ZNF75A Antibody

What is ZNF75A and why is it studied in research?

ZNF75A (Zinc Finger Protein 75A) is a C2H2-type transcription factor that may be involved in transcriptional regulation . As part of the zinc finger protein family, it contains DNA-binding domains that potentially regulate gene expression. While less extensively studied than some other zinc finger proteins, researchers investigate ZNF75A to understand its possible role in normal cellular processes and disease states, particularly given findings about related zinc finger proteins like ZNF275, which has been implicated in cancer pathways .

What types of ZNF75A antibodies are currently available for research?

Currently available ZNF75A antibodies include polyclonal antibodies primarily derived from rabbit hosts. These are available in various forms including unconjugated versions and those conjugated with biotin, HRP, or FITC for different detection methods . Most commercially available antibodies target either the N-terminal region or specific amino acid sequences (AA 1-296, AA 98-124) of human ZNF75A . These antibodies have been validated for techniques including Western blotting, immunohistochemistry, immunofluorescence, and ELISA, though validation extent varies by supplier and specific antibody .

How specific are current ZNF75A antibodies and what cross-reactivity should researchers be aware of?

Current ZNF75A antibodies are primarily developed for human ZNF75A reactivity, with most showing 100% predicted reactivity to human sequences . Limited information exists about cross-reactivity with other species, though some antibodies show reactivity with monkey samples . The specificity of these antibodies depends on their production method, with affinity-purified antibodies generally offering higher specificity. Researchers should be aware that potential cross-reactivity with other zinc finger proteins might occur due to structural similarities in the zinc finger domains. Validation using appropriate controls, including lysates from cells with ZNF75A knockdown, is recommended to confirm specificity for individual experimental systems .

What are the validated applications for ZNF75A antibodies in current research?

ZNF75A antibodies have been validated for several research applications, with varying degrees of optimization across suppliers. The primary validated applications include:

These applications allow researchers to investigate ZNF75A expression, localization, and interactions within cellular contexts. The most robust validation has been performed for Western blotting, making this a reliable starting point for new investigations .

How can ZNF75A antibodies be effectively used in immunohistochemistry for tissue distribution studies?

For effective immunohistochemistry (IHC) using ZNF75A antibodies, researchers should follow these methodological considerations:

• Fixation protocol: Use 4% paraformaldehyde fixation for 15 minutes for cell samples or standard formalin fixation for tissue samples .

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is generally recommended for zinc finger proteins.

• Blocking: Use 5-10% normal serum corresponding to the secondary antibody host.

• Primary antibody: Apply ZNF75A antibody at 1:50 to 1:200 dilution and incubate overnight at 4°C .

• Detection system: Use an appropriate detection system compatible with rabbit primary antibodies.

• Controls: Include positive controls (tissues known to express ZNF75A) and negative controls (primary antibody omission and/or ZNF75A-knockdown samples) .

The extensive validation performed by the Human Protein Atlas project provides reference data for expected ZNF75A expression patterns across 44 normal human tissues, offering valuable comparative data for researchers .

What are the critical considerations for using ZNF75A antibodies in Western blotting?

For optimal Western blotting results with ZNF75A antibodies, researchers should consider:

• Protein extraction: Use RIPA buffer supplemented with protease inhibitors for most cell types.

• Sample loading: Load 20-40 μg of total protein per lane.

• Antibody concentration: Use ZNF75A antibodies at 0.04-0.4 μg/mL for optimal signal-to-noise ratio .

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Incubation: Primary antibody incubation overnight at 4°C, followed by appropriate HRP-conjugated secondary antibody.

• Validation controls: Include positive control lysates and, ideally, ZNF75A knockdown samples as negative controls.

• Detection: Both chemiluminescence and fluorescence-based detection systems are compatible.

For detecting endogenous ZNF75A, which may be expressed at low levels in some cell types, enhanced chemiluminescence reagents and longer exposure times may be necessary. Ensuring complete transfer of high-molecular-weight proteins is important, as ZNF75A contains multiple zinc finger domains .

How can ZNF75A antibodies be utilized for studying protein-protein interactions?

ZNF75A antibodies can be valuable tools for investigating protein-protein interactions through several approaches:

• Co-immunoprecipitation (Co-IP): Using ZNF75A antibodies to pull down ZNF75A and associated protein complexes. This requires:

  • Mild lysis conditions to preserve protein interactions

  • Optimized antibody concentrations (typically 2-5 μg of antibody per mg of protein lysate)

  • Appropriate controls including IgG controls and reverse Co-IP

• Proximity Ligation Assay (PLA): For detecting in situ protein interactions with potential binding partners:

  • Requires two primary antibodies from different host species

  • Can combine ZNF75A antibody with antibodies against suspected interaction partners

  • Provides spatial resolution of interactions within cells

• Chromatin Immunoprecipitation (ChIP): For studying DNA-binding properties of ZNF75A:

  • Use 3-5 μg of ZNF75A antibody per ChIP reaction

  • Fixation with 1% formaldehyde for 10 minutes

  • Sonication to generate 200-500 bp DNA fragments

Each method requires validation of antibody specificity and optimization of experimental conditions. Given ZNF75A's potential role as a transcription factor, ChIP approaches may be particularly informative for understanding its gene regulatory functions .

What are the common challenges when using ZNF75A antibodies and how can they be addressed?

Researchers working with ZNF75A antibodies may encounter several challenges:

• Low signal intensity:

  • Increase antibody concentration or incubation time

  • Use signal amplification methods (TSA for IHC/IF, enhanced chemiluminescence for WB)

  • Enrich for nuclear fraction in Western blotting as ZNF75A is likely concentrated in nuclei

• High background or non-specific binding:

  • Optimize blocking conditions (test BSA vs. normal serum vs. non-fat milk)

  • Increase washing steps duration and number

  • Pre-adsorb antibody with non-specific proteins

  • Use more stringent washing buffers

• Inconsistent results between applications:

  • Different epitopes may be accessible in different applications

  • Try antibodies targeting different regions of ZNF75A

  • Use multiple antibodies to confirm results

• Cross-reactivity with other zinc finger proteins:

  • Validate with ZNF75A knockdown controls

  • Perform peptide competition assays with the immunizing peptide

  • Use more specific detection methods like mass spectrometry to confirm identity

• Variability between lots:

  • Test each new lot against a reference sample

  • Request lot-specific validation data from manufacturers

How can researchers effectively validate ZNF75A knockdown or knockout models?

Proper validation of ZNF75A knockdown or knockout models is essential for ensuring experimental reliability. A comprehensive validation approach includes:

• mRNA level validation:

  • RT-qPCR targeting multiple regions of ZNF75A transcript

  • Reference genes selection based on tissue/cell type stability

  • Multiple primer sets targeting different exons

• Protein level validation:

  • Western blotting with ZNF75A antibodies targeting different epitopes

  • Recommended dilution: 0.04-0.4 μg/mL for Western blotting

  • Include positive controls and wild-type samples for comparison

• Functional validation:

  • Assess phenotypic changes expected based on hypothesized ZNF75A function

  • Rescue experiments by re-expressing ZNF75A to confirm specificity

  • For knockdown models, establish dose-response relationships between knockdown efficiency and phenotype

• Specificity controls:

  • Measure expression of closely related zinc finger proteins to rule out off-target effects

  • Use multiple independent shRNA/siRNA sequences targeting different regions of ZNF75A

  • The sequences used in published research include ZNF275-shRNA-1: CCCATTGAATGCAGCATTA and ZNF275-shRNA-2: GTATTCCTGTTGTGAGGAA, which could inform design strategies for ZNF75A

For stable knockdown models, selection with 2 μg/mL puromycin for 48 hours after lentiviral transduction has been reported as effective for zinc finger proteins .

How does research on ZNF75A compare to studies on other zinc finger proteins like ZNF275?

Research on ZNF75A is currently less extensive than studies on some other zinc finger proteins such as ZNF275. Comparative analysis reveals:

• Expression patterns:

  • ZNF275 shows elevated expression in cervical cancer tissues compared to normal tissue

  • ZNF75A expression patterns across tissues are documented but functional significance remains less characterized

• Functional roles:

  • ZNF275 has demonstrated roles in cancer cell proliferation, migration, and invasion through the AKT/Bcl-2 signaling pathway

  • ZNF75A's functional roles remain to be fully elucidated but, as a transcription factor, likely involves gene regulation

• Therapeutic implications:

  • ZNF275 expression levels may predict response to combination therapy with triciribine and cisplatin in cervical cancer

  • Therapeutic implications of ZNF75A expression or modulation remain unexplored

• Experimental approaches:

  • Similar techniques are applicable to both proteins, including knockdown studies, Western blotting, and immunohistochemistry

  • Researchers interested in ZNF75A could adapt methodologies from ZNF275 studies, including lentiviral knockdown systems and colony formation assays

This comparison suggests that ZNF75A represents an opportunity for novel research, with established methodologies from studies of related proteins providing a foundation for investigation.

What experimental designs are recommended for investigating ZNF75A function in cellular processes?

For researchers investigating ZNF75A function, the following experimental designs are recommended:

• Expression modulation studies:

  • Knockdown using shRNA or siRNA with multiple targeting sequences

  • Overexpression studies using tagged constructs (FLAG, HA, or GFP)

  • CRISPR/Cas9 knockout for complete ablation of expression

• Transcriptional regulatory function assessment:

  • ChIP-seq to identify genomic binding sites

  • RNA-seq following ZNF75A modulation to identify regulated genes

  • Luciferase reporter assays with identified target promoters

• Protein interaction studies:

  • Immunoprecipitation followed by mass spectrometry

  • Yeast two-hybrid screening

  • Proximity-dependent biotin identification (BioID)

• Cellular phenotype analysis:

  • Proliferation assays (CCK-8, BrdU incorporation)

  • Apoptosis assessment (Annexin V/PI staining, caspase activity)

  • Migration and invasion assays (transwell assays)

  • Colony formation assays (1 × 10³ cells/well in 6-well plates)

• Signaling pathway investigation:

  • Western blotting for pathway components like those in AKT signaling

  • Pathway inhibitor studies to establish causality

  • Phosphorylation-specific antibodies to assess activation states

These designs should incorporate appropriate controls, including scrambled shRNA controls for knockdown studies, empty vector controls for overexpression, and multiple cell lines to ensure generalizability of findings.

How can researchers design experiments to investigate potential roles of ZNF75A in disease processes?

To investigate potential roles of ZNF75A in disease processes, researchers should consider these experimental approaches:

• Expression correlation studies:

  • Compare ZNF75A expression between normal and disease tissues using immunohistochemistry (recommended dilution: 1:50-1:200)

  • Analyze public database expression data (TCGA, GTEx) for correlations with disease states

  • Perform survival analysis to determine prognostic significance

• Functional studies in disease models:

  • Modulate ZNF75A expression in disease-relevant cell lines

  • Assess impact on hallmark disease phenotypes

  • Use patient-derived xenograft (PDX) models to evaluate in vivo relevance

• Mechanistic investigations:

  • Identify ZNF75A transcriptional targets in disease contexts using ChIP-seq

  • Investigate pathway alterations following ZNF75A modulation

  • Assess post-translational modifications of ZNF75A in disease states

• Therapeutic relevance:

  • Determine if ZNF75A expression correlates with treatment response

  • Investigate whether ZNF75A modulation sensitizes cells to therapies

  • Screen for molecules that modulate ZNF75A expression or function

• Clinical sample analysis:

  • Tissue microarray analysis of ZNF75A expression across disease stages

  • Correlation with clinical parameters and outcomes

  • Single-cell analysis to identify cell-type specific roles

By applying these approaches systematically, researchers can build a comprehensive understanding of ZNF75A's potential contributions to disease processes and identify possible therapeutic implications.

What new technologies might enhance the study of ZNF75A in the coming years?

Emerging technologies that may enhance ZNF75A research include:

• Advanced genomic and epigenomic approaches:

  • CUT&RUN or CUT&Tag for more efficient and sensitive mapping of ZNF75A binding sites

  • Single-cell ChIP-seq to understand cell-to-cell variability in ZNF75A function

  • HiChIP or PLAC-seq to connect ZNF75A binding with 3D chromatin architecture

• Protein analysis innovations:

  • Advanced proximity labeling methods (TurboID, miniTurbo)

  • Targeted protein degradation (dTAG, AID) for rapid ZNF75A depletion

  • Time-resolved proteomics to track dynamic ZNF75A interactions

• Improved antibody technologies:

  • Recombinant antibodies with enhanced specificity

  • Nanobodies for improved access to protein complexes

  • Intrabodies for live-cell tracking of ZNF75A

• Live-cell analysis:

  • CRISPR-based transcriptional reporters to monitor ZNF75A target gene expression

  • Optogenetic control of ZNF75A activity

  • Live-cell protein tracking with minimal tags (Halo, SNAP)

• Structural biology approaches:

  • Cryo-EM analysis of ZNF75A-containing complexes

  • Integrative structural biology combining multiple data types

  • AlphaFold2 and related AI methods for structure prediction

These technologies will enable more precise, dynamic, and comprehensive understanding of ZNF75A function in cellular contexts and may reveal previously unappreciated roles in normal physiology and disease.

What methodological advances are needed to better understand ZNF75A function in cellular contexts?

To advance understanding of ZNF75A function, several methodological improvements are needed:

• Improved antibody resources:

  • Development of monoclonal antibodies with higher specificity

  • Antibodies validated across multiple applications and cell types

  • ChIP-grade antibodies specifically validated for chromatin studies

• Better model systems:

  • Conditional knockout mouse models for tissue-specific ZNF75A ablation

  • Knock-in reporter systems for endogenous monitoring of ZNF75A expression

  • Organoid systems expressing physiological levels of ZNF75A

• Advanced functional genomics:

  • Domain-specific mutagenesis to dissect functional regions

  • Genome-wide CRISPR screens to identify synthetic interactions

  • High-throughput reporter assays to map DNA binding preferences

• Integration of multiple data types:

  • Computational frameworks combining genomics, proteomics, and phenotypic data

  • Systems biology approaches to position ZNF75A in regulatory networks

  • Machine learning methods to predict ZNF75A functions from existing data

• Standardized protocols:

  • Optimized methods for chromatin immunoprecipitation

  • Consensus approaches for quantifying ZNF75A expression levels

  • Defined cellular systems for comparative studies

These methodological advances would address current limitations in ZNF75A research and enable more comprehensive understanding of its biological roles.