ZNF287 Antibody

What is ZNF287 and what is its role in cellular processes?

ZNF287, also known as zinc finger protein 287, is a member of the KRAB-containing zinc finger protein family. It belongs to the larger Krüppel-like family of transcription factors that regulate various cellular processes. ZNF287 is also referred to by several alternative names including SKAT-2, ZKSCAN13, and ZSCAN45 . The protein functions primarily as a transcriptional regulator, with research suggesting it may play a role in neuronal differentiation through transcriptional repression of key neuronal markers .

What types of ZNF287 antibodies are available for research?

Several types of ZNF287 antibodies are available for research applications:

• Polyclonal antibodies (pAbs) with reactivity to human and/or mouse ZNF287

• Monoclonal antibodies (mAbs) with specific epitope targeting

• Antibodies validated for various applications including Western blot, immunofluorescence, and ELISA

The selection of antibody type depends on the specific research application, with polyclonal antibodies offering broader epitope recognition while monoclonal antibodies provide higher specificity for particular epitopes.

What are the validated applications for ZNF287 antibodies?

ZNF287 antibodies have been validated for multiple research applications including:

• Western blotting (WB)

• Enzyme-linked immunosorbent assay (ELISA)

• Immunofluorescence (IF)

• Immunohistochemistry (IHC)

• Immunocytochemistry (ICC)

Different antibodies show varying performance across these applications, with some optimized for specific techniques. For example, some antibodies (like ABIN6257559) are validated for multiple applications including WB, ELISA, IHC, IF, and ICC, while others (like ABIN528151) are more specifically validated for WB and IF .

How can researchers verify ZNF287 antibody specificity?

Antibody specificity for ZNF287 can be verified through several validation approaches:

• siRNA knockdown experiments to demonstrate decreased antibody signal

• Use of GFP-tagged ZNF287 cell lines to evaluate signal overlap

• Comparison of staining patterns between independent antibodies targeting different epitopes of ZNF287

• Western blot analysis on diverse human tissues and cell lines to confirm band specificity at the expected molecular weight

• Correlation between immunohistochemistry data and RNA expression levels

The Human Protein Atlas categorizes validation results as "Enhanced," "Supported," "Approved," or "Uncertain" based on the level of experimental validation evidence .

What experimental controls should be included when using ZNF287 antibodies?

When designing experiments with ZNF287 antibodies, researchers should include:

• Positive controls: Cell lines or tissues known to express ZNF287

• Negative controls:

  • Primary antibody omission

  • Isotype controls (IgG matched to the ZNF287 antibody)

  • Tissues or cells with ZNF287 knocked down via siRNA/shRNA

• Loading controls: For Western blot experiments, include housekeeping proteins like GAPDH

• Blocking peptide controls: To verify specific epitope binding

A systematic approach to controls helps distinguish true ZNF287 signal from non-specific binding and background, particularly important when studying proteins with multiple isoforms or family members.

What are the optimal sample preparation methods for ZNF287 detection?

Sample preparation significantly impacts ZNF287 antibody performance:

For Western blotting:

• Complete protein extraction requires efficient nuclear lysis since ZNF287 is predominantly nuclear

• Use of protease inhibitors is essential to prevent degradation

• Mild detergents (such as Triton X-100) are effective for solubilizing ZNF287

• Sonication of chromatin fractions may improve extraction efficiency for chromatin-bound ZNF287

For immunofluorescence/immunohistochemistry:

• Fixation with 4% paraformaldehyde typically preserves ZNF287 epitopes

• Antigen retrieval may be necessary, particularly for formalin-fixed tissues

• Permeabilization optimization is crucial for nuclear protein access

How can ZNF287 antibodies be effectively used in chromatin immunoprecipitation (ChIP) experiments?

ChIP experiments with ZNF287 antibodies require special considerations:

• Crosslinking optimization: Usually 1% formaldehyde for 10 minutes at room temperature

• Sonication parameters: Adjust to yield DNA fragments of 200-500bp

• Antibody selection: Choose antibodies validated specifically for ChIP applications

• Controls: Include IgG negative controls and positive controls (known ZNF287 target regions)

• Sequential ChIP considerations: When studying ZNF287 interaction with other proteins like hnRNP K

Research indicates that ZNF287 (or the related ZNF827) associates with specific promoter regions, such as the NGFR and RARA promoters, and this association can be detected via ChIP experiments with appropriate antibodies .

What are typical Western blot conditions for optimal ZNF287 detection?

For optimal Western blot detection of ZNF287:

Multiple ZNF287 antibodies have been validated for Western blot applications with varying degrees of sensitivity and specificity .

How can ZNF287 antibodies be used to study protein-protein interactions?

ZNF287 antibodies are valuable tools for investigating protein-protein interactions through multiple approaches:

• Co-immunoprecipitation (Co-IP): ZNF287 antibodies can pull down interaction partners. Research has demonstrated interactions between the related protein ZNF827 and hnRNP K/L using this approach .

• Proximity ligation assay (PLA): Allows visualization of protein interactions in situ using pairs of antibodies against ZNF287 and potential interacting proteins.

• Chromatin immunoprecipitation (ChIP): Can identify DNA regions where ZNF287 binds, potentially in complex with other proteins.

• Sequential ChIP (ChIP-reChIP): Useful for determining if ZNF287 and another protein simultaneously occupy the same DNA regions .

Studies have shown that ZNF827 (related to ZNF287) interacts strongly with hnRNP K and to a lesser extent with hnRNP L in nucleoplasmic extracts, with these interactions being even stronger in chromatin-enriched fractions .

What is known about the subcellular localization of ZNF287?

ZNF287 demonstrates specific subcellular localization patterns:

• Predominantly nuclear localization, consistent with its role as a transcription factor

• Forms distinct nuclear foci when overexpressed, particularly when studied in relation to its circular RNA counterpart

• May shuttle between nucleus and cytoplasm under certain conditions

• Association with chromatin is likely dynamic and may be regulated by cellular differentiation state

Immunofluorescence studies with validated antibodies have been crucial in determining these localization patterns. The presence of ZNF287 in distinct nuclear foci suggests it may participate in specific nuclear compartments or transcriptional complexes .

How can ZNF287 antibodies be used to study gene regulation mechanisms?

ZNF287 antibodies enable detailed investigation of gene regulatory mechanisms:

• ChIP-seq experiments: To map genome-wide binding sites of ZNF287 and identify target genes

• Co-localization studies: Using immunofluorescence to examine spatial relationship with transcriptional machinery

• Reporter assays: Combining with ZNF287 overexpression/knockdown to assess functional impact on gene expression

• Chromatin conformation capture: When used alongside other techniques to understand 3D genome organization

Research with the related protein ZNF827 has shown it associates with specific promoter regions like NGFR, and this association is diminished upon knockdown of its related circular RNA. Similar approaches can be applied to studying ZNF287's role in gene regulation .

What are the considerations for using ZNF287 antibodies in tissue samples?

When applying ZNF287 antibodies to tissue samples, researchers should consider:

• Tissue-specific expression patterns: ZNF287 expression may vary significantly across tissues

• Fixation protocols: Optimize for preservation of nuclear proteins

• Antigen retrieval methods: May be necessary for formalin-fixed paraffin-embedded samples

• Autofluorescence concerns: Particularly relevant for brain tissue analysis

• Co-staining strategies: To identify cell types expressing ZNF287

• Quantification approaches: Standardized scoring systems for expression levels

The Human Protein Atlas provides standardized protocols for antibody validation in tissue samples, which can guide researchers in optimizing conditions for ZNF287 detection in various tissue types .

How should researchers quantify and normalize ZNF287 expression data?

Quantification and normalization of ZNF287 expression require systematic approaches:

For Western blot quantification:

• Use digital image analysis software (ImageJ, ImageLab, etc.)

• Normalize to housekeeping proteins (GAPDH, β-actin, etc.)

• Include calibration standards when possible

• Average multiple technical and biological replicates

• Apply appropriate statistical analyses

For immunohistochemistry quantification:

• Establish clear scoring criteria (intensity, percentage of positive cells)

• Use digital pathology tools for unbiased quantification

• Consider nuclear vs. cytoplasmic localization separately

• Compare immunohistochemistry data with RNA expression for validation

What are common pitfalls in interpreting ZNF287 antibody results?

Researchers should be aware of several potential pitfalls when interpreting ZNF287 antibody data:

• Cross-reactivity with related zinc finger proteins: The zinc finger protein family has many members with structural similarities (ZNF285, ZNF283, ZNF286B, etc.)

• Isoform-specific detection: Different antibodies may recognize different ZNF287 isoforms

• Post-translational modifications: May affect epitope recognition

• Background signal interpretation: Particularly in tissues with high autofluorescence

• Overinterpretation of overexpression studies: May not reflect physiological conditions

• Inconsistency between protein and RNA levels: Due to post-transcriptional regulation

Validation using multiple independent antibodies and complementary techniques can help avoid misinterpretation of results.

How can antibody validation data from repositories be leveraged for ZNF287 research?

Researchers can maximize the value of antibody validation repositories:

• The Human Protein Atlas: Provides comprehensive validation data including:

  • Standard validation based on UniProtKB/Swiss-Prot database information

  • Enhanced validation using siRNA knockdown, GFP-tagged cell lines, or independent antibodies

  • Consistency assessment between immunohistochemistry and RNA expression

  • Western blot validation on tissues and cell lines

• Antibody vendor validation data: Commercial suppliers provide validation data specific to their ZNF287 antibodies, including:

  • Application-specific validation (WB, IF, IHC, ELISA)

  • Species reactivity information

  • Recommended dilutions and protocols

Critically evaluating this validation data helps researchers select the most appropriate antibodies for their specific research questions.

How do ZNF287 expression patterns correlate with functional outcomes?

Interpreting ZNF287 expression in relation to functional outcomes requires:

• Correlation with target gene expression: For instance, ZNF827 (related to ZNF287) has been shown to repress neuronal markers like NGFR

• Context-dependent interpretation: Expression may have different implications in different cell types

• Integration with other datasets: Combining with transcriptomics, epigenomics, and proteomics data

• Temporal considerations: Expression patterns may change during development or differentiation

• Subcellular localization analysis: Nuclear vs. cytoplasmic distribution may indicate different functional states

Research indicates that zinc finger proteins like ZNF287 can form part of transcriptional repressive complexes, so correlating their expression with target gene repression can provide functional insights .

What new techniques are emerging for studying ZNF287 protein complexes?

Emerging techniques for studying ZNF287 protein complexes include:

• Proximity labeling approaches: BioID or APEX2 fusion proteins to identify proteins in close proximity to ZNF287

• Single-molecule imaging: To track ZNF287 dynamics in living cells

• CUT&RUN or CUT&Tag: Higher resolution alternatives to ChIP for mapping ZNF287 binding sites

• Cryo-electron microscopy: For structural characterization of ZNF287-containing complexes

• CRISPR-based genomic screens: To identify genetic interactions with ZNF287

These techniques could help address outstanding questions about ZNF287's role in transcriptional regulation and protein complex formation.

How might ZNF287 research contribute to understanding neuronal differentiation?

ZNF287 research has potential implications for neuronal biology:

• Transcriptional regulation during differentiation: Related zinc finger proteins like ZNF827 repress expression of key neuronal markers, suggesting a role in regulating neuronal differentiation

• Interaction with epigenetic regulators: ZNF287 may recruit chromatin modifiers to neuronal gene promoters

• Integration with signaling pathways: Understanding how external signals modulate ZNF287 activity could reveal mechanisms of neuronal fate determination

• Potential therapeutic targets: Modulating ZNF287 activity might influence neuronal differentiation in regenerative medicine applications

Research on ZNF827 has shown it represses NGFR expression, which is a key neuronal marker, suggesting similar zinc finger proteins may play important roles in neuronal development .

What are the challenges in developing more specific ZNF287 antibodies?

Developing highly specific ZNF287 antibodies faces several challenges:

• Structural similarity with other zinc finger proteins: The zinc finger protein family includes many members with similar structural domains, making specific epitope identification challenging

• Limited structural information: Incomplete knowledge of the three-dimensional structure of ZNF287

• Post-translational modifications: These may affect epitope accessibility and recognition

• Validation complexity: Comprehensive validation requires multiple techniques and controls

• Species conservation issues: Variations in ZNF287 sequence across species may limit cross-reactivity of antibodies

Future antibody development efforts may benefit from increased structural information and more sophisticated epitope mapping approaches.