ZNF350 Antibody

What is ZNF350 and what cellular functions does it perform?

ZNF350, also known as zinc-finger and BRCA1-interacting protein with a Kruppel-associated box (KRAB) domain (ZBRK1), is a transcriptional suppressor involved in the development of several human tumor types, including breast, colon, and cervical carcinomas. The protein contains an N-terminal A+B box domain, eight C2H2 zinc fingers, and a C-terminal repression domain (CTRD). ZNF350 functions primarily as a transcriptional suppressor that can either form complexes with other proteins or play a direct transcriptional suppressive role in a single-factor form. The protein has been shown to interact with BRCA1 and can repress the expression of genes involved in cancer cell migration and metastasis .

What are the molecular characteristics of ZNF350 antibody?

The ZNF350 antibody (catalog number 84121-5-RR) is a recombinant rabbit IgG antibody that targets human ZNF350 protein. It has been designed to recognize the full zinc finger protein 350 and can be used in multiple applications including Western Blot, Flow Cytometry (intracellular), and ELISA. The antibody detects ZNF350 at its observed molecular weight of approximately 60 kDa. The antibody was developed using a ZNF350 fusion protein immunogen (Ag23982) and has been purified using Protein A purification methods .

What are the recommended dilutions for ZNF350 antibody across different applications?

The optimal dilution of ZNF350 antibody varies depending on the specific application and sample type. Based on validation studies, the following dilutions are recommended for different applications:

It is important to note that these recommendations serve as starting points, and the antibody should be titrated in each testing system to obtain optimal results. The optimal dilution may be sample-dependent, so researchers should check validation data before finalizing their experimental protocols .

How is ZNF350 implicated in cancer cell migration and metastasis?

ZNF350 has been identified as a potential metastasis suppressor in various cancer types. Research demonstrates that ZNF350 can inhibit cancer cell migration and suppress metastatic activity. In colon cancer studies, subpopulations of cells with accelerated baseline motility (MG cells) showed significant downregulation of ZNF350 expression compared to immotile cells (non-MG cells). Specifically, ZNF350 mRNA levels in highly motile cells were reduced to approximately 13% of those observed in non-motile cells .

Experimental evidence indicates that ZNF350 knockdown accelerates migration of immotile cancer cells, while overexpression of ZNF350 in motile cells significantly impairs their migration capabilities. The mechanism appears to involve transcriptional repression of genes involved in epithelial-mesenchymal transition (EMT) and cell migration. ZNF350 acts as a transcriptional corepressor with nuclear villin and represses Slug (SNAI2) expression, resulting in inhibition of EMT. Additional ZNF350-regulated EMT-related genes include MMP9 and KAP1 .

What role does DNA methylation play in regulating ZNF350 expression in cancer cells?

DNA methylation plays a crucial role in the regulation of ZNF350 expression, particularly in cancer cells. Research on colon cancer cells has demonstrated that hypermethylation of the ZNF350 promoter correlates with decreased ZNF350 expression and increased migratory capacity of the cells. Treatment with a DNA methyltransferase inhibitor, 5-azacytidine, restored ZNF350 expression and significantly reduced cell migration in highly motile cancer cells .

Pyrosequencing analysis of the ZNF350 promoter region (from -297 to +14 bp) revealed specific differences in methylation patterns between motile and non-motile cancer cells, with several hypermethylated CpG sites identified in the motile cells. Further investigation using serially truncated fragments of the ZNF350 promoter showed that fragments containing three hypermethylated CpG sites (CpG 9, 10, and 11) were particularly important for basal promoter activity .

Analysis of The Cancer Genome Atlas (TCGA) data showed that DNA methylation levels of ZNF350 were significantly and negatively correlated with ZNF350 gene expression in clinical samples, suggesting that this regulatory mechanism is relevant to human cancer progression .

How should researchers design controls for ZNF350 antibody validation experiments?

For proper validation of ZNF350 antibody experiments, researchers should implement multiple control strategies:

• Positive Controls: Use cell lines known to express ZNF350, such as THP-1 cells for Western blotting and A431 cells for flow cytometry, as documented in validation studies .

• Negative Controls: Include samples where ZNF350 is knocked down using siRNA or CRISPR-Cas9 gene editing. Based on published research, effective ZNF350 knockdown can be achieved with siRNA, though complete knockdown may be challenging (typical reduction is approximately 50%) .

• Isotype Controls: Include a matched isotype control (rabbit IgG) to assess non-specific binding, particularly in flow cytometry and immunohistochemistry applications.

• Blocking Peptide Controls: When available, use the immunizing peptide or recombinant ZNF350 protein to compete for antibody binding, confirming signal specificity.

• Cross-Reactivity Assessment: Test the antibody on samples from non-human species to confirm specificity for human ZNF350, as the antibody is documented to be human-reactive .

How can researchers effectively study the interaction between ZNF350 and BRCA1 in cancer models?

Studying the ZNF350-BRCA1 interaction in cancer models requires a multi-faceted approach:

• Co-Immunoprecipitation (Co-IP): Use ZNF350 antibody to pull down protein complexes, followed by Western blotting for BRCA1, or vice versa. This can confirm physical interaction between these proteins in your specific cancer model.

• Proximity Ligation Assay (PLA): This technique allows visualization of protein-protein interactions in situ with high specificity and sensitivity. Use ZNF350 and BRCA1 antibodies from different species to detect interactions at the single-molecule level.

• Chromatin Immunoprecipitation (ChIP): Since the ZNF350/BRCA1 complex functions as a transcriptional repressor, ChIP experiments can identify genomic regions where this complex binds. Sequential ChIP (ChIP-reChIP) can confirm co-occupancy of both proteins at specific loci.

• Functional Assays: Compare the effects of individual knockdowns of ZNF350 and BRCA1 versus double knockdown on cancer cell phenotypes such as migration, invasion, and gene expression profiles. Pay particular attention to genes regulated by the ZNF350/BRCA1 complex, including ANG1, HMGA2, and SNAI2 .

• Domain Mapping: Use truncated versions of ZNF350 to identify which domains are essential for BRCA1 interaction. The C-terminal repression domain (CTRD) of ZNF350 forms tetrameric oligomers that allow selective interaction with BRCA1 and other proteins .

Importantly, analysis of differentially expressed BRCA1-regulated genes (such as BCL2, VIM, NFE2L2, and IFITM1) in motile versus non-motile cancer cells can provide insights into how the ZNF350/BRCA1 complex contributes to cancer cell migration .

What methodological approaches are recommended for investigating ZNF350 promoter methylation in clinical samples?

Investigating ZNF350 promoter methylation in clinical samples requires robust methodological approaches:

• Bisulfite Sequencing: After bisulfite conversion (which converts unmethylated cytosines to uracils while methylated cytosines remain unchanged), perform targeted sequencing of the ZNF350 promoter region. Focus particularly on the region from -297 to +14 bp, which contains critical CpG sites identified in research studies .

• Pyrosequencing: This quantitative method can determine the methylation percentage at individual CpG sites. Focus on CpG sites 9, 10, and 11, which have been identified as particularly important for ZNF350 promoter activity .

• Methylation-Specific PCR (MSP): Design primers specific for methylated and unmethylated versions of the ZNF350 promoter to quickly screen clinical samples.

• Correlation Analysis: Compare ZNF350 promoter methylation levels with mRNA expression levels in the same clinical samples to establish functional relevance, similar to what has been done with TCGA data showing significant negative correlation between methylation and expression .

• Functional Validation: For clinical samples showing hypermethylation, isolate cells when possible and treat with demethylating agents such as 5-azacytidine to confirm that demethylation restores ZNF350 expression.

• Prognostic Analysis: Assess whether ZNF350 promoter methylation status correlates with clinical parameters such as tumor stage, metastasis, or patient survival.

When analyzing results, remember that while ZNF350 levels are generally reduced in colon cancer tissues compared to surrounding normal tissues, stage-dependent changes in ZNF350 expression may not be significant, as indicated by TCGA data analysis (Pearson's r = +0.0549) .

How can researchers distinguish between ZNF350-dependent and ZNF350-independent mechanisms in cancer cell migration studies?

Distinguishing between ZNF350-dependent and ZNF350-independent mechanisms in cancer cell migration requires careful experimental design:

• Gene Rescue Experiments: After ZNF350 knockdown increases migration, re-introduce wild-type ZNF350 to confirm migration suppression. Then introduce domain-specific mutants (e.g., zinc finger mutations, CTRD mutations) to identify which domains are essential for migration suppression.

• Pathway Analysis: Compare transcriptome profiles between wild-type, ZNF350-knockdown, and ZNF350-overexpressing cells to identify differentially regulated pathways. Focus on known ZNF350 target genes including GADD45A, ANG1, HMGA1, p21, MMP9, FGF2, SNAI2, and KAP1 .

• Dual Modulation Experiments: Simultaneously modulate ZNF350 and its downstream targets. For example, if ZNF350 knockdown increases SNAI2 expression and cell migration, simultaneous knockdown of both ZNF350 and SNAI2 would help determine whether migration is dependent on the ZNF350-SNAI2 axis.

• Temporal Analysis: Use inducible systems to modulate ZNF350 expression and monitor the kinetics of migration changes and target gene expression changes to establish cause-effect relationships.

• Context-Dependent Studies: Test ZNF350 modulation in different cell types and under different conditions (e.g., with/without growth factors, on different extracellular matrices) to identify context-dependent effects.

It's important to note that research shows ZNF350 is likely one of multiple factors regulating cancer cell migration. Even partial (50%) knockdown of ZNF350 can facilitate migration of immotile cells, but ZNF350 overexpression has only a limited inhibitory effect on migration, suggesting involvement of additional regulators in this complex phenotype .

What are the best experimental approaches for investigating the relationship between ZNF350 and epithelial-mesenchymal transition (EMT) in cancer progression?

Investigating the relationship between ZNF350 and EMT in cancer progression requires a comprehensive experimental approach:

• EMT Marker Analysis: Assess how ZNF350 modulation affects canonical EMT markers including E-cadherin, N-cadherin, vimentin, fibronectin, and EMT transcription factors (SNAI1, SNAI2, ZEB1, ZEB2, TWIST1). This should be done at both protein and mRNA levels using Western blot, immunofluorescence, and qRT-PCR.

• Morphological Assessment: Document cellular morphology changes associated with ZNF350 modulation, as EMT involves transition from cobblestone-like epithelial morphology to spindle-shaped mesenchymal morphology.

• Functional Assays: Beyond migration, assess other EMT-associated behaviors including:

  • Invasion through Matrigel

  • Resistance to anoikis

  • Formation of mammospheres/tumorspheres

  • Stem cell marker expression (CD44, CD133)

• Signaling Pathway Analysis: Determine how ZNF350 interfaces with known EMT-inducing pathways (TGF-β, Wnt, Notch, etc.) through Western blotting for phosphorylated signaling intermediates and reporter assays for pathway activity.

• ChIP-seq Analysis: Identify global ZNF350 binding sites, particularly at promoters of EMT-related genes, to distinguish direct from indirect regulation.

• EMT Hybrid State Analysis: Given that gene expression signatures from motile cancer cells indicate they may represent an EMT hybrid state , use single-cell approaches to characterize heterogeneity in ZNF350 expression and EMT marker expression within a cell population.

• In vivo Models: Assess how ZNF350 modulation affects tumor-initiating capacity, metastasis, and circulating tumor cell generation in animal models.

Focus particularly on SNAI2 (Slug) regulation, as research has shown that ZNF350 represses SNAI2 expression, and SNAI2 upregulation was observed in motile colon cancer cells with low ZNF350 expression .

How can researchers troubleshoot inconsistent results when using ZNF350 antibody in different experimental systems?

When encountering inconsistent results with ZNF350 antibody across different experimental systems, consider the following troubleshooting steps:

• Antibody Validation: Confirm antibody specificity through:

  • Western blot analysis showing a single band at the expected molecular weight (60 kDa)

  • Comparison with ZNF350 knockdown samples

  • Testing in known positive cell lines (THP-1 for Western blot, A431 for flow cytometry)

• Protocol Optimization:

  • Adjust antibody dilution within the recommended range (1:1000-1:8000 for Western blot)

  • Optimize incubation times and temperatures

  • Test different blocking agents to reduce background

  • For challenging samples, consider antigen retrieval methods

• Sample Preparation:

  • Ensure complete cell lysis for intracellular proteins

  • Use protease inhibitors to prevent degradation

  • Standardize protein quantification methods

  • Consider subcellular fractionation as ZNF350 is primarily nuclear

• Expression Level Considerations:

  • ZNF350 expression can vary significantly between cell types and cancer subtypes

  • In some cancer tissues, ZNF350 levels may be reduced to 13% of normal levels

  • DNA methylation status can dramatically affect expression levels

• Storage and Handling:

  • Store the antibody at -20°C

  • Avoid repeated freeze-thaw cycles

  • For long-term storage, consider aliquoting the antibody

  • Check for precipitates before use and centrifuge if necessary

• Detection System Optimization:

  • For low-abundance targets, consider more sensitive detection methods (enhanced chemiluminescence for Western blot)

  • For flow cytometry, optimize fixation and permeabilization procedures for nuclear proteins

Remember that the antibody has been validated for human samples only, so cross-reactivity with other species should not be expected .

What are the emerging areas of research regarding ZNF350's role in cancer beyond its interaction with BRCA1?

Several emerging research areas regarding ZNF350 extend beyond its established interaction with BRCA1:

• Epigenetic Regulation Network: Research suggests ZNF350 is part of a broader epigenetic regulatory network. Beyond its own regulation by promoter methylation, ZNF350 may influence global epigenetic patterns by recruiting histone deacetylases and other chromatin modifiers to specific genomic loci.

• Cancer Stem Cell Regulation: Given ZNF350's role in suppressing EMT, which is associated with stemness properties, investigating its function in cancer stem cell maintenance and differentiation represents a promising research direction.

• Therapeutic Targeting: Developing approaches to restore ZNF350 expression or function in cancers where it is downregulated could represent a novel therapeutic strategy. This might involve demethylating agents specifically targeting the ZNF350 promoter or peptide mimetics that replicate ZNF350's interaction with key partner proteins.

• Biomarker Potential: ZNF350 promoter methylation status could serve as a biomarker for cancer aggressiveness or metastatic potential, particularly in colorectal cancer where its methylation correlates with increased cell migration.

• Context-Dependent Functions: Research indicates that ZNF350's role may vary across cancer types. While it suppresses migration in colon cancer cells, its functions in other cancer types and in normal tissue homeostasis remain to be fully characterized.

• Regulation of Non-Coding RNAs: As a transcriptional regulator, ZNF350 may influence expression of non-coding RNAs including miRNAs and lncRNAs that contribute to cancer progression.

• Integration with Metabolic Reprogramming: Exploring connections between ZNF350 activity and cancer metabolic reprogramming could reveal new insights, particularly given the emerging links between EMT and metabolic changes in cancer.