ZNF174 Antibody

What is ZNF174 and why is it a target for antibody development?

ZNF174, also known as ZSCAN8 or AW-1, is a transcriptional repressor belonging to the Krueppel C2H2-type zinc-finger protein family. It specifically represses promoter activities of PDGF-B and TGF beta1. ZNF174 localizes to the nucleus and contains three C2H2-type zinc fingers at the C-terminus and one SCAN domain near the N-terminus . The protein has a molecular weight of approximately 46 kDa and is expressed in various tissues, with highest expression in adult ovary and testis . Due to its role in transcriptional regulation and tissue-specific expression patterns, antibodies against ZNF174 are valuable tools for studying gene regulation mechanisms and tissue-specific functions.

What types of ZNF174 antibodies are currently available for research?

Based on the search results, several types of ZNF174 antibodies are available for research:

The antibodies vary in their epitope specificity, with some targeting specific regions like amino acids 201-250 or 37-132 of the ZNF174 protein.

How should I optimize Western blot protocols specifically for ZNF174 detection?

For optimal Western blot detection of ZNF174:

• Sample preparation: ZNF174 has been successfully detected in various cell lines including BxPC-3, HeLa, and HepG2 cells .

• Antibody dilution: Use the following recommended dilutions:

  • Polyclonal antibody (Proteintech 18157-1-AP): 1:500-1:2000

  • Nordic Biosite polyclonal: 1:500-1:2000

  • Sigma Aldrich antibody: 0.04-0.4 μg/mL

• Expected molecular weight: Look for a band at approximately 46 kDa, which corresponds to the calculated and observed molecular weight of ZNF174 .

• Controls: Include positive controls from tissues with high ZNF174 expression (testis, ovary) and negative controls to verify antibody specificity.

• Buffer system: Based on the antibody storage conditions, PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 may provide a compatible environment for the antibody during incubation .

Always perform antibody titration in your specific experimental system to determine the optimal concentration for your samples.

What are the recommended protocols for immunohistochemistry using ZNF174 antibodies?

For IHC applications using ZNF174 antibodies:

• Tissue preparation: ZNF174 has been successfully detected in human testis, brain, heart, kidney, lung, ovary, placenta, skin, and spleen tissues .

• Antigen retrieval: Use either:

  • TE buffer pH 9.0 (primary recommendation)

  • Citrate buffer pH 6.0 (alternative method)

• Antibody dilution:

  • Proteintech polyclonal: 1:20-1:200

  • Nordic Biosite polyclonal: 1:100-1:300

  • Sigma Aldrich antibody: 1:50-1:200

• Detection system: Choose an appropriate secondary antibody and visualization system compatible with rabbit or mouse primary antibodies, depending on which antibody you're using.

• Positive controls: Include tissues known to express ZNF174, such as testis and ovary, which show the highest expression levels .

• Negative controls: Include samples processed without primary antibody to assess non-specific binding.

How can I use ZNF174 antibodies to study the SCAN domain dimerization properties?

The SCAN domain of ZNF174 forms homodimers, which is critical for its function in mediating protein-protein interactions . To study SCAN domain dimerization:

• Co-immunoprecipitation: Use ZNF174 antibodies that recognize the SCAN domain (aa 37-132) to immunoprecipitate ZNF174 and its binding partners. The monoclonal antibody PCRP-ZNF174-1D12 has been specifically validated for immunoprecipitation applications .

• Size exclusion chromatography with immunodetection: Follow the method described by Williams et al., which demonstrated that the ZNF174 SCAN domain forms a homodimer :

  • Purify the SCAN domain (residues 37-132)

  • Perform size exclusion chromatography

  • Analyze fractions by Western blot using ZNF174 antibodies

• Mammalian two-hybrid assay: As described by Stone et al., this assay can detect interactions between SCAN domains:

  • Use SCAN domain regions fused to GAL4 and VP16 expression vectors

  • Measure CAT reporter gene activation when interactions occur

  • Evaluate interactions between different SCAN domains

• Circular dichroism (CD) spectroscopy: Use this technique to analyze the secondary structure of the SCAN domain and compare wild-type with mutant forms:

  • Prepare recombinant ZNF174 SCAN domain at 5-25 μM concentration

  • Record CD spectra at 4-25°C from 260 to 195/205 nm

  • Analyze data for characteristic α-helical signatures (dips at 208 and 222 nm)

The research by Williams et al. demonstrated that "both size exclusion chromatography and equilibrium sedimentation analysis demonstrate that the ZNF174 SCAN domain forms a homodimer" .

How can ZNF174 antibodies be used to investigate its role in transcriptional repression?

ZNF174 functions as a transcriptional repressor. To investigate this function:

• Chromatin immunoprecipitation (ChIP):

  • Use ZNF174 antibodies to immunoprecipitate ZNF174-bound chromatin

  • Sequence the immunoprecipitated DNA to identify genome-wide binding sites

  • Focus on promoter regions of PDGF-B and TGF beta1, which are known targets of ZNF174 repression

• Reporter gene assays:

  • Similar to studies on ZNF217 by Vandevenne et al., construct reporter genes containing ZNF174 binding sites

  • Co-express ZNF174 and measure repression of reporter gene activity

  • Use ZNF174 antibodies in parallel assays to confirm expression and nuclear localization

• Co-immunoprecipitation with co-repressors:

  • SCAN domain proteins often recruit transcriptional co-repressors

  • Use ZNF174 antibodies to immunoprecipitate protein complexes

  • Identify co-repressors by Western blot or mass spectrometry

• Immunofluorescence co-localization:

  • Use ZNF174 antibodies (dilution 1:10-1:100 for IF/ICC ) to visualize nuclear localization

  • Co-stain with antibodies against transcriptional co-repressors

  • Analyze co-localization patterns in different cell types

How can I resolve issues with non-specific binding when using ZNF174 antibodies?

When encountering non-specific binding with ZNF174 antibodies:

• Antibody validation: Verify antibody specificity using:

  • Recombinant expression validation as performed for Sigma's antibody

  • Positive control tissues with known ZNF174 expression (testis, ovary)

  • Negative controls (primary antibody omission)

• Blocking optimization:

  • For Western blot: Use 5% non-fat dry milk or BSA in TBST

  • For IHC/IF: Use 0.5% BSA as indicated in some ZNF174 antibody buffer formulations

• Dilution series:

  • Test a range of antibody dilutions beyond the recommended ranges

  • For WB: 1:500 to 1:5000

  • For IHC: 1:20 to 1:500

  • For IF/ICC: 1:10 to 1:500

• Cross-reactivity assessment:

  • Consider potential cross-reactivity with other ZNF family members

  • ZNF7 is an important paralog that could potentially cross-react

  • Validate results with a second antibody targeting a different epitope

• Sample preparation:

  • Ensure proper fixation for IHC (antigen retrieval with TE buffer pH 9.0 is recommended )

  • For WB, use fresh samples with protease inhibitors to prevent degradation

How should I interpret ZNF174 expression data in different tissues and disease states?

When interpreting ZNF174 expression data:

• Normal tissue expression baseline:

  • Highest expression occurs in adult testis and ovary

  • Moderate expression in small intestine, colon, prostate, thymus, spleen, pancreas, skeletal muscle

  • Lower expression in heart, brain, and kidney

  • Positive IHC detection has been confirmed in human testis, brain, heart, kidney, lung, ovary, placenta, skin, and spleen tissues

• Cell type specificity:

  • ZNF174 is expressed in umbilical vein endothelial cells, foreskin fibroblast, and HepG2 cells

  • Positive WB detection in BxPC-3, HeLa, and HepG2 cells

  • Positive IF/ICC detection in HeLa cells

• Cancer expression patterns:

  • The Human Protein Atlas provides data on ZNF174 expression across 17 different cancer types

  • Consider correlation between mRNA expression and patient survival when available

• Subcellular localization:

  • ZNF174 primarily localizes to the nucleus

  • Confirm nuclear localization with DAPI co-staining in IF/ICC experiments

• Quantification methods:

  • For IHC: Consider both staining intensity and percentage of positive cells

  • For WB: Normalize ZNF174 band intensity to loading controls like β-actin or GAPDH

  • For qPCR validation: Use specific primers for ZNF174 to confirm antibody results

How can ZNF174 antibodies be used in single-cell applications to study cell heterogeneity?

For single-cell applications using ZNF174 antibodies:

• Single-cell immunofluorescence:

  • Use ZNF174 antibodies at optimized dilutions (1:10-1:100 or 1:200-1:1000 )

  • Combine with markers for specific cell types within heterogeneous tissues

  • Quantify expression at single-cell level using high-content imaging

• Mass cytometry (CyTOF):

  • Conjugate ZNF174 antibodies with rare earth metals

  • Combine with other antibodies to identify cell populations

  • Analyze expression patterns across different cell types

• Single-cell Western blot:

  • Adapt traditional Western blot protocols to microfluidic platforms

  • Use recommended antibody dilutions (1:500-1:2000 )

  • Quantify ZNF174 expression in individual cells

• Integration with transcriptomics:

  • Correlate protein-level detection of ZNF174 with single-cell RNA-seq data

  • Validate expression patterns observed at transcriptomic level

  • Identify potential post-transcriptional regulation mechanisms

What are the most effective approaches for studying ZNF174 mutations and their effect on protein function?

To study ZNF174 mutations and their functional impacts:

• Site-directed mutagenesis coupled with functional assays:

  • Generate specific mutations in the SCAN domain (residues 43-126) or zinc finger regions

  • Express mutant proteins in appropriate cell systems

  • Use ZNF174 antibodies to confirm expression and localization

  • Assess impact on dimerization and transcriptional repression

• Circular dichroism (CD) spectroscopy for structural assessment:

  • Compare wild-type and mutant ZNF174 SCAN domains

  • As shown by Stone et al., CD spectroscopy revealed that the characteristic α-helical structure is disrupted by mutations (e.g., L→P at positions 44-45)

  • Prepare recombinant proteins at 5 μM concentration in PBS with 0.2 mM DTT

  • Record CD spectra from 260 to 205 nm using established protocols

• Mammalian two-hybrid system for interaction studies:

  • Assess how mutations affect the ability of ZNF174 to form dimers

  • Stone et al. demonstrated that the SCAN domain mediates self-association through this assay system

  • Mutations in key residues disrupt this interaction

• Co-immunoprecipitation with mutant proteins:

  • Express tagged wild-type and mutant ZNF174 proteins

  • Perform co-IP using ZNF174 antibodies

  • Assess impact of mutations on protein-protein interactions