ZNF844 Antibody

What is ZNF844 and what is its biological significance?

ZNF844 is a member of the KRAB-Zinc Finger Protein (KRAB-ZFP) family, which represents the largest family of mammalian transcription regulators. These proteins are differentially expressed in various tissues during cellular development and phenotypic differentiation. ZNF844 is located on chromosome 19p13.2 and is a paralog of ZNF433 .

Within the kidney, ZNF844 expression is primarily associated with proximal convoluted tubules and immune cells, including macrophages, B-cells, and T-cells . Recent research has indicated that ZNF844 may function as a putative immune-related tumor suppressor gene, particularly in clear-cell renal cell carcinoma (ccRCC) .

What are the validated applications for ZNF844 antibody in experimental settings?

ZNF844 antibodies have been validated for several experimental applications:

• Immunohistochemistry (IHC)

• Immunohistochemistry-Paraffin (IHC-P) at dilutions of 1:50 - 1:200

When designing experiments using ZNF844 antibodies, researchers should consider the specific application requirements and optimize protocols accordingly. The specificity of commercially available antibodies has been verified on protein arrays containing the target protein plus 383 other non-specific proteins .

What are the optimal storage conditions for ZNF844 antibodies?

For maximum stability and antibody performance, ZNF844 antibodies should be stored according to the following recommendations:

• Short-term storage: 4°C

• Long-term storage: -20°C (aliquoted to minimize freeze-thaw cycles)

• Avoid repeated freeze-thaw cycles as these can degrade antibody quality

Most commercial preparations are supplied in PBS (pH 7.2) with 40% glycerol and 0.02% sodium azide as preservative . When handling the antibody, use aseptic technique to prevent contamination.

How does ZNF844 expression differ between normal and cancer tissues?

Studies using The Cancer Genome Atlas (TCGA) dataset and immunohistochemistry have demonstrated significant differences in ZNF844 expression between normal kidney tissue and renal carcinoma:

• ZNF844 is significantly down-regulated in clear-cell renal cell carcinoma (ccRCC) compared to normal kidney tissue (p=1.624×10^-12)

• Expression levels are consistently lower across all histological grades and pathological stages of ccRCC

• Immunohistochemistry confirms that ZNF844 protein expression is lower in renal carcinoma tissues ("low" or "not detected" staining with "weak" intensities) compared to normal tissues ("medium" staining with "moderate" intensities)

• Analysis of metastatic versus normal tissues (GSE12606 dataset) corroborates that ZNF844 is significantly down-regulated (adjusted p-value=0.025928)

This expression pattern suggests ZNF844 may have tumor suppressor properties in renal tissues.

What experimental controls should be included when working with ZNF844 antibodies?

When designing experiments using ZNF844 antibodies, include the following controls to ensure reliable results:

• Positive control: Tissue or cell lysates known to express ZNF844 (normal kidney tissue sections are recommended)

• Negative control: Samples with confirmed absence or knockdown of ZNF844

• Technical controls:

  • Primary antibody omission control

  • Isotype control (using matched IgG at the same concentration)

  • Blocking peptide control (pre-incubation of antibody with immunizing peptide)

For IHC applications, antibodies have been developed against a specific recombinant protein sequence, which can be used for blocking experiments: LPHTFKCMKGLTLESNCMNLNNVKKPLDLSETFKFMKRHTLERNPIRNMEKHSTISLPFKYMQQCTEDRMPMNVKSVTKHSYLPRSFEYMQEHTLE .

How is ZNF844 related to other KRAB-ZFPs on chromosome 19p13.2?

ZNF844 is part of a cluster of KRAB-ZFPs located on chromosome 19p13.2. Analysis of this cluster reveals:

This clustering of related KRAB-ZFPs suggests potential functional redundancy or coordinated regulation, which should be considered when designing experiments targeting ZNF844.

What is the prognostic value of ZNF844 in cancer research?

ZNF844 has demonstrated significant prognostic value in ccRCC research:

• Decreased ZNF844 expression is associated with poor patient survival (HR=0.41; 95% CI=0.3-0.56; p<0.0001)

• Survival outcomes for patients with low expression (<7.512 transcripts/million) were significantly lower than those with high expression (>7.512 transcripts/million)

• ROC analysis indicated an AUC of 0.642 (95% CI=0.599 to 0.683), suggesting ZNF844 expression is a moderate predictor of survival outcomes

• In multivariate Cox regression analysis, ZNF844 expression retained predictive status alongside age and stage, indicating it may be an independent predictive biomarker for ccRCC

These findings suggest that researchers studying cancer prognosis should consider incorporating ZNF844 expression analysis into their experimental designs, particularly for renal cancer studies.

What methodologies are recommended for investigating the relationship between ZNF844 and immune cell infiltration?

To investigate ZNF844's relationship with immune cell infiltration, researchers can employ several complementary approaches:

• Correlation analysis with T-cell signatures: Using GEPIA2 database to correlate ZNF844 expression with established T-cell signatures:

  • Naïve T-cell signature (genes: CCR7, LEF1, TCF7, SELL)

  • Helper T-cell Type 1 signature (genes: CXCL13, HAVCR2, IFNG, CXCR3, CD4, BHLHE40)

  • Helper T-cell Type 2 signature (genes: CCL26, IL5, IL13, GATA3, STAT6)

  • Effector T-reg signature (genes: FOXP3, CTLA4, CCR8, TNFRSF9)

  • Effector T-cell signature (genes: CX3CR1, FGFBP2, FCGR3A)

  • Exhausted T-cell signature (genes: HAVCR2, TIGIT, LAG3, PDCD1, CXCL13, LAYN)

• Immune infiltration analysis: Using TIMER2.0 with the XCell algorithm to examine ZNF844 expression versus immune infiltration levels

• Double immunofluorescence/immunohistochemistry: Co-staining of ZNF844 with immune cell markers in tissue sections to visualize spatial relationships

Previous research has shown that ZNF844 expression has a moderate, inverse correlation to Helper T-cell (CD4 or Th1) subtype 1 infiltration (R=–0.558, p=5.15×10^–39) and with the exhausted T-cell phenotype (R=–0.37; p=4.1×10^–21) .

What experimental approaches can validate the tumor suppressor role of ZNF844 in cancer models?

To validate the putative tumor suppressor role of ZNF844, researchers should consider these experimental approaches:

• Loss and gain of function studies:

  • siRNA or shRNA-mediated knockdown of ZNF844 in normal kidney cell lines to assess phenotypic changes

  • CRISPR-Cas9 knockout of ZNF844 to evaluate effects on cell proliferation, migration, and invasion

  • Overexpression of ZNF844 in ccRCC cell lines to determine if cancer phenotypes are reversed

• Mechanistic studies:

  • ChIP-seq to identify DNA binding sites of ZNF844

  • RNA-seq following ZNF844 manipulation to identify downstream targets

  • Co-immunoprecipitation to identify protein-protein interactions

• In vivo models:

  • Xenograft models using ZNF844-manipulated cell lines

  • Patient-derived xenografts with varying levels of ZNF844 expression

  • Correlation of tumor growth and metastasis with ZNF844 expression levels

These approaches would provide comprehensive validation of ZNF844's role as a tumor suppressor and could potentially identify molecular mechanisms through which it exerts its effects.

What is the optimal protocol for immunohistochemical detection of ZNF844 in FFPE tissue sections?

For optimal immunohistochemical detection of ZNF844 in formalin-fixed paraffin-embedded (FFPE) tissue sections, the following protocol is recommended:

• Sample preparation:

  • Cut 4-6 μm thick sections from FFPE blocks

  • Mount on positively charged slides

  • Dry overnight at 37°C

• Deparaffinization and rehydration:

  • Xylene: 2 × 5 minutes

  • 100% ethanol: 2 × 3 minutes

  • 95% ethanol: 1 × 3 minutes

  • 70% ethanol: 1 × 3 minutes

  • Rinse in distilled water

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Pressure cooker method: 125°C for 30-40 seconds, followed by cooling

• Blocking and antibody incubation:

  • Block endogenous peroxidase: 3% H₂O₂ for 10 minutes

  • Protein block: 5% normal goat serum for 1 hour at room temperature

  • Primary antibody: ZNF844 antibody at 1:50 - 1:200 dilution overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-rabbit IgG for 1 hour at room temperature

• Detection and visualization:

  • DAB chromogen: 5-10 minutes (monitor microscopically)

  • Counterstain: Hematoxylin for 30 seconds

  • Dehydrate, clear, and mount with permanent mounting medium

Note that optimization may be required for specific tissue types, and comparison with normal kidney tissue (known to express ZNF844) is recommended as a positive control.

How can researchers quantitatively analyze ZNF844 expression in experimental studies?

For quantitative analysis of ZNF844 expression, researchers should consider these methodological approaches:

• Quantitative RT-PCR:

  • Design primers specific to ZNF844 mRNA

  • Use reference genes appropriate for the tissue being studied

  • Apply the 2^-ΔΔCt method for relative quantification

  • Compare expression levels to established datasets such as TCGA-KIRC

• Immunohistochemistry quantification:

  • Digital image analysis using software like ImageJ or QuPath

  • H-score calculation: Σ(percentage of cells at each intensity level × intensity level)

    • Where intensity is scored as 0 (negative), 1+ (weak), 2+ (moderate), or 3+ (strong)

  • Compare staining patterns to those observed in normal kidney tissues

• Western blot analysis:

  • Densitometric analysis of band intensity

  • Normalization to housekeeping proteins (β-actin or GAPDH)

• RNA-seq data analysis:

  • Use established pipelines and normalization methods

  • Compare to public datasets (TCGA, GEO) for validation

  • Apply appropriate statistical tests based on data distribution

Previous studies have established baseline expression levels for comparison: normal kidney tissues show "medium" staining with "moderate" intensities, while renal carcinoma tissues show "low" or "not detected" staining with "weak" intensities .

What are common challenges when working with ZNF844 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with ZNF844 antibodies:

• Background staining in IHC:

  • Solution: Optimize blocking conditions (try 5% BSA or 10% normal serum)

  • Increase washing steps duration and frequency

  • Optimize antibody dilution (start with manufacturer's recommended range of 1:50 - 1:200)

  • Consider using polymer-based detection systems to reduce non-specific binding

• Weak or absent signal:

  • Solution: Optimize antigen retrieval (test both citrate and EDTA buffers)

  • Increase antibody concentration or incubation time

  • Use signal amplification systems (tyramide signal amplification)

  • Ensure proper storage of antibody to maintain activity (avoid freeze-thaw cycles)

• Inconsistent results between samples:

  • Solution: Standardize fixation times for tissues

  • Process all samples simultaneously when possible

  • Include positive controls (normal kidney tissue) in each experiment

  • Use automated staining platforms if available

• Cross-reactivity concerns:

  • Solution: Validate antibody specificity using knockdown/knockout controls

  • Perform blocking peptide experiments with the immunizing peptide

  • Check for potential cross-reactive proteins using sequence alignment tools

How can researchers interpret discrepancies between ZNF844 mRNA and protein expression data?

When faced with discrepancies between ZNF844 mRNA and protein expression data, researchers should consider:

• Post-transcriptional regulation:

  • Analyze potential miRNA regulation using prediction tools

  • Investigate RNA stability using actinomycin D chase experiments

  • Assess alternative splicing patterns by RT-PCR with isoform-specific primers

• Post-translational modifications:

  • Examine ubiquitination status to assess protein degradation

  • Investigate phosphorylation states that might affect protein stability

  • Consider acetylation or other modifications that could affect antibody recognition

• Technical considerations:

  • Evaluate antibody specificity for different protein isoforms

  • Assess subcellular localization (nuclear vs. cytoplasmic fractions)

  • Compare sensitivity limits of RNA vs. protein detection methods

• Experimental validation approaches:

  • Pulse-chase experiments to measure protein half-life

  • Proteasome inhibitors to assess degradation pathways

  • Combined RNA-protein extraction from the same sample for direct correlation

Research has shown that ZNF844 expression can vary significantly across different pathological stages and grades of ccRCC, so context-specific regulation should be considered when interpreting discrepant results .