ZNF586 Antibody

What is ZNF586 and to which protein family does it belong?

ZNF586 (Zinc finger protein 586) is a member of the KRAB domain-containing zinc finger proteins (KZFP) family. It functions primarily as a transcriptional regulator and is believed to play a role in heterochromatin maintenance at transposable elements (TEs) . ZNF586 is encoded by gene ID 54807 and contains 402 amino acids with a calculated molecular weight of 46 kDa . Like other KZFPs, ZNF586 enables sequence-specific DNA binding activity and contributes to transcriptional regulation mechanisms within the nucleus.

What are the primary functions of ZNF586 in cellular biology?

ZNF586 is primarily involved in transcriptional regulation processes . As part of the larger KZFP family, it likely participates in heterochromatin maintenance at transposable elements, helping to regulate their activity and prevent genomic instability . While specific functions of ZNF586 are still being characterized, research on related KZFPs suggests potential roles in development, tissue-specific gene regulation, and maintenance of genomic stability. The broader KZFP family is increasingly recognized for facilitating the domestication of transposable elements' regulatory potential rather than merely silencing them .

How conserved is ZNF586 across species?

ZNF586 shows moderate conservation across mammalian species. The human ZNF586 protein shares approximately 46% sequence identity with both mouse and rat orthologs . This level of conservation suggests that while the protein maintains some core functional domains across mammalian evolution, it has also undergone significant species-specific adaptations. This divergence is consistent with the broader pattern observed in KZFP evolution, where many family members show primate-specific features or expansions .

What are the key specifications of commercially available ZNF586 antibodies?

Currently available ZNF586 antibodies include polyclonal options that are validated for specific research applications. Below is a comprehensive overview of a leading ZNF586 antibody product:

This antibody has been specifically validated for Western blot applications in human samples, with optimal results in HepG2 and COLO 320 cell lines .

What considerations should be made when selecting a ZNF586 antibody for specific applications?

When selecting a ZNF586 antibody, researchers should consider several critical factors:

• Experimental application: Ensure the antibody is validated for your specific application (e.g., Western blot, immunoprecipitation). The currently available antibody (25023-1-AP) is validated for WB and ELISA but not for other applications like immunohistochemistry or immunofluorescence .

• Species reactivity: Verify that the antibody recognizes ZNF586 in your experimental species. The available antibody reacts with human samples but may have limited cross-reactivity with other species .

• Epitope specificity: Consider the immunogen used to generate the antibody and whether it might cross-react with other zinc finger proteins with similar domains.

• Validation data: Review available validation data in cell lines or tissues similar to your experimental system. For example, the 25023-1-AP antibody has been validated in HepG2 and COLO 320 cells .

• Controls: Plan appropriate positive and negative controls. For blocking experiments, recombinant protein fragments may be available as controls .

How should Western blot protocols be optimized for ZNF586 detection?

For optimal Western blot detection of ZNF586, consider the following protocol adjustments:

• Sample preparation: Use cell lines with confirmed ZNF586 expression (e.g., HepG2 or COLO 320 cells) as positive controls .

• Gel percentage: Use 10-12% polyacrylamide gels suitable for resolving proteins around 46 kDa.

• Primary antibody dilution: Start with the recommended dilution range (1:500-1:1000) and optimize based on signal-to-noise ratio .

• Blocking conditions: Use 5% non-fat dry milk or BSA in TBST for blocking (usually 1 hour at room temperature).

• Incubation times: Incubate primary antibody overnight at 4°C for optimal binding.

• Detection method: Choose a detection system appropriate for your expected expression level. For lower expression, consider more sensitive detection methods such as enhanced chemiluminescence (ECL).

• Exposure times: Test multiple exposure times to capture the optimal signal without background.

• Molecular weight verification: Confirm detection at approximately 46 kDa, which is the observed molecular weight for ZNF586 .

Remember that optimal conditions may vary based on your specific experimental system and should be determined empirically.

What controls should be included when working with ZNF586 antibodies?

Proper controls are essential for validating ZNF586 antibody results:

• Positive controls: Include lysates from cell lines with confirmed ZNF586 expression, such as HepG2 or COLO 320 cells .

• Negative controls: Consider using:

  • Cell lines with low or no ZNF586 expression

  • ZNF586 knockdown samples (using siRNA or shRNA)

  • Primary antibody omission control

• Blocking peptide control: Pre-incubate the antibody with a ZNF586 recombinant protein fragment (such as the available human ZNF586 aa 92-119 control fragment) at 100x molar excess for 30 minutes at room temperature before application to verify specificity .

• Loading controls: Include appropriate housekeeping proteins (e.g., β-actin, GAPDH) to normalize protein loading across samples.

• Molecular weight markers: Always include molecular weight markers to confirm the detected band is at the expected size (46 kDa for ZNF586) .

These controls help distinguish specific antibody binding from background or non-specific signals, increasing confidence in experimental results.

What are common issues when detecting ZNF586 and how can they be resolved?

Researchers may encounter several challenges when detecting ZNF586:

• No signal detected:

  • Verify ZNF586 expression in your samples using positive control cell lines (HepG2, COLO 320)

  • Increase antibody concentration or incubation time

  • Check protein transfer efficiency

  • Consider using a more sensitive detection system

  • Verify sample preparation protocol preserves nuclear proteins

• Multiple bands:

  • Increase blocking stringency

  • Optimize antibody dilution

  • Perform peptide competition assays to identify specific bands

  • Consider post-translational modifications or splice variants

  • Use fresh samples to minimize degradation products

• High background:

  • Increase washing duration and frequency

  • Decrease primary antibody concentration

  • Optimize blocking conditions (try different blocking agents)

  • Ensure membrane is completely covered during antibody incubation

  • Check secondary antibody specificity and concentration

• Inconsistent results between experiments:

  • Standardize lysate preparation methods

  • Maintain consistent antibody batches

  • Implement rigorous protocol documentation

  • Consider variations in culture conditions affecting ZNF586 expression

Each of these issues requires systematic troubleshooting, changing one variable at a time to determine the optimal conditions for your experimental system.

How can researchers distinguish between ZNF586 and other closely related zinc finger proteins?

Distinguishing ZNF586 from related zinc finger proteins requires careful experimental design:

• Antibody selection: Choose antibodies raised against unique regions of ZNF586 rather than conserved zinc finger domains. Review the immunogen sequence information to ensure minimal overlap with related proteins.

• Blocking peptide experiments: Perform competition assays using specific ZNF586 recombinant protein fragments to confirm antibody specificity .

• Molecular weight verification: Confirm that detected bands precisely match the expected molecular weight of ZNF586 (46 kDa) . Compare with predicted weights of related zinc finger proteins.

• Genetic approaches: Use ZNF586-specific siRNA/shRNA knockdown to confirm specificity of antibody signal. If the signal diminishes with ZNF586 knockdown, this supports antibody specificity.

• Mass spectrometry verification: For critical experiments, consider immunoprecipitation followed by mass spectrometry to confirm protein identity.

• Expression pattern comparison: Compare the expression pattern with known distribution patterns of ZNF586 versus related zinc finger proteins across tissues or cell types.

• Recombinant protein standards: Run purified recombinant ZNF586 alongside your samples as a definitive size reference.

These approaches collectively increase confidence in the specific detection of ZNF586 rather than related KZFP family members.

How might ZNF586 function in relation to cancer biology based on studies of related KZFPs?

While ZNF586-specific cancer research is limited, studies on related KZFP family members provide valuable insights:

Recent research identifies a cluster of primate-specific KZFPs (including ZNF587 and ZNF417) that are upregulated in various cancers, including diffuse large B-cell lymphoma (DLBCL) . These KZFPs target evolutionarily recent transposable elements and appear to protect cancer cells by:

• Maintaining heterochromatin distribution: Preventing disruptive heterochromatin redistribution that would lead to replicative stress .

• Preventing inflammatory responses: Suppressing cGAS-STING mediated interferon/inflammatory pathways that would enhance immune surveillance .

• Immune evasion: Potentially reducing HLA-I surface expression and presentation of immunogenic peptides, thereby reducing cancer cell susceptibility to immune recognition .

While ZNF586's specific role hasn't been directly demonstrated in cancer, its membership in the KZFP family suggests it may participate in similar mechanisms. Researchers investigating ZNF586 in cancer contexts should consider designing experiments that examine:

• ZNF586 expression levels across cancer types

• Correlation with patient prognosis

• Effects of ZNF586 depletion on cancer cell proliferation

• Impacts on heterochromatin distribution and inflammatory responses

• Potential effects on immune evasion mechanisms

Such investigations could determine whether ZNF586 follows patterns similar to other KZFPs in cancer biology.

What cutting-edge techniques could advance our understanding of ZNF586 function?

Several advanced methodologies could significantly enhance our understanding of ZNF586:

• ChIP-seq and CUT&RUN: Identify genome-wide binding sites of ZNF586, particularly at transposable elements, to map its regulatory network. These techniques would reveal which specific DNA sequences ZNF586 recognizes and what genes it potentially regulates.

• HiChIP or CUT&Tag followed by sequencing: Characterize three-dimensional chromatin interactions mediated by ZNF586 to understand its role in genome organization.

• CRISPR-Cas9 genome editing: Generate ZNF586 knockout or knock-in cell lines to study functional consequences of ZNF586 loss or mutation. This could be coupled with RNA-seq to identify differentially expressed genes.

• Proximity labeling (BioID or APEX): Identify protein interaction partners of ZNF586 to place it within cellular signaling networks and complexes.

• Single-cell approaches: Examine ZNF586 expression and function at single-cell resolution to uncover potential heterogeneity in its activity across different cell states or populations.

• ATAC-seq following ZNF586 manipulation: Determine how ZNF586 affects chromatin accessibility genome-wide, particularly at transposable elements and heterochromatic regions.

• Functional genomics screens: Perform CRISPR screens in the context of ZNF586 overexpression or knockout to identify synthetic interactions and pathways.

• Structural biology approaches: Determine the three-dimensional structure of ZNF586, particularly its DNA-binding domains, to understand its sequence specificity and potential for therapeutic targeting.

These advanced approaches would provide comprehensive insights into ZNF586's molecular functions and biological significance beyond what conventional techniques can reveal.