ZNF335 Antibody

What is ZNF335 and what are its key molecular characteristics?

ZNF335 is a nuclear protein with a canonical length of 1342 amino acid residues and a molecular weight of approximately 144.9 kDa in humans. It belongs to the Krueppel C2H2-type zinc-finger protein family and functions as a critical regulator of neurogenesis and neuronal differentiation. The protein is also known by synonyms including NRC-interacting factor 1 (NIF1) and zinc-finger/leucine-zipper co-transducer NIF1 . ZNF335 contains multiple conserved zinc finger domains that are essential for its function, with certain residues (like Cys467) being particularly critical within these domains .

Where is ZNF335 protein localized in cells and what is its expression pattern?

ZNF335 predominantly exhibits nuclear subcellular localization, consistent with its role in transcriptional regulation . It is ubiquitously expressed across many tissue types, though its expression is particularly significant in neural tissues given its critical role in neurogenesis and brain development . This nuclear localization is important to consider when designing extraction protocols for ZNF335 detection experiments.

What applications are ZNF335 antibodies commonly used for in research?

Common research applications for ZNF335 antibodies include:

Most commercially available antibodies are validated for Western blot and ELISA applications, with fewer options for IHC, ICC, or IP methodologies .

How should researchers validate ZNF335 antibodies before experimental use?

Proper validation is essential for generating reliable data with ZNF335 antibodies:

• Perform Western blot analysis using positive control lysates from tissues/cells known to express ZNF335 (expect a band at approximately 144.9 kDa)

• Conduct knockdown/knockout validation using siRNA, shRNA, or CRISPR techniques to confirm specificity

• Implement peptide competition assays to verify epitope specificity

• Compare results using multiple antibodies targeting different ZNF335 epitopes

• Test on samples from multiple species if cross-reactivity is claimed

• Validate across multiple applications if the antibody will be used in diverse experimental contexts

What are the critical considerations for Western blot detection of ZNF335?

When performing Western blots for ZNF335:

• Use appropriate nuclear extraction methods, as standard whole-cell lysis may yield insufficient protein

• Include positive controls (brain tissue lysates or neural cell lines)

• Be aware of the expected molecular weight (144.9 kDa) and potential post-translational modifications

• Optimize transfer conditions for high molecular weight proteins

• Consider longer exposure times, as ZNF335 may be expressed at lower levels in some tissues

• Use gradient gels (4-12%) to achieve better resolution of this large protein

What controls should be included in ZNF335 antibody experiments?

Essential controls include:

• Positive tissue controls (brain tissue with known ZNF335 expression)

• Negative controls (tissues with minimal ZNF335 expression or knockdown samples)

• Secondary antibody-only controls to assess background

• Isotype controls to evaluate non-specific binding

• For developmental studies, include samples from multiple timepoints

• When studying variants, include wild-type samples for comparison

How do ZNF335 isoforms impact antibody selection and experimental design?

ZNF335 has two reported isoforms resulting from alternative splicing . When selecting antibodies:

• Identify the epitope location on ZNF335 and determine if it's present in all isoforms

• Choose antibodies that target common regions if general ZNF335 detection is desired

• For isoform-specific detection, select antibodies targeting unique regions

• Be aware that isoforms may have different expression patterns across tissues or developmental stages

• Consider that functional differences between isoforms may affect interpretation of results

What are the best practices for studying ZNF335 in neurological research?

Given ZNF335's critical role in neurogenesis:

• Include brain region-specific controls when examining expression patterns

• Consider developmental timepoints carefully, as ZNF335 function varies throughout neurodevelopment

• When studying microcephaly models, compare ZNF335 expression/localization between normal and affected tissues

• Implement co-staining with neuronal markers to assess ZNF335's relationship with specific neural populations

• For studies involving ZNF335 variants, verify that your antibody still recognizes the mutated protein

How can researchers differentiate between ZNF335 and related zinc finger proteins?

To ensure specificity:

• Select antibodies that target unique regions of ZNF335 rather than conserved zinc finger domains

• Perform sequence alignments to identify regions with low homology to other family members

• Validate specificity by testing on samples with overexpressed ZNF335 versus related family members

• Use knockdown controls to confirm signal specificity

• Analyze molecular weight carefully, as many zinc finger proteins have distinct sizes

How do ZNF335 variants impact antibody selection and detection?

The literature describes several pathogenic variants in ZNF335, including p.Cys467Arg, p.Tyr502Cys, p.Phe724del, and others . These variants affect antibody selection:

• Variants within the antibody's epitope may reduce or eliminate binding

• Some variants affect splicing (e.g., c.3332G>A and potentially c.3998A>G), potentially removing entire epitope regions

• Conformational changes from mutations may mask epitopes distant from the mutation site

For studying samples with known ZNF335 variants:

• Select antibodies targeting regions distant from known mutation sites

• Use multiple antibodies targeting different epitopes

• Verify antibody recognition using recombinant proteins containing the specific variants

What are the challenges in detecting ZNF335 in brain tissue samples?

Brain tissue analysis presents unique challenges:

• Fixation sensitivity: Over-fixation may mask epitopes

• High background due to brain tissue autofluorescence

• Nuclear localization requiring proper permeabilization

• Varying expression across brain regions and developmental stages

• In disease states like microcephaly, protein expression/localization may be altered

For optimal results:

• Test multiple antigen retrieval methods

• Use appropriate blocking solutions to reduce non-specific binding

• Consider fresh-frozen versus fixed tissue preparation methods

What strategies should be employed when studying ZNF335 in microcephaly models?

When investigating ZNF335 in microcephaly research:

Additionally, combine protein detection with genetic analysis since biallelic ZNF335 variants cause a spectrum of microcephaly phenotypes ranging from severe (with structural brain anomalies) to milder presentations with hypomyelination .

How can researchers optimize immunoprecipitation for ZNF335 interaction studies?

For successful ZNF335 immunoprecipitation:

• Use nuclear extraction protocols since ZNF335 is primarily nuclear

• Consider cross-linking to capture transient interactions

• Test different lysis/wash buffers to maintain protein-protein interactions

• Select antibodies that don't interfere with known protein interaction domains (e.g., the C-terminal portion integral for DBC-1 interaction)

• Thoroughly pre-clear lysates to reduce non-specific binding

• For co-IP studies, include RNase treatment controls if RNA-mediated interactions are suspected

What methodologies are recommended for studying ZNF335 in neurodevelopmental processes?

When investigating ZNF335's role in neurodevelopment:

• Combine protein detection with functional assays (e.g., reporter assays for transcriptional activity)

• Consider temporal analysis across developmental stages

• Use in vitro differentiation models (such as neural progenitor cells) to study ZNF335's role in neurogenesis

• Implement knockdown/overexpression studies to assess functional consequences

• For microcephaly studies, analyze both cell proliferation and differentiation endpoints

• Consider the effects of ZNF335 variants on protein-protein interactions, as mouse models with null mutations are embryonically lethal

How should researchers approach contradictory data when studying ZNF335?

When encountering contradictory results:

• Verify antibody specificity using multiple validation methods

• Consider that different ZNF335 isoforms may be detected by different antibodies

• Assess if differences in sample preparation could affect results (fixation, extraction methods)

• Determine if developmental timing or brain region differences could explain disparate findings

• Examine if the specific ZNF335 variants being studied have different functional consequences

• Compare results with published phenotypes from patients with biallelic ZNF335 variants, which range from severe congenital microcephaly with structural brain anomalies to milder presentations