ZNF227 Antibody

What is ZNF227 and what is its biological function?

ZNF227 (Zinc Finger Protein 227) is a protein-coding gene that functions as a candidate transcription factor. Among its related pathways is Gene Expression, and its GO annotations include nucleic acid binding and transcription factor activity with sequence-specific DNA binding capabilities . ZNF227 is likely involved in transcriptional regulation processes, similar to other members of the zinc finger protein family. ZNF229 has been identified as an important paralog of this gene .

ZNF227 belongs to the broader family of zinc finger proteins, which often contain C2H2 zinc finger motifs and function as components in various transcriptional regulatory complexes. While specific ZNF227 functions are still being elucidated, research on related zinc finger proteins suggests potential roles in chromatin remodeling and transcriptional control .

What types of ZNF227 antibodies are currently available for research?

Based on current research resources, polyclonal antibodies against ZNF227 are predominantly available. The most characterized include:

The immunogen typically used for these antibodies is a synthesized peptide derived from human ZNF227 (UniProt ID: Q86WZ6), corresponding to amino acid residues N199-P249 . This specific epitope region ensures detection of endogenous levels of total ZNF227 protein in experimental applications .

What validated applications exist for ZNF227 antibodies in research?

ZNF227 antibodies have been validated for several experimental applications:

It is important to note that optimal working dilutions should be determined experimentally by each investigator based on specific sample types and experimental conditions . The antibody has demonstrated reactivity against human and mouse species , but validation in other organisms may require additional testing.

How should researchers optimize experimental conditions when using ZNF227 antibodies?

When designing experiments with ZNF227 antibodies, consider these methodological recommendations:

For Western Blot:

• Start with recommended dilution range (1:500-1:2000) and optimize based on signal strength

• Use PVDF membrane for optimal protein binding

• Include appropriate positive control (human or mouse cell lysates known to express ZNF227)

• Block with 5% non-fat milk or BSA in TBST

• Incubate primary antibody overnight at 4°C for optimal binding

For IHC-p:

• Begin with 1:100-300 dilution range and titrate as needed

• Antigen retrieval methods should be optimized (typically heat-induced epitope retrieval in citrate buffer pH 6.0)

• Include appropriate negative controls (omitting primary antibody)

• Consider counterstaining to provide cellular context

The antibody is supplied in PBS with 50% glycerol and 0.02% sodium azide at pH 7.4 , which should be considered when designing blocking and washing conditions to minimize background.

What controls are essential when conducting experiments with ZNF227 antibodies?

For rigorous scientific investigation, the following controls should be implemented:

• Positive Controls: Lysates or tissues known to express ZNF227 (based on available data, human cell lines are appropriate)

• Negative Controls:

  • Primary antibody omission

  • Samples from ZNF227 knockout models (if available)

  • Pre-absorption of antibody with immunizing peptide

• Loading Controls: For Western blots, include housekeeping proteins (GAPDH, β-actin) to normalize expression

• Specificity Validation: Consider performing siRNA knockdown of ZNF227 to confirm antibody specificity

These controls help distinguish specific signal from background and ensure experimental validity, particularly important when investigating a transcription factor with potential family member cross-reactivity.

How can researchers troubleshoot common issues when working with ZNF227 antibodies?

For persistent issues with ZNF227 antibody applications, researchers should consider that ZNF227 has multiple potential isoforms. The antibody is raised against a specific epitope (N199-P249) , which may not be present in all isoforms or may be masked under certain experimental conditions.

How does ZNF227 relate to other zinc finger proteins in transcriptional regulation?

ZNF227 belongs to the zinc finger protein family, many members of which function as transcription factors with sequence-specific DNA binding capabilities. While specific ZNF227 functions are still being characterized, research on related zinc finger proteins provides context:

• Structural Similarities: Like other family members, ZNF227 likely contains C2H2 zinc finger motifs that mediate DNA binding

• Transcriptional Regulation: ZNF proteins can function as:

  • Transcriptional activators

  • Transcriptional repressors

  • Components of chromatin remodeling complexes

• Evolutionary Conservation: ZNF227 gene is present across mammalian species, indicating a potentially conserved function

Researchers investigating ZNF227 should consider the established mechanisms of other zinc finger proteins, particularly ZNF229 (an important paralog) , while recognizing that each family member has unique biological functions.

What are the current limitations in ZNF227 antibody research and how can they be addressed?

Current research with ZNF227 antibodies faces several limitations:

• Limited Validation Data: While the antibody detects endogenous levels of ZNF227, comprehensive validation across diverse applications and conditions remains limited

• Isoform Specificity: The presence of multiple ZNF227 isoforms (as seen in the Cape elephant shrew with isoforms X1 and X2) suggests human ZNF227 may also have multiple isoforms, potentially with different functions

• Cross-Reactivity Concerns: The antibody's specificity against other zinc finger proteins requires rigorous validation

To address these limitations, researchers should:

• Conduct systematic validation with knockout/knockdown controls

• Compare multiple antibodies targeting different epitopes when possible

• Include mass spectrometry analysis to confirm target identity

• Pair antibody-based approaches with orthogonal techniques (e.g., RNA analysis)

How can ZNF227 antibodies be effectively used in co-immunoprecipitation studies?

For successful co-immunoprecipitation (Co-IP) studies investigating ZNF227 protein interactions:

• Buffer Optimization: Use gentle lysis buffers (containing 0.5% NP-40 or 1% Triton X-100) to preserve protein complexes

• Antibody Selection: The polyclonal nature of available ZNF227 antibodies provides advantages for Co-IP by recognizing multiple epitopes

• Protocol Recommendations:

  • Pre-clear lysates with protein A/G beads

  • Incubate antibody with lysate overnight at 4°C

  • Include appropriate negative controls (IgG from same species)

  • Elute under non-denaturing conditions if subsequent activity assays are planned

• Validation Steps:

  • Confirm successful IP by Western blot for ZNF227

  • Analyze co-precipitated proteins by mass spectrometry or Western blot for suspected interactors

This approach can help identify novel ZNF227 binding partners, potentially illuminating its role in transcriptional regulation complexes.

What considerations should be made when using ZNF227 antibodies in chromatin immunoprecipitation (ChIP) experiments?

Given ZNF227's likely function as a transcription factor, ChIP experiments can provide valuable insights into its genomic binding sites. For optimal ChIP results:

• Crosslinking Optimization:

  • Test different formaldehyde concentrations (0.5-1%) and times (10-15 minutes)

  • For potential indirect DNA interactions, consider dual crosslinking with DSG followed by formaldehyde

• Sonication Parameters:

  • Optimize to achieve DNA fragments of 200-500 bp

  • Verify fragmentation efficiency by agarose gel electrophoresis

• Antibody Considerations:

  • Current ZNF227 antibodies are polyclonal, which can increase ChIP efficiency but may introduce variability

  • Use 2-5 μg antibody per ChIP reaction

  • Include appropriate controls (IgG, input)

• Data Analysis:

  • Perform qPCR for suspected target genes before proceeding to ChIP-seq

  • For ChIP-seq analysis, include appropriate bioinformatic approaches to identify enriched motifs

Through careful experimental design and methodological optimization, researchers can generate valuable insights into ZNF227's genomic targets and transcriptional regulatory networks.