ZNF175 Antibody

What is ZNF175 and what are its key biological functions?

ZNF175, also known as Zinc finger protein OTK18, functions primarily as a transcriptional suppressor that down-regulates the expression of several chemokine receptors. This protein plays a critical role in interfering with HIV-1 replication by suppressing Tat-induced viral LTR promoter activity . ZNF175 expression in brain mononuclear phagocytes serves as a molecular signature for advanced HIV-1 encephalitis, indicating its importance in neuroimmunological responses during viral infection . The protein is encoded by the ZNF175 gene (gene ID: 7728) and has a predicted molecular weight of approximately 82 kDa . Understanding these functions is essential for researchers designing experiments to investigate transcriptional regulation mechanisms or viral-host interactions involving ZNF175.

What is the structural composition of ZNF175?

ZNF175 is characterized by multiple zinc finger domains, which are common structural motifs in transcription factors that facilitate DNA binding. According to the recombinant protein information, one important fragment corresponds to amino acids QNQIQPMSHSAFFNKKTLNTESNCEYKDPGKMIRTRPHLASSQKQPQKCCLFTESLKLNLEVNGQNESNDTEQLDDVVGSGQLFSHSSSDACSKNIHTGETFCKGNQCRKVCGHKQSLKQHQ, which likely contains functional domains critical for its activity . The full-length protein consists of 711 amino acids, as indicated by antibodies targeting the full length (AA 1-711) . When working with ZNF175 antibodies, researchers should consider which epitopes or structural regions are being targeted, as this can affect experimental outcomes depending on protein folding, accessibility, and post-translational modifications.

How evolutionarily conserved is ZNF175 across species?

ZNF175 shows variable conservation across species, which is an important consideration when selecting appropriate experimental models. BLAST analysis reveals 100% identity between human and chimpanzee ZNF175, with decreasing conservation in other primates: gorilla and monkey (92%), galago (90%), gibbon and marmoset (85%) . The recombinant protein fragment (amino acids 59-128) shows only 51% identity with both mouse and rat orthologs . This evolutionary divergence should inform researchers' choices of model organisms and interpretation of cross-species experiments. When studying ZNF175 in non-human models, researchers should select antibodies validated for cross-reactivity or consider species-specific antibodies to ensure reliable results.

What types of ZNF175 antibodies are available for research applications?

Researchers have multiple options when selecting ZNF175 antibodies, including:

• Clonality variations:

  • Polyclonal antibodies (e.g., rabbit polyclonal targeting N-terminal regions)

  • Monoclonal antibodies (e.g., mouse monoclonal clone 1C2)

  • Recombinant monoclonal antibodies (e.g., rabbit recombinant monoclonal EPR8391(2))

• Host species options:

  • Rabbit-derived antibodies (predominant in available products)

  • Mouse-derived antibodies

• Target region specificity:

  • N-terminal targeting antibodies

  • C-terminal targeting antibodies

  • Full-length protein targeting antibodies

  • Specific amino acid region antibodies (e.g., AA 150-187, AA 81-178)

Each antibody type offers distinct advantages depending on experimental requirements. Selection should be guided by the specific research question, application method, and sample type to be analyzed.

What considerations should guide the selection between polyclonal and monoclonal ZNF175 antibodies?

When choosing between polyclonal and monoclonal ZNF175 antibodies, researchers should consider:

Polyclonal advantages:

• Broader epitope recognition, potentially increasing detection sensitivity

• More resilient to protein denaturation or modifications

• Often suitable for applications like Western blotting and IHC where proteins may be partially denatured

• Available for various species reactivity (human, cow, horse, dog, mouse, pig, rat)

Monoclonal advantages:

• Highly specific to a single epitope

• Reduced batch-to-batch variation

• Superior for applications requiring precise epitope targeting

• Recombinant monoclonal antibodies like EPR8391(2) combine specificity with reproducibility

For initial characterization or detection in complex samples, polyclonal antibodies might be preferable. For experiments requiring high specificity or longitudinal studies where consistency is critical, monoclonal antibodies are often more suitable.

How should researchers validate ZNF175 antibody specificity for their experimental systems?

Proper validation of ZNF175 antibodies is crucial for experimental reliability. Recommended validation approaches include:

• Positive control testing: Use cell lines known to express ZNF175, such as Neuro-2a, SH-SY5Y, 293T, or HepG2 cells, which have demonstrated detectable ZNF175 expression by Western blot .

• Peptide competition assays: Utilize recombinant protein fragments (such as human ZNF175 aa 59-128) in blocking experiments to confirm antibody specificity. For optimal results, pre-incubate the antibody with 100x molar excess of the protein fragment control for 30 minutes at room temperature before application .

• Cross-platform validation: Confirm antibody performance across multiple techniques (e.g., WB, IHC, IF) to ensure consistent target recognition in different contexts.

• Knockout/knockdown controls: When possible, include ZNF175 knockout or knockdown samples as negative controls.

• Orthogonal methods: Validate findings using multiple antibodies targeting different epitopes of ZNF175 or through complementary nucleic acid-based detection methods.

Proper validation not only ensures experimental reliability but also helps troubleshoot unexpected results that might arise from antibody cross-reactivity or non-specific binding.

What are the optimal applications for ZNF175 antibodies?

ZNF175 antibodies have been validated for several applications, each with specific optimization requirements:

The application selection should be guided by the specific research question. For protein expression quantification, Western blotting is appropriate; for localization studies in tissue or subcellular contexts, IHC or IF/ICC approaches are more suitable.

What is the recommended protocol for Western blot detection of ZNF175?

For optimal Western blot detection of ZNF175, researchers should follow this methodological approach:

• Sample preparation:

  • Prepare cell/tissue lysates using RIPA buffer with protease inhibitors

  • Load 10-20 μg of total protein per lane (as referenced in successful detections with Neuro-2a, SH-SY5Y, 293T, and HepG2 cell lysates)

• Electrophoresis and transfer:

  • Separate proteins using 8-10% SDS-PAGE (appropriate for the 82 kDa predicted size)

  • Transfer to PVDF or nitrocellulose membrane at 100V for 60-90 minutes

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary ZNF175 antibody at 1:100-1:500 dilution overnight at 4°C

  • Wash 3-5 times with TBST

  • Incubate with appropriate HRP-conjugated secondary antibody at 1:2000-1:5000 for 1 hour at room temperature

• Detection and interpretation:

  • Develop using ECL substrate

  • Expected band size is approximately 82 kDa

  • Consider including positive control lysates such as Neuro-2a, SH-SY5Y, 293T, or HepG2 cell lines

To increase specificity, antibody dilution and incubation conditions may need optimization. For challenging samples, consider using recombinant monoclonal antibodies like EPR8391(2) which may offer greater specificity.

How should researchers optimize immunohistochemical detection of ZNF175?

For successful immunohistochemical detection of ZNF175 in paraffin-embedded tissues, follow these methodological guidelines:

• Tissue preparation and antigen retrieval:

  • Use 4-6 μm thick paraffin sections

  • Perform heat-mediated antigen retrieval with citrate buffer pH 6, which is essential for epitope accessibility

  • Optimal antigen retrieval has been demonstrated in human colon and pancreas tissues

• Blocking and antibody application:

  • Block endogenous peroxidase with 3% H₂O₂

  • Apply protein block (serum-free) for 10-15 minutes

  • Incubate with ZNF175 antibody at appropriate dilution (1:50 for recombinant monoclonal, 1:50-1:200 for polyclonal antibodies)

  • Overnight incubation at 4°C typically yields optimal results

• Detection systems and visualization:

  • Use appropriate detection system (HRP-polymer or biotin-streptavidin)

  • Develop with DAB and counterstain with hematoxylin

  • Mount with permanent mounting medium

• Controls and validation:

  • Include positive tissue controls (colon, pancreas have been validated)

  • Use isotype controls to confirm specificity

  • Consider peptide competition assays to validate staining patterns

Optimization may be required for different tissue types, fixation conditions, or when studying tissues from different species due to variable cross-reactivity.

How can researchers address non-specific binding issues with ZNF175 antibodies?

Non-specific binding is a common challenge when working with antibodies. For ZNF175 antibodies, consider these methodological solutions:

• Antibody selection optimization:

  • Consider using immunoaffinity-purified antibodies, which undergo additional purification steps to reduce non-specific binding

  • For critical applications, recombinant monoclonal antibodies may offer higher specificity

• Blocking protocol enhancement:

  • Extend blocking time (2-3 hours instead of standard 1 hour)

  • Test different blocking agents (BSA, casein, commercial blockers) to identify optimal conditions

  • Add 0.1-0.3% Triton X-100 to blocking buffer for membrane permeabilization in ICC/IF applications

• Dilution optimization:

  • Perform antibody titration experiments to determine optimal concentration

  • For Western blotting, test dilutions between 0.2-1 μg/mL

  • For IHC-P, dilutions between 1:50-1:200 are recommended starting points

• Validation with competition assays:

  • Use the ZNF175 recombinant protein fragment (aa 59-128) for blocking experiments

  • Pre-incubate antibody with 100x molar excess of the protein fragment for 30 minutes at room temperature

  • Compare blocked versus unblocked antibody staining patterns

• Buffer optimization:

  • Add 0.1-0.5% Tween-20 or 0.1% SDS to wash buffers to reduce hydrophobic interactions

  • Consider using specialized low-background antibody diluents

These methodological approaches should be systematically tested to identify the optimal conditions for each specific experimental system.

What controls are essential when using ZNF175 antibodies?

Rigorous experimental design requires appropriate controls. For ZNF175 antibody experiments, include:

• Positive controls:

  • Cell lines with confirmed ZNF175 expression: Neuro-2a, SH-SY5Y, 293T, HepG2

  • Tissue samples: human colon and pancreas tissues have shown positive staining

• Negative controls:

  • Primary antibody omission control (all reagents except primary antibody)

  • Isotype control (irrelevant antibody of same isotype and concentration)

  • If available, ZNF175 knockout or knockdown samples

• Specificity controls:

  • Peptide competition assay using recombinant ZNF175 protein fragment

  • Multiple antibodies targeting different ZNF175 epitopes to confirm staining patterns

  • Cross-validation with orthogonal detection methods (e.g., in situ hybridization)

• Technical controls:

  • Loading controls for Western blotting (e.g., β-actin, GAPDH)

  • Internal tissue controls for IHC (tissues with known positive and negative regions)

Proper experimental controls not only validate results but also help troubleshoot unexpected findings and enhance the rigor and reproducibility of research involving ZNF175.

How should researchers interpret contradictory results with different ZNF175 antibodies?

When facing contradictory results using different ZNF175 antibodies, consider these methodological approaches:

• Epitope mapping analysis:

  • Compare target regions of the contradicting antibodies (N-term vs. C-term)

  • Different binding sites may have variable accessibility depending on protein conformation or post-translational modifications

  • Antibodies targeting different regions (AA 150-187 vs. AA 81-178) may yield different results based on epitope exposure

• Antibody validation assessment:

  • Review validation data for each antibody

  • Check antibody specificity validation methods (Western blot, peptide competition, etc.)

  • Consider antibody purification method (immunoaffinity purification may provide higher specificity)

• Technical consideration review:

  • Evaluate differences in sample preparation methods

  • Assess potential differences in antigen retrieval techniques (critical for IHC applications)

  • Compare experimental conditions (buffer compositions, incubation times, temperatures)

• Confirmatory approaches:

  • Employ orthogonal methods to validate results (mRNA expression, mass spectrometry)

  • Test additional antibodies targeting different epitopes

  • Consider genetic approaches (overexpression, knockdown) to confirm specificity

• Contextual interpretation:

  • Different cell types or tissues may express variable ZNF175 isoforms

  • Post-translational modifications may affect epitope accessibility

  • Environmental conditions (stress, infection) may alter protein conformation or localization

How can ZNF175 antibodies be utilized to study HIV-1 pathogenesis mechanisms?

ZNF175 antibodies can be instrumental in elucidating HIV-1 pathogenesis through these methodological approaches:

• Cellular expression profiling during infection:

  • Quantify ZNF175 expression levels in infected versus uninfected cells by Western blotting

  • Map expression patterns in brain tissue sections from HIV-1 encephalitis patients using IHC, as ZNF175 expression in brain mononuclear phagocytes serves as a signature for advanced HIV-1 encephalitis

  • Track temporal changes in ZNF175 expression during disease progression

• Mechanistic studies of viral suppression:

  • Investigate ZNF175's role in suppressing Tat-induced viral LTR promoter activity using chromatin immunoprecipitation (ChIP) with ZNF175 antibodies

  • Combine with reporter assays to quantify the impact of ZNF175 on viral transcription

  • Identify ZNF175 binding partners using co-immunoprecipitation followed by mass spectrometry

• Cellular localization studies:

  • Use immunofluorescence to track ZNF175 nuclear translocation during infection

  • Perform co-localization studies with viral proteins to identify potential interaction sites

  • Employ super-resolution microscopy with fluorescently conjugated ZNF175 antibodies to map precise subcellular distribution

• Therapeutic intervention assessment:

  • Evaluate changes in ZNF175 expression/activity following antiviral treatment

  • Screen for compounds that modulate ZNF175 activity using cellular assays

  • Monitor ZNF175 as a potential biomarker for disease progression or treatment response

These approaches can provide critical insights into how ZNF175 functions in the context of HIV-1 infection and could potentially reveal novel therapeutic targets.

What considerations are important for using ZNF175 antibodies in co-immunoprecipitation studies?

For successful co-immunoprecipitation (Co-IP) studies with ZNF175 antibodies, researchers should consider these methodological guidelines:

• Antibody selection criteria:

  • Choose antibodies that recognize native protein conformations

  • Polyclonal antibodies may capture more target protein due to recognition of multiple epitopes

  • Consider antibodies with demonstrated affinity purification qualities

  • Test both N-terminal and C-terminal targeting antibodies as epitope accessibility may differ in protein complexes

• Lysis buffer optimization:

  • Use gentle, non-denaturing lysis buffers (e.g., NP-40 or Triton X-100 based)

  • Include protease and phosphatase inhibitors to preserve protein interactions

  • Adjust salt concentration to maintain specific interactions while reducing non-specific binding

  • Consider adding protein stabilizers such as glycerol (10%) if necessary

• Experimental controls:

  • Include isotype control antibody to identify non-specific binding

  • Perform reverse Co-IP to confirm interactions

  • Consider using ZNF175 knockdown/knockout cells as negative controls

  • Include input samples for quantitative comparison

• Detection strategies:

  • Use clean detection antibodies from different host species than the IP antibody

  • Consider antibodies targeting different ZNF175 epitopes for detection versus capture

  • For complex samples, consider mass spectrometry for unbiased interaction partner identification

• Technical considerations:

  • Pre-clear lysates to reduce non-specific binding

  • Optimize antibody-to-lysate ratios (typically 2-5 μg antibody per 500 μg total protein)

  • Consider crosslinking approaches for transient or weak interactions

  • Optimize wash stringency to balance between preserving specific interactions and removing non-specific binding

These methodological considerations can significantly improve the success and specificity of ZNF175 co-immunoprecipitation experiments.

How can researchers utilize ZNF175 antibodies to investigate its role in transcriptional regulation?

To investigate ZNF175's role as a transcriptional regulator, researchers can employ these advanced methodological approaches:

• Chromatin immunoprecipitation (ChIP) studies:

  • Use ZNF175 antibodies to immunoprecipitate chromatin complexes containing ZNF175

  • Follow with sequencing (ChIP-seq) to map genome-wide binding sites

  • Validate with ChIP-qPCR for specific target genes, particularly chemokine receptors and HIV-1 LTR regions

  • Consider the recombinant monoclonal antibody for enhanced specificity in ChIP applications

• Transcriptional activity assessment:

  • Combine ZNF175 immunoprecipitation with mass spectrometry to identify co-factors

  • Perform sequential ChIP (re-ChIP) to study co-occupancy with other transcription factors

  • Correlate ZNF175 binding with histone modifications using dual ChIP approaches

  • Map ZNF175 binding relative to transcription start sites to understand regulatory mechanisms

• Functional validation experiments:

  • Use ZNF175 antibodies in electrophoretic mobility shift assays (EMSA) to confirm direct DNA binding

  • Correlate ZNF175 binding patterns with gene expression changes using RNA-seq

  • Employ reporter gene assays to quantify transcriptional suppression capacities at specific promoters

  • Perform CRISPR-mediated mutagenesis of ZNF175 binding sites and assess effects on gene expression

• Dynamic regulation studies:

  • Track ZNF175 occupancy changes during cellular activation or viral infection

  • Perform time-course studies to correlate ZNF175 binding with transcriptional changes

  • Investigate post-translational modifications of ZNF175 that might regulate its activity

These approaches can provide comprehensive insights into how ZNF175 functions as a transcriptional suppressor and help elucidate its role in regulating chemokine receptors and viral gene expression.