ZNF195 Antibody

What is ZNF195 and why is it significant for research?

ZNF195 (Zinc Finger Protein 195) belongs to the Krueppel C2H2-type zinc-finger protein family and functions as a transcription factor implicated in various cellular processes. Its significance stems from its structural features including an N-terminal KRAB domain and multiple C2H2-type zinc fingers (10-14) at its C-terminus . The gene is strategically located near the centromeric border of chromosome 11p15.5, adjacent to an imprinted domain associated with maternal-specific loss of heterozygosity in Wilms' tumors, making it potentially relevant for cancer research .

What tissue expression patterns characterize ZNF195?

ZNF195 displays distinct tissue-specific expression patterns:

• Adult tissues: Predominantly expressed in heart, brain, placenta, skeletal muscle, and pancreas with a 4.3 kb transcript

• Limited expression in adult lung, liver, and kidney

• Fetal tissues: The predominant transcript in fetal lung, liver, kidney, and brain is 3.5 kb

• Fetal brain uniquely expresses both 3.5 kb and 4.3 kb transcripts

When designing experiments to study ZNF195, these expression patterns are crucial for selecting appropriate cell lines or tissue samples and interpreting results contextually.

What are the main applications for ZNF195 antibodies?

ZNF195 antibodies are utilized in multiple experimental techniques:

Most commercial antibodies have been validated for at least one of these applications, with Western blotting being the most commonly validated technique .

How should researchers approach alternative splicing when studying ZNF195?

Alternative splicing produces multiple ZNF195 transcript variants, requiring careful experimental design:

• Isoform awareness: RT-PCR analysis has demonstrated that exons 4a (containing an inverted Alu sequence) and 4b are differentially spliced and absent from the major transcript .

• Antibody targeting strategy: Select antibodies targeting regions conserved across isoforms for pan-ZNF195 detection, or target unique regions for isoform-specific studies. The calculated molecular weights vary significantly: 10 kDa, 64 kDa, 69 kDa, 70 kDa, and 72 kDa depending on the isoform .

• Validation approach: When validating antibody specificity, use positive controls expressing known isoforms and include negative controls where ZNF195 is not expressed or has been knocked down.

• Data interpretation: The observed molecular weight of approximately 65 kDa on Western blots may not align with theoretical predictions due to post-translational modifications or the specific isoform being detected .

What considerations are important when studying ZNF195's role in transcriptional regulation?

ZNF195's KRAB domain is thought to interact with KAP1, thereby recruiting histone-modifying proteins , suggesting the following methodological approaches:

• Protein complex analysis: Co-immunoprecipitation using ZNF195 antibodies can identify interacting partners like KAP1 or histone modifiers.

• Chromatin studies: Combine ChIP-seq with ZNF195 antibodies to identify genomic binding sites, followed by gene expression analysis after ZNF195 manipulation.

• Domain-specific functions: Use antibodies targeting different domains (N-terminal KRAB domain versus C-terminal zinc fingers) to distinguish domain-specific interactions and functions.

• Epigenetic regulation: Integrate studies of histone modifications at ZNF195 binding sites to understand its impact on chromatin structure.

How does ZNF195's chromosomal location influence experimental design?

ZNF195's proximity to an imprinted domain associated with Wilms' tumors on chromosome 11p15.5 necessitates:

• Allele-specific approach: Design experiments that can distinguish maternal and paternal allele expression.

• Disease model selection: When studying ZNF195 in cancer contexts, include Wilms' tumor samples to investigate potential roles in tumor suppression or progression.

• Genetic linkage: Consider neighboring genes and regulatory elements when interpreting results of genetic manipulation experiments.

• Epigenetic landscape: Analyze DNA methylation patterns and chromatin accessibility in this chromosomal region when studying ZNF195 regulation.

What criteria should guide ZNF195 antibody selection for research applications?

When selecting a ZNF195 antibody, consider these critical factors:

• Target epitope location: Antibodies targeting different regions (N-terminal, C-terminal, or internal) may yield different results. For example, ABIN2780522 targets the N-terminal region with the sequence "EWKCLDLAQQNLYRDVMLENYRNLFSVGLTVCKPGLITCLEQRKEPWNVK" .

• Species reactivity: Most ZNF195 antibodies react with human samples, while cross-reactivity with mouse and rat varies. Specific reactivity percentages include: Dog: 86%, Horse: 86%, Human: 100%, Mouse: 86%, Rabbit: 86%, and Rat: 86% for some antibodies .

• Validated applications: Ensure the antibody has been validated for your specific application. Most are validated for Western blot, but validation for IHC, IF, or ELISA varies significantly .

• Clonality: Most available ZNF195 antibodies are polyclonal, which may provide broader epitope recognition but potentially more batch-to-batch variability .

What is the optimal Western blotting protocol for ZNF195 detection?

For optimal ZNF195 detection by Western blot:

• Sample preparation:

  • Include protease inhibitors to prevent degradation

  • Use RIPA or NP-40 buffer for nuclear protein extraction

  • Denature samples at 95°C for 5 minutes in reducing conditions

• Gel selection and transfer:

  • Use 10% SDS-PAGE gels for optimal separation

  • Transfer to PVDF membranes at 100V for 60-90 minutes

• Antibody incubation:

  • Block with 5% non-fat milk in TBST for 1 hour

  • Incubate with primary ZNF195 antibody at 1:500-1:2000 dilution overnight at 4°C

  • Wash 3×10 minutes with TBST

  • Incubate with appropriate secondary antibody at 1:5000-1:10000 for 1 hour

• Detection and troubleshooting:

  • Expect bands at approximately 65 kDa, though this may vary by isoform

  • Positive controls should include samples known to express ZNF195 (e.g., HL-60, SW620, A549 cells, mouse brain)

  • If multiple bands appear, validate with knockout/knockdown controls

How can immunofluorescence for ZNF195 be optimized?

For optimal immunofluorescence detection of ZNF195:

• Fixation and permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 for 10 minutes

  • For tissue sections, antigen retrieval may be necessary (citrate buffer pH 6.0)

• Antibody parameters:

  • Use 1:50-1:200 dilution of primary antibody

  • Incubate overnight at 4°C in a humidified chamber

  • Use fluorophore-conjugated secondary antibodies at 1:500

• Controls and counterstaining:

  • Include nuclear counterstain (e.g., DAPI) to confirm nuclear localization

  • Use cell lines with known ZNF195 expression as positive controls (A549 cells are validated)

  • Include secondary-only controls to assess background

• Interpretation:

  • Expect primarily nuclear localization with potential cytoplasmic signal

  • Co-localization with other nuclear markers can confirm specificity

Why might there be discrepancies between calculated and observed molecular weights for ZNF195?

The calculated molecular weights for ZNF195 range from 10-72 kDa depending on the isoform, while the observed molecular weight is typically around 65 kDa . This discrepancy may result from:

• Post-translational modifications: Phosphorylation, SUMOylation, or other modifications common in transcription factors can alter migration patterns.

• Protein structure: The high number of zinc finger domains (10-14) can affect protein mobility in SDS-PAGE.

• Isoform detection: Different antibodies may detect different isoforms. For example, antibodies targeting N-terminal regions won't detect isoforms lacking that region.

• Technical factors: Gel percentage, running conditions, and molecular weight markers can all influence apparent molecular weight.

To address this issue, researchers should validate with multiple antibodies targeting different epitopes and consider using isoform-specific positive controls .

What strategies can minimize non-specific binding of ZNF195 antibodies?

To reduce non-specific binding:

• Blocking optimization:

  • Test different blocking agents: 5% BSA may be superior to milk for phospho-specific antibodies

  • Increase blocking time to 2 hours at room temperature

  • Add 0.1% Tween-20 to blocking buffer

• Antibody dilution:

  • Optimize primary antibody dilution (start with manufacturer's recommendation then adjust)

  • For Western blot: test 1:500, 1:1000, and 1:2000 dilutions

  • For IF/IHC: test 1:50, 1:100, and 1:200 dilutions

• Incubation conditions:

  • Longer incubation at 4°C (overnight) with more dilute antibody often reduces background

  • Use gentle rocking/rotation to ensure even antibody distribution

• Washing steps:

  • Increase wash duration and number (5×5 minutes instead of 3×5 minutes)

  • Add additional salt (up to 500 mM NaCl) to wash buffer for high-background antibodies

How can researchers validate ZNF195 antibody specificity?

Comprehensive validation should include:

• Genetic controls:

  • siRNA/shRNA knockdown: Reduction in signal intensity proportional to knockdown efficiency

  • CRISPR knockout: Complete absence of specific signal

  • Overexpression: Increased signal intensity at the expected molecular weight

• Peptide competition:

  • Pre-incubation of antibody with immunizing peptide should abolish specific signal

  • Non-competing peptides should have no effect on signal

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of ZNF195

  • Consistent results across antibodies increase confidence in specificity

• Tissue/cell type controls:

  • Test tissues with known expression patterns (e.g., positive: brain, heart; negative: adult kidney)

  • Developmentally regulated expression (fetal vs. adult tissue) can provide additional validation

How can ZNF195 antibodies be utilized in chromatin immunoprecipitation (ChIP) studies?

While none of the antibodies in the search results explicitly mention ChIP validation, researchers can adapt ZNF195 antibodies for this application:

• Antibody selection criteria:

  • Choose antibodies that work well in immunoprecipitation assays

  • Target epitopes unlikely to be masked by DNA binding (N-terminal antibodies may be preferable)

  • Polyclonal antibodies often perform better in ChIP than monoclonals

• Protocol optimization:

  • Crosslinking time may need optimization (start with 10 minutes of 1% formaldehyde)

  • Sonication conditions must be optimized to generate 200-500 bp fragments

  • Use higher antibody concentrations than for Western blotting (5-10 μg per IP)

  • Include appropriate controls (IgG negative control, histone H3 positive control)

• Data analysis considerations:

  • Look for enrichment at promoters and enhancers given ZNF195's role as a transcription factor

  • Integrate with gene expression data after ZNF195 manipulation

  • Consider the 10 C2H2-type zinc fingers when analyzing DNA binding motifs

How can protein-protein interactions of ZNF195 be studied using antibodies?

Given ZNF195's KRAB domain interaction with KAP1 and recruitment of histone modifiers , these approaches are recommended:

• Co-immunoprecipitation (Co-IP):

  • Use 1-2 mg of nuclear extract per IP

  • Include appropriate wash controls to remove non-specific binders

  • Validate interactions bidirectionally (IP ZNF195 and probe for partners; IP partners and probe for ZNF195)

  • Test interactions in multiple cell types relevant to ZNF195 expression

• Proximity ligation assay (PLA):

  • This technique can detect protein-protein interactions in situ

  • Requires two antibodies raised in different species (one for ZNF195, one for potential interactor)

  • Provides spatial information about where interactions occur within cells

• Mass spectrometry after IP:

  • IP ZNF195 using validated antibodies

  • Analyze by mass spectrometry to identify novel binding partners

  • Validate hits by targeted Co-IP or functional studies

What approaches can integrate ZNF195 antibody use with functional genomics?

To comprehensively understand ZNF195 function:

• ChIP-seq with RNA-seq integration:

  • Use ZNF195 antibodies for ChIP-seq to identify binding sites

  • Perform RNA-seq after ZNF195 depletion/overexpression

  • Integrate datasets to identify direct transcriptional targets

• CRISPR screening with antibody validation:

  • Perform CRISPR screens to identify genetic interactions with ZNF195

  • Use ZNF195 antibodies to validate effects on protein levels and localization

  • Study downstream effects on target gene expression

• Proteomics approaches:

  • Study ZNF195 interactome changes under different conditions

  • Analyze post-translational modifications using modification-specific antibodies

  • Correlate with functional outcomes and transcriptional changes

• Disease model applications:

  • Given ZNF195's location near a tumor-associated locus , analyze expression and localization in Wilms' tumor samples

  • Study correlation between ZNF195 binding patterns and epigenetic alterations in disease states