ZNF644 Antibody, HRP conjugated

What is ZNF644 and what is its primary function in cellular processes?

ZNF644 (Zinc finger protein 644), also known as Zinc finger motif enhancer-binding protein 2 (Zep-2) or KIAA1221, is a protein that likely plays a crucial role in transcriptional regulation . As a zinc finger protein, it contains domains that can bind to DNA and potentially regulate gene expression. While the complete characterization of its function remains ongoing, research suggests it acts as a transcriptional regulator through its DNA-binding capacity . The protein has a predicted molecular weight of approximately 150 kDa . In experimental settings, ZNF644 has been detected in multiple human cell lines including HeLa, 293T, and Jurkat cells, suggesting widespread expression in different cell types .

How do HRP-conjugated antibodies differ from unconjugated antibodies for ZNF644 detection?

HRP-conjugated ZNF644 antibodies have horseradish peroxidase (HRP) enzyme directly attached to the antibody molecule, enabling direct detection through enzymatic reactions without requiring secondary antibodies . This differs significantly from unconjugated ZNF644 antibodies, which require a secondary detection system typically involving species-specific secondary antibodies that recognize the primary antibody .

The key methodological differences are:

• Detection workflow: HRP-conjugated antibodies simplify protocols by eliminating the secondary antibody incubation step, reducing experimental time and potential sources of background noise.

• Applications: While unconjugated ZNF644 antibodies are validated for applications like Western blot (WB) and immunoprecipitation (IP) , HRP-conjugated versions are particularly optimized for ELISA-based detection systems .

• Sensitivity considerations: Direct conjugation can sometimes affect antibody avidity, but properly optimized HRP-conjugated antibodies can provide improved signal-to-noise ratios in appropriate applications.

What epitopes of ZNF644 do commercially available antibodies typically recognize?

Available ZNF644 antibodies target different epitopes of the protein, which affects their binding specificity and experimental applications. According to the search results, antibodies have been developed against:

• N-terminal regions: The Abcam antibody (ab117777) was generated using a synthetic peptide within the first 50 amino acids (aa 1-50) of human ZNF644 .

• Mid-region epitopes: The A88338 antibody was developed using a recombinant fusion protein corresponding to amino acids 1-300 of human ZNF644 (NP_958357.1) .

• C-terminal regions: The HRP-conjugated A39117 antibody was generated using a recombinant human ZNF644 protein fragment spanning amino acids 752-913 .

When selecting an antibody for specific experimental applications, researchers should consider which protein domain they wish to target, particularly if studying specific isoforms or if certain domains might be masked in experimental conditions.

What are the optimal experimental applications for ZNF644 Antibody, HRP conjugated?

The HRP-conjugated ZNF644 antibody (A39117) is specifically optimized for ELISA applications according to the manufacturer's data . The direct HRP conjugation makes this antibody particularly suitable for:

• Quantitative ELISA: For measuring ZNF644 levels in cell or tissue lysates with high sensitivity.

• High-throughput screening: The simplified workflow (eliminating secondary antibody steps) makes it ideal for processing multiple samples.

• Chromatin immunoprecipitation followed by ELISA (ChIP-ELISA): For investigating ZNF644 binding to specific DNA sequences without the interference of secondary antibodies.

For non-ELISA applications, researchers should consider that while HRP-conjugated antibodies can theoretically work for immunoblotting, the unconjugated variants have been specifically validated for Western blot and immunoprecipitation applications . The Abcam antibody (ab117777) has been validated for IP and WB applications with human samples , while the A88338 antibody has been validated for WB with mouse samples .

How should I optimize ELISA protocols when using ZNF644 Antibody, HRP conjugated?

When optimizing ELISA protocols with HRP-conjugated ZNF644 antibody, follow these methodological guidelines:

• Antibody titration: Begin with a broad range of antibody dilutions (e.g., 1:500, 1:1000, 1:2000, 1:5000) to determine the optimal concentration that provides the best signal-to-noise ratio.

• Buffer optimization: The antibody is supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . For optimal performance:

  • Use a blocking buffer with 1-5% BSA or casein to minimize non-specific binding

  • Include 0.05% Tween-20 in wash buffers to reduce background

  • Consider adding carrier proteins (0.1-0.5% BSA) to dilution buffers to prevent antibody loss through adsorption

• Substrate selection: For HRP-conjugated antibodies, select an appropriate substrate based on desired sensitivity:

  • TMB (3,3',5,5'-Tetramethylbenzidine) for colorimetric detection

  • Enhanced chemiluminescent (ECL) substrates for higher sensitivity

  • Fluorescent substrates (e.g., Amplex Red) for multiplex applications

• Incubation conditions: Optimize both temperature and duration:

  • Test both room temperature (1-2 hours) and 4°C (overnight) incubations

  • Include gentle agitation to improve binding kinetics while minimizing washing artifacts

• Data normalization: When quantifying ZNF644 levels, include appropriate controls for normalization, particularly when comparing different cell types or conditions.

What cross-reactivity should be considered when using ZNF644 antibodies across species?

Cross-reactivity considerations are crucial when planning experiments with ZNF644 antibodies across different species. Based on the search results:

• The Abcam antibody (ab117777) has been validated specifically for human samples , though cross-reactivity with other species may be possible due to sequence conservation.

• The A88338 antibody has been validated for mouse samples , representing a different species specificity profile.

• The HRP-conjugated antibody (A39117) appears to be reactive with human samples, generated against human ZNF644 protein fragment .

When considering cross-reactivity:

• Sequence homology analysis: Compare the ZNF644 sequence between your species of interest and the immunogen species. Higher homology in the epitope region increases the likelihood of cross-reactivity.

• Validation experiments: Always perform preliminary validation when using the antibody in a species different from the stated reactivity, including:

  • Western blot with positive and negative controls

  • Inclusion of competing peptides to confirm specificity

  • Side-by-side comparison with other validated antibodies

• Optimization for non-validated species: When working with species not explicitly validated, consider:

  • Increasing antibody concentration (typically 2-5 fold)

  • Extending incubation times

  • Modifying blocking conditions to reduce background

How can I validate the specificity of ZNF644 Antibody, HRP conjugated in my experimental system?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For ZNF644 Antibody, HRP conjugated, implement these methodological approaches:

• Positive and negative controls:

  • Positive controls: Use cell lines known to express ZNF644, such as HeLa, 293T, or Jurkat cells, which have been shown to express detectable ZNF644 levels .

  • Negative controls: Consider ZNF644 knockdown cells (siRNA or CRISPR-edited) or tissues/cells known not to express the protein.

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide (if available) before application

  • A specific antibody will show significantly reduced signal when pre-blocked with its target peptide

• Molecular weight verification:

  • ZNF644 has a predicted molecular weight of approximately 150 kDa

  • In western blots using non-conjugated antibodies, bands have been detected at the expected size in various human cell lines

• Correlation with orthogonal methods:

  • Compare results with alternative detection methods (e.g., mRNA expression via qPCR)

  • Use multiple antibodies targeting different epitopes of ZNF644

• Signal specificity testing:

  • For ELISA applications, implement dilution linearity tests to confirm signal proportionality to sample concentration

  • Include testing across different sample types to confirm consistent detection specificity

What are common sources of background or false positives when using HRP-conjugated antibodies, and how can they be minimized?

HRP-conjugated antibodies can present specific technical challenges. When working with ZNF644 Antibody, HRP conjugated, address these common issues:

• Endogenous peroxidase activity:

  • Problem: Endogenous peroxidases in samples can react with HRP substrates

  • Solution: Include a peroxidase quenching step (e.g., 0.3% H₂O₂ in methanol for 15-30 minutes) before antibody application in protocols

• Non-specific binding:

  • Problem: Direct HRP conjugation may alter antibody binding characteristics

  • Solutions:

    • Optimize blocking (test different blocking agents: BSA, casein, normal serum)

    • Include carrier proteins in antibody dilution buffers

    • Consider longer blocking times (2-3 hours at room temperature or overnight at 4°C)

• Cross-reactivity with similar proteins:

  • Problem: ZNF644 belongs to the zinc finger protein family, which has numerous members with similar domains

  • Solution: Validate specificity using ZNF644-depleted samples or recombinant protein competition assays

• Buffer composition interference:

  • Problem: Components like sodium azide can inhibit HRP activity

  • Solution: Ensure all buffers are compatible with HRP enzymatic activity; avoid sodium azide in working solutions

• Signal development optimization:

  • Problem: Over-development can increase background

  • Solution: Carefully optimize substrate incubation times; consider kinetic reading for ELISA to determine optimal signal development window

How should I store and handle ZNF644 Antibody, HRP conjugated to maintain its activity?

Proper storage and handling are critical for maintaining antibody activity, particularly for conjugated antibodies. For ZNF644 Antibody, HRP conjugated:

• Storage conditions:

  • Upon receipt, store at -20°C or -80°C as recommended by the manufacturer

  • Avoid repeated freeze-thaw cycles by preparing small, single-use aliquots

  • For working solutions, store at 4°C for short term (1-2 weeks) use only

• Buffer considerations:

  • The antibody is supplied in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

  • Maintain these conditions when making dilutions to ensure stability

  • When diluting, use buffers containing stabilizing proteins (0.1-1% BSA) to prevent adsorption loss

• HRP activity preservation:

  • Avoid repeated exposure to strong light

  • Minimize exposure to oxidizing agents or extreme pH conditions

  • Keep diluted antibody solutions on ice when not in use during experimental procedures

• Quality control:

  • Include positive controls in each experiment to monitor antibody performance over time

  • Document lot numbers and performance to track any potential lot-to-lot variations

  • Consider implementing antibody validation tests after extended storage periods

• Reconstitution guidance:

  • If lyophilized (though the search results indicate the product is supplied as a liquid), reconstitute using sterile water or buffer

  • Allow the antibody to reach room temperature before opening to prevent condensation

How can ZNF644 Antibody, HRP conjugated be integrated into chromatin immunoprecipitation (ChIP) studies?

While traditional ChIP protocols typically use unconjugated primary antibodies, HRP-conjugated antibodies can be adapted for specialized ChIP workflows:

• ChIP-ELISA approaches:

  • The HRP-conjugated ZNF644 antibody is particularly suitable for detecting ChIP-enriched ZNF644-bound DNA fragments in ELISA format

  • This approach is useful for targeted analysis of specific genomic regions

• Modified ChIP protocol for HRP-conjugated antibodies:

  • Pre-clear chromatin with protein A/G beads to reduce non-specific binding

  • Use biotinylated secondary antibodies against rabbit IgG followed by streptavidin beads for immunoprecipitation

  • Alternatively, consider chemical crosslinking of the HRP-conjugated antibody to protein A/G beads prior to immunoprecipitation

• Integration with iPOND (isolation of Proteins On Nascent DNA) methodology:

  • Similar to the approach described in search result , ZNF644 antibodies can be used to investigate whether ZNF644 associates with nascent DNA

  • This would provide insights into its potential role in DNA replication or repair processes

• Validation recommendations:

  • When adapting HRP-conjugated antibodies for ChIP, always validate results against established unconjugated antibodies

  • Confirm enrichment of known ZNF644 binding sites using qPCR

  • Consider sequential ChIP (re-ChIP) to investigate co-binding with other transcription factors

• Data analysis considerations:

  • Include appropriate controls (input, IgG, positive and negative genomic regions)

  • Normalize enrichment data using methods similar to those described in result , such as calculating fold enrichment over background

What is the relationship between ZNF644 and transcriptional regulation, and how can antibodies help elucidate these mechanisms?

ZNF644 is suggested to be involved in transcriptional regulation , and antibodies against ZNF644, including HRP-conjugated versions, can be instrumental in elucidating these mechanisms:

• Investigating protein-protein interactions:

  • Co-immunoprecipitation (Co-IP) using ZNF644 antibodies can identify binding partners in transcriptional complexes

  • When using HRP-conjugated antibodies for subsequent detection, ensure the conjugation doesn't interfere with epitope recognition in complex samples

• Spatiotemporal regulation analysis:

  • Examine cellular localization and expression patterns under different cellular states

  • Combine with cell fractionation to determine nuclear vs. cytoplasmic distribution under various conditions

• Transcriptional activity correlation:

  • Use ZNF644 antibodies in conjunction with RNA expression profiling to correlate ZNF644 binding with gene expression changes

  • This can be achieved through integrated ChIP-seq and RNA-seq approaches

• Post-translational modification (PTM) investigation:

  • Combine ZNF644 antibodies with PTM-specific antibodies to determine how modifications affect transcriptional activity

  • This could include phosphorylation, ubiquitination, or SUMOylation studies

• Methodological approach for mechanistic studies:

  • Perform ZNF644 ChIP-seq to identify genome-wide binding sites

  • Correlate binding with chromatin accessibility data (ATAC-seq)

  • Validate functional impact through reporter assays or CRISPR-mediated genomic editing of binding sites

What emerging technologies can be combined with ZNF644 antibodies for advanced epigenetic research?

Emerging technologies can significantly enhance research using ZNF644 antibodies, including HRP-conjugated versions:

• CUT&RUN/CUT&Tag alternatives to traditional ChIP:

  • These techniques offer higher signal-to-noise ratios and require fewer cells

  • ZNF644 antibodies can be adapted for these protocols by:

    • Using biotinylated secondary antibodies against the primary ZNF644 antibody

    • Optimizing wash conditions to maintain specificity while reducing background

• Proximity labeling approaches:

  • BioID or APEX2 fusions with ZNF644 can identify proximal proteins in living cells

  • ZNF644 antibodies are essential for validating expression and localization of these fusion proteins

• Single-cell epigenomics integration:

  • Combine with single-cell technologies to examine ZNF644 binding heterogeneity in cell populations

  • ZNF644 antibodies can be used for cell sorting prior to single-cell analysis or in situ approaches

• CRISPR screening with antibody-based readouts:

  • Use ZNF644 antibodies to assess how perturbation of different genes affects ZNF644 binding or activity

  • This can identify regulatory networks controlling ZNF644 function

• Multiplex imaging approaches:

  • Combine with cyclic immunofluorescence or mass cytometry for spatial organization studies

  • When using HRP-conjugated antibodies, tyramide signal amplification can provide enhanced sensitivity for imaging applications

The methodological integration of these technologies requires careful optimization, particularly when adapting protocols designed for unconjugated antibodies to work with HRP-conjugated versions.

How can I troubleshoot weak or absent signals when using ZNF644 Antibody, HRP conjugated in ELISA?

When facing weak or absent signals in ELISA using HRP-conjugated ZNF644 antibody, implement this systematic troubleshooting approach:

• Antibody activity verification:

  • Test the antibody with a positive control sample known to contain ZNF644 (e.g., HeLa cell lysate)

  • Verify HRP activity using a direct enzyme activity test with substrate

• Protocol optimization:

  • Adjust antibody concentration: Try a titration series (e.g., 1:250, 1:500, 1:1000, 1:2000)

  • Extend incubation times: Increase from standard 1-2 hours to overnight at 4°C

  • Modify wash protocol: Ensure thorough washing while preventing excessive loss of bound antibody

• Sample preparation assessment:

  • Ensure proper sample preparation with appropriate lysis buffers that maintain protein integrity

  • Check for presence of proteases or denaturants that might affect antibody recognition

  • Consider sample pre-clearing to remove components that might interfere with antibody binding

• Detection system evaluation:

  • Test alternative substrates with different sensitivity levels

  • Ensure substrate is fresh and properly stored

  • Check plate reader settings and functionality

• Methodological matrix:

  Issue	Possible Cause	Solution
  No signal	Inactive enzyme	Use fresh substrate; verify HRP activity with control
  No signal	Target absent	Validate sample preparation; use positive control
  Weak signal	Insufficient antibody	Increase concentration; extend incubation time
  Weak signal	Inefficient capture	Optimize coating conditions; try alternative capture method
  High background	Insufficient blocking	Extend blocking time; try alternative blocking reagents

How should I interpret contradictory results between different detection methods using ZNF644 antibodies?

When faced with contradictory results between different detection methods (e.g., ELISA vs. Western blot, or between different antibodies), follow these analytical approaches:

• Epitope availability analysis:

  • Different antibodies target different regions of ZNF644 (aa 1-50, aa 1-300, or aa 752-913)

  • These regions may have differential accessibility in various experimental conditions

  • Consider whether protein folding, complex formation, or post-translational modifications might affect epitope recognition

• Method-specific considerations:

  • ELISA: Typically detects native protein; may be affected by protein-protein interactions

  • Western blot: Detects denatured protein; exposes internal epitopes but may lose conformational epitopes

  • IP: Maintains native structure; depends on epitope accessibility in solution

• Validation through orthogonal approaches:

  • Confirm protein expression at the mRNA level using RT-qPCR

  • Use multiple antibodies targeting different epitopes

  • Consider mass spectrometry-based proteomic validation for definitive identification

• Reconciliation strategy for conflicting data:

  • Document specific experimental conditions that produce each result

  • Test whether specific buffer conditions, detergents, or sample preparation methods affect outcomes

  • Determine whether results correlate with functional assays or expected biological contexts

• Advanced troubleshooting for persistent contradictions:

  • Consider isoform-specific detection: ZNF644 may have multiple isoforms with different domain compositions

  • Investigate potential post-translational modifications affecting epitope recognition

  • Examine whether protein complexes or interacting partners might mask specific epitopes

What controls should I include when studying ZNF644 in the context of epigenetic regulation?

When investigating ZNF644 in epigenetic regulation contexts, robust controls are essential for data interpretation:

• Expression controls:

  • Positive control: Cell lines with confirmed ZNF644 expression (e.g., HeLa, 293T, Jurkat)

  • Negative control: ZNF644 knockdown or knockout cells

  • Isotype control: Non-specific IgG of the same species and isotype as the ZNF644 antibody

• Chromatin association controls:

  • Input sample: Total chromatin before immunoprecipitation

  • Negative genomic regions: Loci not expected to bind ZNF644

  • Positive genomic regions: Previously validated ZNF644 binding sites if available

  • Technical controls: Similar to those used in the iPOND-MS methodology described in result , including "no click" and thymidine chase conditions

• Functional validation controls:

  • Gene expression correlation: Compare ZNF644 binding with transcriptional changes

  • Perturbation validation: Assess effects of ZNF644 depletion or overexpression on epigenetic marks

  • Temporal controls: Time-course experiments to establish causality in epigenetic changes

• Methodological controls specific to epigenetic studies:

  • Chromatin shearing/digestion controls: Verify appropriate fragment size distribution

  • ChIP-seq quality controls: Include spike-in normalization standards

  • Cross-validation: Compare results between different antibodies or detection methods

• Analytical considerations:

  • Normalization approach: Similar to the methods described in result , using appropriate references (e.g., housekeeping genes, spike-in controls)

  • Statistical thresholds: Apply multiple testing correction as described in the iPOND-MS analysis

  • Biological replicates: Include sufficient replication to account for biological variability