ZNF689 Antibody

What is the basic structure and function of ZNF689 protein?

ZNF689 (Zinc Finger Protein 689) is a 500-amino-acid protein containing 12 zinc-finger domains and a Kruppel-associated box domain . It functions as a transcription factor and has been implicated in suppressing apoptosis of hepatocellular carcinoma cells via downregulation of pro-apoptotic factors . The protein contains specific sequence regions that are targeted by various antibodies, with different antibodies recognizing distinct epitopes such as amino acids 1-300 or 1-500 .

What types of ZNF689 antibodies are available for research applications?

Multiple ZNF689 antibodies are available targeting different epitopes:

These antibodies differ in their binding specificity, host animal, and experimental applications, allowing researchers to select the most appropriate antibody based on their specific experimental requirements .

What is the optimal protocol for using ZNF689 antibody in Western Blotting applications?

For Western Blotting applications using ZNF689 antibodies, the following protocol has been effectively utilized:

• Extract protein from tissues using radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail

• Measure protein concentration using a bicinchoninic acid protein assay kit

• Denature samples at 95°C for 10 minutes

• Separate 50 μg of each protein sample using SDS-PAGE (10% gel)

• Transfer proteins to a polyvinylidene difluoride membrane

• Block with 5% nonfat dry milk in TBS with 0.1% Tween-20 (TBST) for 1 hour at room temperature

• Incubate membrane with ZNF689 primary antibody (dilution 1:1,000) at 4°C overnight

• Wash three times with TBST buffer (10 minutes per wash)

• Incubate with HRP-conjugated secondary antibody (dilution 1:5,000) for 1 hour at room temperature

• Develop using an ECL substrate and image using a suitable detection system

This protocol has been validated for detecting ZNF689 in human tissue samples and provides reliable results.

What are the recommended procedures for immunohistochemical detection of ZNF689?

For immunohistochemistry (IHC) staining of ZNF689 in tissue samples, the following procedure is recommended:

• Fix tissues in 10% formalin and embed in paraffin

• Cut into 4 μm-thick sections

• De-paraffinize with xylene and rehydrate through a graded alcohol series (100%, 90%, 70%, and 50% ethyl alcohol) for 10 minutes at room temperature

• Perform antigen retrieval by incubating samples in sodium citrate buffer (pH 6.0) for 30 minutes at 98°C

• Inactivate endogenous peroxidase with 3% hydrogen peroxide solution for 20 minutes at room temperature

• Block with 5% normal goat serum for 30 minutes at room temperature

• Incubate with rabbit polyclonal anti-ZNF689 primary antibodies (dilution 1:100) overnight at 4°C

• Wash and incubate with HRP-conjugated secondary antibody for 40 minutes at 37°C (dilution 1:500)

• Evaluate IHC staining using an immunoreactivity score (IRS), calculated by multiplying the staining intensity and extent

This protocol enables reliable detection and quantification of ZNF689 protein expression in tissue samples.

How should RT-qPCR be performed to accurately quantify ZNF689 mRNA expression?

For accurate quantification of ZNF689 mRNA expression using RT-qPCR:

• Extract total RNA from specimens using TRIzol® reagent

• Determine RNA concentration with a spectrophotometer

• Verify RNA integrity by separating 3 μg of RNA on a 1% denatured agarose gel

• Design primers specific to ZNF689 (see table below for validated primers)

• Synthesize cDNA using a first-strand cDNA synthesis kit

• Perform qPCR in triplicate for each sample using SYBR Green qPCR Master mix

• Use the following thermal cycling conditions: 3 minutes at 95°C for initial denaturation, followed by 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds

• Calculate relative expression levels using the 2^(-ΔΔCq) method with GAPDH as reference gene

This validated protocol ensures reliable and reproducible quantification of ZNF689 mRNA expression levels.

How does ZNF689 expression correlate with hepatocellular carcinoma prognosis?

Studies have shown that positive expression of ZNF689 protein in hepatocellular carcinoma (HCC) is significantly associated with poor prognosis . Specifically, positive ZNF689 expression correlates with:

• Larger tumor size (≥10 cm)

• Tumor capsule infiltration

• Microvascular invasion

Statistical analysis has identified positive expression of ZNF689 as a prognostic factor for:

These findings suggest that ZNF689 may be a novel predictor for prognosis in patients with HCC, potentially useful for risk stratification and treatment planning.

What is the mechanistic role of ZNF689 in hepatocellular carcinoma pathogenesis?

Research indicates that ZNF689 plays a role in HCC development through:

• Suppression of apoptosis in HCC cells by downregulating pro-apoptotic factors

• Potential involvement in epithelial-mesenchymal transition (EMT) pathways, as suggested by studies examining its relationship with markers like E-cadherin, β-catenin, and Snail1

The elevated expression of ZNF689 in both HCC tissues and adjacent non-cancerous tissues compared to normal liver suggests it may contribute to creating a permissive microenvironment for tumor development even before histological changes are apparent .

How should researchers address potential cross-reactivity when using ZNF689 antibodies?

When using ZNF689 antibodies, researchers should consider potential cross-reactivity with other proteins, particularly other zinc finger family members. To address this:

• Validate antibody specificity using positive and negative controls

• Consider using multiple antibodies targeting different epitopes of ZNF689

• Perform blocking peptide experiments to confirm specificity

• For human samples, select antibodies specifically validated for human ZNF689 detection

• When studying other species, verify cross-reactivity claims with appropriate controls

Different ZNF689 antibodies have varying cross-reactivity profiles. For instance, some antibodies react only with human ZNF689, while others show cross-reactivity with mouse, rat, or cow ZNF689 . Careful antibody selection based on the experimental model is crucial for accurate results.

What strategies can improve detection sensitivity when ZNF689 is expressed at low levels?

When ZNF689 is expressed at low levels, consider these approaches to improve detection sensitivity:

• For Western blotting:

  • Increase protein loading (up to 100 μg per lane)

  • Use enhanced chemiluminescence substrates designed for higher sensitivity

  • Extend primary antibody incubation time to overnight at 4°C

  • Optimize antibody concentration through titration experiments

• For immunohistochemistry:

  • Optimize antigen retrieval conditions (buffer type, pH, temperature, duration)

  • Use signal amplification systems like tyramide signal amplification

  • Consider using more sensitive detection systems

• For RT-qPCR:

  • Increase input RNA amount

  • Optimize primer design for maximum efficiency

  • Consider digital PCR for absolute quantification of low-abundance transcripts

These strategies can significantly improve detection of ZNF689 when expressed at levels near the limit of detection.

How can researchers effectively design ZNF689 knockdown or overexpression experiments to study its function?

For effective ZNF689 knockdown or overexpression experiments:

• Knockdown strategies:

  • Design multiple siRNAs or shRNAs targeting different regions of ZNF689 mRNA

  • Target regions with minimal sequence homology to other zinc finger proteins

  • Include scrambled RNA controls and validate knockdown efficiency by Western blot and RT-qPCR

  • Consider inducible knockdown systems for studying temporal effects

• Overexpression approaches:

  • Use expression vectors containing the full-length ZNF689 cDNA (1-500 aa) for complete functional studies

  • Consider tagged constructs (FLAG, HA, GFP) to distinguish exogenous from endogenous protein

  • Create domain-specific constructs to identify functional regions

  • Use appropriate empty vector controls

• Functional readouts:

  • Assess effects on cell proliferation, apoptosis, and migration

  • Examine changes in downstream target genes

  • Investigate potential effects on EMT markers (E-cadherin, β-catenin, Snail1)

  • Consider in vivo models for comprehensive functional assessment

These experimental designs allow for robust investigation of ZNF689 function in both normal and pathological contexts.

What novel techniques are being applied to study ZNF689 interactions with DNA or other proteins?

Emerging techniques for studying ZNF689 interactions include:

• ChIP-seq (Chromatin Immunoprecipitation followed by sequencing):

  • Identifies genome-wide DNA binding sites of ZNF689

  • Requires highly specific ZNF689 antibodies suitable for ChIP applications

  • Enables identification of direct transcriptional targets

• Proximity-dependent biotin labeling (BioID, TurboID):

  • Identifies proteins that interact with ZNF689 in living cells

  • Provides spatial and temporal resolution of protein-protein interactions

  • Offers advantages over traditional co-immunoprecipitation methods

• CRISPR-Cas9 genome editing:

  • Creates precise ZNF689 knockout models

  • Enables tagging of endogenous ZNF689 for live-cell imaging

  • Allows for introduction of specific mutations to study structure-function relationships

These advanced techniques provide deeper insights into the molecular mechanisms of ZNF689 function beyond traditional antibody-based detection methods.

How do different ZNF689 antibodies perform in detecting post-translational modifications of the protein?

The detection of post-translational modifications (PTMs) of ZNF689 requires careful consideration:

• Standard ZNF689 antibodies may not specifically recognize PTMs such as phosphorylation, ubiquitination, or SUMOylation

• For PTM studies, consider:

  • Using PTM-specific antibodies in combination with ZNF689 immunoprecipitation

  • Mass spectrometry analysis of immunoprecipitated ZNF689 to identify PTMs

  • Phosphatase or deubiquitinase treatments to confirm specificity of PTM detection

• When designing experiments to study ZNF689 PTMs:

  • Include appropriate positive controls

  • Consider the dynamic nature of PTMs and optimize cell lysis conditions to preserve modifications

  • Be aware that different antibodies recognizing different epitopes may have varying abilities to detect modified forms of ZNF689

Understanding the post-translational regulation of ZNF689 may provide important insights into its function in both normal and pathological contexts.