ZNF513 Antibody

What is ZNF513 and what is its functional significance in retinal research?

ZNF513 (Zinc Finger Protein 513) is a presumptive transcription factor that plays a critical role in retinal development and maintenance. Research has demonstrated that ZNF513 is expressed in multiple retinal layers, including the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) . The protein contains three Cys2His2-type zinc finger binding domain signatures starting at residues Cys208, Cys418, and Cys446, suggesting its function in transcriptional regulation . Studies using zebrafish models have shown that knockdown of znf513 results in reduced eye size, decreased retinal thickness, diminished expression of rod and cone opsins, and specific loss of photoreceptors, indicating its essential role in photoreceptor development and function .

What are the appropriate antibody selection criteria for ZNF513 research?

When selecting ZNF513 antibodies for research, consider the following methodological criteria:

• Species reactivity: Determine if the antibody recognizes ZNF513 in your experimental model. Available antibodies demonstrate reactivity with human, mouse, rat, and other species including bat, cow, horse, and monkey ZNF513 .

• Application compatibility: Verify the antibody is validated for your intended application. Current ZNF513 antibodies are available for various techniques including:

  • Western blotting (WB)

  • Enzyme-linked immunosorbent assay (ELISA)

  • Immunohistochemistry (IHC)

• Validation data: Examine the validation information provided by manufacturers. Look for antibodies with multiple validation points to ensure specificity and reliability .

• Target region: Consider whether the antibody recognizes regions that may be affected by known mutations such as p.C339R, particularly for comparative studies of wild-type and mutant ZNF513 .

What protocols are recommended for immunolocalization studies of ZNF513?

For successful immunolocalization of ZNF513, the following methodological approach is recommended based on published protocols:

• Fixation: Fix cells or tissue sections with 4% paraformaldehyde. For retinal tissue, ensure proper fixation time to maintain structural integrity while preserving epitope accessibility.

• Blocking: Block with 1% normal goat serum/1% BSA to reduce nonspecific binding, as successfully employed in zebrafish retinal studies .

• Primary antibody incubation: Incubate with anti-ZNF513 antibody at appropriate dilution (typically 1:300-1:1000 range). For co-localization studies, antibodies such as anti-PKCβ1 (1:300) have been successfully used alongside ZNF513 detection .

• Secondary antibody selection: Choose fluorophore-conjugated secondary antibodies appropriate for your imaging system. For nuclear proteins like ZNF513, counterstaining with DAPI is recommended to visualize nuclear localization .

• Mounting and visualization: Mount with anti-fade mounting medium containing DAPI and visualize using confocal microscopy for optimal resolution of nuclear localization patterns .

Note that both wild-type and mutant ZNF513 proteins localize to the nucleus as dispersed foci but do not co-localize with nucleolin, suggesting exclusion from the nucleolus .

How can I optimize Western blot analysis for ZNF513 detection?

For optimal Western blot detection of ZNF513, follow these research-validated steps:

• Sample preparation: Extract proteins using standard lysis buffers containing protease inhibitors. For ZNF513 analysis, 30 μg of reduced protein has been successfully used in published protocols .

• Gel selection: Use 10% Bis-Tris gels for proper separation. ZNF513 has an expected molecular weight of approximately 82 kDa when expressed as a GFP-fusion protein .

• Transfer conditions: Optimize transfer conditions based on protein size; for ZNF513, standard transfer protocols for proteins >50 kDa are suitable.

• Blocking and antibody incubation: Block membranes with 5% non-fat milk or BSA in TBST. Incubate with ZNF513 antibody at manufacturer-recommended dilutions (typically 1:500-1:2000).

• Detection system: Use enhanced chemiluminescence (ECL) or fluorescence-based detection systems compatible with your secondary antibody.

• Controls: Include positive controls such as tissues known to express ZNF513 (e.g., retinal tissue) and negative controls (tissues with minimal ZNF513 expression such as lens or cornea) .

What are the key considerations for chromatin immunoprecipitation (ChIP) with ZNF513 antibodies?

ChIP analysis with ZNF513 antibodies requires specific optimization steps to identify transcriptional targets:

• Crosslinking and sonication: Optimize formaldehyde crosslinking time (typically 10-15 minutes) and sonication conditions to generate DNA fragments of 200-500 bp.

• Antibody selection: Use ChIP-validated antibodies. For tagged ZNF513 constructs, anti-tag antibodies (e.g., GFP antibody for GFP-ZNF513 fusion proteins) have been successfully employed .

• Washing modifications: Implement modified washing conditions to minimize high background and nonspecific reactions, as noted in published ZNF513 ChIP protocols .

• Controls: Include:

  • Input DNA control (pre-immunoprecipitation chromatin)

  • Negative control (normal serum IgG)

  • Positive control (antibody against a known transcription factor)

• Target validation: Verify binding to potential target promoters such as Pax6, Sp4, Arr3, Irbp, and photoreceptor opsin promoters, which have been identified as ZNF513 binding targets in published research .

• Mutant comparison: When studying functional consequences of mutations such as p.C339R, perform parallel ChIP experiments with wild-type and mutant proteins to compare binding capabilities .

How can ZNF513 antibodies be used to investigate retinal development and disease mechanisms?

ZNF513 antibodies can be instrumental in elucidating retinal development and disease mechanisms through several advanced approaches:

• Developmental expression profiling: Quantify ZNF513 expression throughout retinal development using antibodies in immunohistochemistry and Western blot analyses. Research has shown that retinal expression of ZNF513 increases progressively with age, beginning to level off between 180 and 300 days in mouse models .

• Mutation impact assessment: Compare localization and binding properties of wild-type versus mutant ZNF513 (e.g., p.C339R) to understand how mutations affect protein function. While both wild-type and mutant proteins localize to the nucleus, ChIP analysis has revealed that only wild-type ZNF513 binds effectively to target promoters .

• Protein-protein interaction studies: Use co-immunoprecipitation with ZNF513 antibodies to identify interaction partners in the transcriptional regulatory network governing photoreceptor development.

• Therapeutic target validation: Employ ZNF513 antibodies in rescue experiments similar to those performed with wild-type znf513 mRNA in zebrafish models to evaluate potential therapeutic approaches for retinal diseases associated with ZNF513 dysfunction .

What methods can be used to study the functional consequences of ZNF513 mutations?

To investigate functional consequences of ZNF513 mutations such as p.C339R, researchers can implement the following methodological approaches:

• Comparative binding analysis: Perform ChIP assays with wild-type and mutant ZNF513 to compare binding to target promoters. Published research demonstrates that the p.C339R mutation inhibits binding to photoreceptor gene promoters including Pax6, Sp4, Arr3, Irbp, and opsins .

• Rescue experiments: In animal models like zebrafish, perform knockdown of endogenous znf513 using morpholinos and attempt rescue with either wild-type or mutant mRNA. Studies have shown that wild-type znf513 mRNA successfully rescues the normal phenotype in 86% of fish, while p.C339R mutant mRNA rescues only 31% .

• Reporter assays: Develop luciferase reporter constructs containing ZNF513 target promoters to quantitatively assess the transcriptional regulatory capacity of wild-type versus mutant ZNF513.

• Structural analysis: Use structural prediction tools to understand how mutations affect zinc finger domains and DNA binding capability. The p.C339R mutation occurs in a highly conserved region present across species from humans to zebrafish .

• Photoreceptor marker analysis: Implement immunofluorescence studies with photoreceptor-specific antibodies (e.g., rhodopsin, cone opsins) to assess the impact of ZNF513 mutations on photoreceptor development and maintenance .

What are the key technical considerations when using ZNF513 antibodies in zebrafish models?

Zebrafish models have proven valuable for studying ZNF513 function. When using ZNF513 antibodies in zebrafish research, consider these technical aspects:

Table 4.1: Applications and Antibody Selection for ZNF513 Research

How can I distinguish between basic and advanced applications of ZNF513 antibodies in retinal research?

The distinction between basic and advanced applications of ZNF513 antibodies can be characterized as follows:

Basic applications focus on:

• Expression detection and localization in tissues and cells

• Confirmation of protein presence in experimental systems

• Standardized protocols using established methods (Western blot, basic IHC)

• Qualitative assessment of protein levels

Advanced applications involve:

• Mechanistic studies of ZNF513's role in transcriptional regulation

• Comparative analysis of wild-type versus mutant protein function

• ChIP and other protein-DNA interaction studies to identify target genes

• Complex interventional experiments (knockdown/rescue) combined with antibody detection

• Quantitative assessment of binding affinities and transcriptional effects

• Multi-species comparative studies leveraging evolutionary conservation of ZNF513

What are common challenges in ZNF513 antibody experiments and how can they be addressed?

Common challenges and their solutions include:

• Non-specific binding:

  • Solution: Increase blocking time/concentration (use 1% normal goat serum/1% BSA as successfully employed in published protocols)

  • Perform additional washing steps with modified conditions to reduce background

• Weak or absent signal in Western blot:

  • Solution: Ensure sufficient protein loading (30 μg recommended)

  • Optimize transfer conditions for higher molecular weight proteins

  • Consider alternative extraction methods to preserve protein integrity

• Poor nuclear staining:

  • Solution: Implement antigen retrieval methods optimized for nuclear proteins

  • Use confocal microscopy to clearly visualize nuclear foci pattern characteristic of ZNF513

• ChIP efficiency issues:

  • Solution: Optimize crosslinking and sonication conditions

  • Implement modified washing conditions as noted in successful ZNF513 ChIP protocols

  • Verify antibody ChIP competency before experimental use

• Inconsistent results in zebrafish models:

  • Solution: Carefully control morpholino dosage

  • Perform rescue experiments to confirm specificity of observed phenotypes

  • Use standardized scoring systems for phenotypic evaluation