zbtb18 Antibody

What are the most reliable applications for ZBTB18 antibodies in research?

ZBTB18 antibodies have been validated for multiple experimental applications with varying degrees of reliability. Based on published literature, the following applications have demonstrated consistent results:

For optimal results, antibody validation using positive and negative controls is essential before proceeding with critical experiments .

How do I optimize ZBTB18 immunohistochemistry in brain tissue samples?

Optimization of ZBTB18 immunohistochemistry in brain samples requires attention to several parameters:

• Fixation method: 4% paraformaldehyde for 24 hours has proven effective for maintaining ZBTB18 epitope integrity

• Antigen retrieval: Data supports using TE buffer at pH 9.0, although citrate buffer at pH 6.0 can be an alternative

• Blocking solution: 5-10% normal serum (matching the secondary antibody host) with 0.3% Triton X-100

• Primary antibody dilution: Begin with 1:100 and optimize based on signal-to-noise ratio

• Incubation conditions: Overnight incubation at 4°C generally yields best results

• Detection system: Polymer-based detection systems show superior sensitivity compared to avidin-biotin methods

Nuclear localization is typically observed in normal neuronal tissues, while cytoplasmic enrichment may indicate pathological states in some contexts .

How can ZBTB18 antibodies be applied to study glioblastoma progression?

ZBTB18 has significant implications in glioblastoma (GBM) as a tumor suppressor. Methodological approaches include:

• Immunohistochemical profiling: Compare ZBTB18 expression and localization across GBM samples of different grades and patient outcomes

• ChIP-seq analysis: Map ZBTB18 binding sites on cytokine promoters (particularly CCL2, CX3CL1, CXCL14)

• Co-localization studies: Combine ZBTB18 immunostaining with microglia/macrophage markers to assess tumor-immune interactions

• Functional validation: Compare ZBTB18 antibody staining with cytokine secretion profiles in the same GBM samples

Research has demonstrated that ZBTB18 represses key cytokines involved in recruiting tumor-associated macrophages (GAMs) to GBM. In particular, ZBTB18 directly binds to the CCL2 and GDF15 promoters, inhibiting their expression and subsequent secretion . This approach can help identify potential therapeutic targets for modulating the immune microenvironment in GBM.

What methodological approaches are recommended for studying ZBTB18's role in cancer metastasis?

Recent studies have revealed ZBTB18's function as a metastasis suppressor. When investigating this role:

• Nuclear-cytoplasmic fractionation: Use ZBTB18 antibodies to quantify subcellular distribution, as cytoplasmic sequestration correlates with metastatic potential

• ATAC-seq combined with ZBTB18 ChIP-seq: Map changes in chromatin accessibility at ZBTB18 binding sites

• Sequential immunofluorescence: Compare ZBTB18 localization in matched primary and metastatic samples

• Proximity ligation assays: Detect ZBTB18 interactions with other chromatin modifiers like LSD1

Research has demonstrated that ZBTB18 prevents metastasis through widespread chromatin closing, particularly at the promoters of genes like TGFBR2 that drive metastatic behavior. Notably, while ZBTB18 expression levels may remain unchanged between metastatic and non-metastatic cells, its nuclear localization is significantly reduced in highly metastatic cells .

How can I distinguish between full-length ZBTB18 and its N-terminal variant in experiments?

The full-length ZBTB18 and its N-terminal short variant (ZBTB18 Nte-SF) have distinct functional properties that require careful experimental design to differentiate:

• Antibody selection: Choose antibodies targeting epitopes specific to the full-length protein (C-terminal region)

• Western blot optimization: Use gradient gels (4-15%) to clearly separate the full-length (~59 kDa) from the N-terminal variant

• Control samples: Include samples with known expression of each variant as references

• Functional validation: The N-terminal variant lacks the tumor-suppressive and anti-cytokine properties of full-length ZBTB18

Experimentally, only the full-length ZBTB18 (not the N-terminal variant) reduced CCL2 secretion when expressed in glioblastoma cell lines, providing a functional readout to distinguish their activities .

Why do I observe different molecular weights for ZBTB18 in Western blots?

Researchers commonly report variations in ZBTB18's apparent molecular weight:

These discrepancies may result from:

• Post-translational modifications: Phosphorylation alters migration patterns

• Tissue-specific processing: Different cell types may process ZBTB18 differently

• Antibody specificity: Different antibodies may recognize specific isoforms or modified forms

• Sample preparation: Denaturing conditions can affect migration patterns

To address this variability, always include positive control samples and consider performing peptide competition assays to confirm specificity .

How can ChIP-seq with ZBTB18 antibodies reveal its transcriptional regulatory networks?

Optimizing ChIP-seq protocols for ZBTB18 requires:

• Crosslinking optimization: 1% formaldehyde for 10 minutes is typically effective

• Sonication parameters: Target DNA fragments of 200-500 bp

• Antibody selection: Use ChIP-validated antibodies with demonstrated specificity

• Input normalization: Include input controls at multiple sequencing depths

• Peak calling algorithms: Use algorithms suited for transcriptional repressors

• Motif analysis: Identify ZBTB18 binding motifs, which often contain consensus sequences found in the hPDI and HOCOMOCO databases

Research has successfully used ChIP to confirm direct binding of ZBTB18 at the CCL2 and GDF15 promoters close to their transcription start sites, specifically 365 bp downstream of the TSS for CCL2 and 105 bp upstream for GDF15 .

What approaches are most effective for investigating ZBTB18's interaction with epigenetic regulators like LSD1?

ZBTB18 interacts with the histone demethylase LSD1 in context-dependent ways. To study these interactions:

• Sequential ChIP (re-ChIP): Precipitate with ZBTB18 antibodies followed by LSD1 antibodies to identify co-occupied regions

• Co-immunoprecipitation with protein crosslinking: Use formaldehyde or DSS to stabilize transient interactions

• Proximity ligation assay: Visualize ZBTB18-LSD1 interactions in situ in different cell types

• Histone modification ChIP: Correlate ZBTB18 binding with changes in H3K4me1/2 (LSD1 targets)

Research indicates that ZBTB18's repressive activity on CCL2 may occur through inhibition of LSD1's activating function, while LSD1 appears to act as a repressor of GDF15 independently of ZBTB18 . This context-dependent interaction highlights the complexity of ZBTB18's regulatory mechanisms.

How can spatial transcriptomics be integrated with ZBTB18 immunostaining?

Combining ZBTB18 immunodetection with spatial transcriptomics offers insights into its local regulatory effects:

• Sequential immunofluorescence and spatial transcriptomics: Perform ZBTB18 immunostaining followed by spatial RNA profiling on the same tissue section

• Computational integration: Correlate ZBTB18 protein localization with local gene expression patterns

• Multi-omic spatial analysis: Combine with chromatin accessibility assays to correlate ZBTB18 binding, chromatin state, and gene expression

• Single-cell spatial proteomics: Detect ZBTB18 alongside its targets at single-cell resolution

This approach is particularly valuable for studying tumor heterogeneity in glioblastoma, where ZBTB18 expression varies between different tumor compartments and influences local immune cell recruitment .

How can ZBTB18 antibodies contribute to understanding its role in neurological disorders?

ZBTB18 has been implicated in neurodevelopmental disorders, including autosomal dominant non-syndromic intellectual disability 22. Research approaches include:

• Developmental timing analysis: Track ZBTB18 expression across brain development stages

• Cell type-specific profiling: Use multiplexed immunofluorescence to identify cell populations expressing ZBTB18

• Post-mortem brain tissue studies: Compare ZBTB18 distribution in control versus disease samples

• Variant-specific antibodies: Develop antibodies that specifically recognize disease-associated ZBTB18 variants

ZBTB18 represses several proneuronogenic genes including PAX6, NEUROG2, and NEUROD1, affecting the differentiation and migration of intermediate neurogenic progenitors. It is highly expressed in the cortex and involved in the Ngn2-Rnd2 pathway crucial for cortical neuron migration .

What protocols are recommended for studying ZBTB18 in liver and metabolic disease models?

Recent research has identified ZBTB18's role in hepatic metabolism and non-alcoholic fatty liver disease (NAFLD):

• Tissue processing: Flash-freezing followed by cryosectioning preserves ZBTB18 epitopes in liver tissue

• Antibody validation: Confirm specificity in hepatic tissues using ZBTB18 knockout controls

• Co-staining panels: Combine ZBTB18 with metabolic markers (SREBP, fatty acid synthase)

• Quantification methods: Use digital pathology for quantitative analysis of nuclear ZBTB18 in hepatocytes

ZBTB18 expression is significantly decreased in liver biopsies from NAFLD patients and in multiple NAFLD mouse models, including db/db diabetic mice, ob/ob obese mice, and high-fat diet-induced obese mice . This suggests potential applications for ZBTB18 antibodies in metabolic disease research.