath) to quantify staining intensity across multiple fields. At the mRNA level, perform quantitative RT-PCR with validated reference genes for normalization (ACTB, GAPDH, or 18S rRNA). For higher throughput analysis, consider droplet digital PCR, which provides absolute quantification without standard curves. Always include appropriate positive and negative controls, and use statistical methods appropriate for your experimental design (t-test, ANOVA, or non-parametric alternatives) .

How should researchers interpret conflicting data regarding ZNF268 expression in different cancer types?

When confronted with conflicting data on ZNF268 expression across cancer types, consider several important factors. First, evaluate methodological differences between studies, including antibody specificity, detection methods, and quantification approaches. Second, determine whether studies distinguished between ZNF268 isoforms, as ZNF268a and ZNF268b2 may have opposite functions in different contexts. Third, analyze the tumor microenvironment and tissue-specific factors that might influence ZNF268 regulation. Critically assess patient cohort characteristics, as differences in cancer stage, grade, treatment history, and genetic background can significantly impact results. To resolve contradictions, conduct meta-analyses of existing data and design experiments that directly compare ZNF268 function across multiple cancer cell lines under identical conditions. This comprehensive approach will help determine whether ZNF268 functions as a tumor suppressor or promoter in a context-dependent manner .

What bioinformatic approaches can be used to predict functional domains within ZNF268 for targeted antibody development?

For predicting functional domains within ZNF268 to guide targeted antibody development, implement a multi-layered bioinformatic strategy. Begin with primary sequence analysis using tools like SMART, Pfam, and InterPro to identify conserved domains beyond the known KRAB domain and zinc fingers. Apply protein structure prediction algorithms (AlphaFold2, I-TASSER) to generate 3D structural models identifying surface-exposed regions suitable for antibody targeting. Conduct conservation analysis across species using multiple sequence alignments to identify evolutionarily constrained regions likely to be functionally important. Perform disorder prediction (PONDR, IUPred) to identify flexible regions that might undergo conformational changes. Additionally, use epitope prediction tools (BepiPred, Discotope) to identify antigenic determinants with high immunogenicity. These analyses will help identify optimal regions for developing antibodies that recognize specific functional domains or isoforms of ZNF268 .