ZNF624 Antibody

What is ZNF624 and what are its primary biological functions?

ZNF624 (Zinc Finger Protein 624) is a nuclear protein that belongs to the zinc finger protein family. It is encoded by a gene located on chromosome 17 in humans and has a molecular weight of approximately 100 kDa . The protein contains multiple C2H2-type zinc finger motifs, which are DNA-binding domains commonly found in transcription factors. Based on its structure, ZNF624 is believed to be involved in transcriptional regulation, though specific target genes and detailed molecular pathways remain to be fully characterized .

As a member of the zinc finger protein family, ZNF624 likely functions by binding to specific DNA sequences through its zinc finger domains to regulate gene expression. Other zinc finger proteins, such as EZI, have been shown to interact with transcription factors like STAT3 to enhance their activity , suggesting that ZNF624 might function through similar protein-protein interactions in transcriptional regulatory complexes.

What types of ZNF624 antibodies are available for research applications?

Several types of ZNF624 antibodies are available for research purposes, varying in their host species, clonality, target epitopes, and conjugation status:

The availability of antibodies targeting different epitopes allows researchers to select the most appropriate reagent based on their specific experimental requirements and the structural conformation of ZNF624 in their experimental system .

How specific are commercially available ZNF624 antibodies?

Commercial ZNF624 antibodies demonstrate varying degrees of specificity depending on the target epitope and purification methods. Most available antibodies are polyclonal, produced in rabbits or mice immunized with synthetic peptides corresponding to specific regions of human ZNF624 .

These antibodies undergo validation testing through techniques such as Western blotting (WB), immunofluorescence (IF), and ELISA to ensure specificity and high affinity. For example, the antibody produced by Boster Bio (catalog # A15028) is specifically validated for Western blot applications with human samples .

Some antibodies show cross-reactivity with ZNF624 from multiple species. For instance, certain middle region-targeting antibodies react not only with human ZNF624 but also with homologs from cow, dog, mouse, rat, horse, pig, bat, monkey, guinea pig, and rabbit samples . This cross-reactivity can be advantageous for comparative studies across species but may require additional validation for studies requiring absolute human specificity.

What are the optimal conditions for Western blotting with ZNF624 antibodies?

For Western blotting applications with ZNF624 antibodies, the following optimized protocol is recommended:

• Sample preparation: Prepare cell or tissue lysates using standard protocols with protease inhibitors to prevent protein degradation.

• Protein loading: Load 20-50 μg of total protein per lane for cell lysates.

• Antibody dilution: Use a 1:1000 dilution for primary ZNF624 antibodies as a starting point for optimization . For the ABIN2460621 antibody, a concentration of 1.0 μg/mL is recommended .

• Secondary antibody: For HRP-conjugated secondary antibodies, a dilution range of 1:50,000-1:100,000 is suggested when using unconjugated ZNF624 primary antibodies .

• Detection: Given the high molecular weight of ZNF624 (approximately 100 kDa), use a longer transfer time and lower percentage SDS-PAGE gels (7-8%) for optimal separation and transfer efficiency.

• Membranes: PVDF membranes are recommended for detecting ZNF624 due to their higher protein binding capacity compared to nitrocellulose.

• Blocking: Use 5% non-fat dry milk or BSA in TBST for blocking to reduce background.

The actual working concentration for both primary and secondary antibodies should be determined empirically, as optimal conditions can vary depending on the specific antibody batch and experimental system .

How should ZNF624 antibodies be stored to maintain optimal activity?

Proper storage of ZNF624 antibodies is crucial for maintaining their activity and specificity over time. Based on manufacturer recommendations:

• Short-term storage (up to 2 weeks): Store at 2-8°C (refrigerated) .

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles .

• Lyophilized antibodies: Reconstitute in distilled water to a final concentration of 1 mg/mL. After reconstitution, the antibody should be stored according to the short-term or long-term guidelines above .

• Working solutions: Diluted antibody solutions should be prepared fresh for each experiment.

• Freeze-thaw cycles: Minimize repeated freeze-thaw cycles as these can lead to denaturation and loss of antibody activity .

Most commercial ZNF624 antibodies are supplied as purified polyclonal antibodies in PBS buffer, sometimes with small amounts of preservatives such as sodium azide (0.09% W/V) . The presence of preservatives helps maintain antibody integrity during storage but should be considered when designing experiments sensitive to these additives.

What are the challenges in studying subcellular localization of ZNF624 using immunofluorescence?

Studying the subcellular localization of ZNF624 using immunofluorescence presents several technical challenges that researchers should consider:

• Nuclear protein detection: As a zinc finger protein likely involved in transcriptional regulation, ZNF624 is expected to be predominantly nuclear. Based on studies of related zinc finger proteins like EZI, which has been shown to localize in the nucleus , optimizing nuclear permeabilization protocols is crucial for accessing nuclear ZNF624.

• Epitope masking: The zinc finger domains may be engaged in DNA binding or protein-protein interactions that mask antibody epitopes. Using antibodies targeting different regions (N-terminal, central, etc.) can help overcome this limitation .

• Signal-to-noise ratio: Nuclear proteins often require more stringent washing conditions to reduce background fluorescence. Titrating primary antibody concentrations and using appropriate blocking solutions are essential for optimal results.

• Fixation methods: Cross-linking fixatives like paraformaldehyde may alter the conformation of zinc finger domains. Comparing different fixation methods (paraformaldehyde, methanol, or combinations) can help identify the optimal approach for ZNF624 detection.

• Validation controls: Using cells with known ZNF624 expression levels (including knock-down or knock-out controls) is essential for confirming antibody specificity in immunofluorescence applications.

• Co-staining compatibility: When performing co-localization studies, ensure that the ZNF624 antibody is compatible with antibodies against other proteins of interest in terms of species origin and detection systems.

Drawing from studies of other zinc finger proteins, researchers might also want to investigate whether ZNF624 localization changes under different cellular conditions, such as during cell cycle progression or in response to specific signaling pathways .

How can researchers resolve inconsistent results with ZNF624 antibodies across different applications?

When facing inconsistent results with ZNF624 antibodies across different applications, researchers should consider several factors:

• Epitope accessibility: The accessibility of epitopes can vary significantly between applications. For instance, an antibody that works well in Western blotting (where proteins are denatured) might not work in immunoprecipitation or immunofluorescence (where proteins maintain native conformation). Consider using antibodies targeting different regions of ZNF624 for different applications .

• Antibody validation: Ensure that the antibody has been validated for your specific application. Some ZNF624 antibodies are only validated for certain applications (e.g., Boster Bio's A15028 is guaranteed for Western blotting) .

• Protein expression levels: ZNF624 expression may vary across cell types or tissues. If detection is inconsistent, consider:

  • Using positive control samples with known ZNF624 expression

  • Concentrating proteins when expression levels are low

  • Optimizing exposure times for Western blots

• Post-translational modifications: ZNF624 may undergo post-translational modifications that affect antibody recognition. Similar zinc finger proteins show bands of larger molecular weight than predicted, suggesting modification . When inconsistent band patterns are observed, consider:

  • Using phosphatase or other enzymatic treatments to remove modifications

  • Using different antibodies targeting regions less likely to be modified

• Cross-reactivity: Some ZNF624 antibodies show cross-reactivity with homologs from multiple species . If working across species, ensure the antibody has been validated for your species of interest.

• Protocol optimization: Systematically optimize key parameters:

  • For Western blotting: Transfer conditions, blocking agents, antibody concentrations

  • For immunofluorescence: Fixation method, permeabilization, antibody incubation time

• Lot-to-lot variability: Particularly with polyclonal antibodies, lot-to-lot variability can occur. Maintain records of antibody lots that work consistently in your experimental system.

What are the potential causes of multiple bands in Western blots with ZNF624 antibodies?

Multiple bands in Western blots using ZNF624 antibodies can occur for several reasons, and distinguishing between specific and non-specific signals requires careful analysis:

• Isoforms and splice variants: ZNF624 may exist in multiple isoforms due to alternative splicing. Check protein databases for documented isoforms to determine if additional bands correspond to predicted molecular weights of known variants.

• Post-translational modifications: Similar zinc finger proteins have been observed to produce bands of higher molecular weight due to modifications. For instance, a related zinc finger protein EZI showed a larger molecular weight band at ~90 kDa in addition to the expected band, which was attributed to modifications in the N-terminal region . Common modifications include:

  • Phosphorylation

  • SUMOylation

  • Ubiquitination

  • Glycosylation

• Proteolytic degradation: Incomplete protease inhibition during sample preparation can lead to partial degradation of ZNF624, resulting in smaller fragments that are detected by the antibody. Ensure that fresh protease inhibitors are added to lysis buffers and samples are kept cold during processing.

• Cross-reactivity: Antibodies may cross-react with other zinc finger proteins that share sequence homology with ZNF624, particularly if the antibody targets conserved zinc finger domains.

• Non-specific binding: High antibody concentrations can lead to non-specific binding. Optimize antibody dilutions and blocking conditions to minimize this issue.

To distinguish between these possibilities:

• Use positive and negative control samples (e.g., ZNF624 overexpression or knockdown)

• Perform peptide competition assays to confirm specificity

• Test multiple antibodies targeting different epitopes of ZNF624

• Use gradient gels for better separation of high molecular weight proteins

• Consider phosphatase treatment of samples to eliminate bands due to phosphorylation

How should researchers design experiments to compare ZNF624 expression across different cell types or tissues?

When designing experiments to compare ZNF624 expression across different cell types or tissues, researchers should consider a comprehensive approach incorporating multiple techniques:

• Antibody selection:

  • Choose antibodies with broad species reactivity if comparing across species

  • Select antibodies validated for quantitative applications

  • Consider using antibodies targeting different epitopes to confirm results

• Western blot analysis:

  • Include positive controls with known ZNF624 expression

  • Use equal protein loading (confirmed by housekeeping proteins)

  • Consider quantitative Western blotting with standard curves

  • Recommended dilution for primary antibody: 1:1000

  • For HRP-conjugated secondary antibodies: 1:50,000-1:100,000

• qRT-PCR validation:

  • Design primers specific to conserved regions of ZNF624 transcripts

  • Validate transcript levels to complement protein data

  • Normalize to appropriate reference genes for each tissue/cell type

• Immunohistochemistry/Immunofluorescence:

  • Use standardized fixation and staining protocols across all samples

  • Process and image all samples in parallel to minimize technical variation

  • Employ quantitative image analysis for objective comparison

  • Include appropriate controls for autofluorescence (particularly important for tissues)

• Controls and normalization:

  • Include gradient of known ZNF624 concentrations (using recombinant protein) for semi-quantitative analysis

  • Normalize expression to appropriate housekeeping genes/proteins for each tissue

  • Include biological replicates (different donors/animals) to account for individual variation

• Statistical analysis:

  • Apply appropriate statistical tests for comparing expression across multiple samples

  • Consider hierarchical clustering to identify patterns of ZNF624 expression

  • Correlate ZNF624 levels with relevant phenotypic or functional parameters

By combining these approaches, researchers can obtain more reliable and comprehensive data on ZNF624 expression patterns that account for potential technical and biological variables.

What are the best approaches for validating ZNF624 antibody specificity in new experimental systems?

Validating ZNF624 antibody specificity is crucial when working with new experimental systems or applying antibodies to novel applications. A comprehensive validation strategy should include:

• Genetic validation:

  • CRISPR/Cas9 knockout of ZNF624: The most definitive validation method; signal should be absent in knockout samples

  • siRNA/shRNA knockdown: Reduction in signal intensity should correlate with knockdown efficiency

  • Overexpression systems: Increased signal should be observed in cells overexpressing ZNF624

• Peptide competition assays:

  • Pre-incubate the antibody with the immunizing peptide

  • If the antibody is specific, the peptide will block binding and eliminate or reduce the signal

  • Consider testing blocking peptides corresponding to the immunogen region (e.g., amino acids 264-298 for the Boster Bio antibody)

• Multiple antibody concordance:

  • Test multiple antibodies targeting different epitopes of ZNF624

  • Consistent results across antibodies increase confidence in specificity

  • Compare antibodies raised in different host species or with different clonality

• Western blot validation:

  • Verify that the detected band matches the expected molecular weight (~100 kDa)

  • Check for additional bands that might indicate cross-reactivity

  • Perform validation across different sample types relevant to your research

• Mass spectrometry validation:

  • Immunoprecipitate ZNF624 using the antibody

  • Confirm the identity of the precipitated protein by mass spectrometry

  • This approach provides unbiased confirmation of antibody specificity

• Heterologous expression systems:

  • Express ZNF624 with epitope tags (e.g., FLAG, HA) in cells with low endogenous expression

  • Compare antibody signal with tag-specific antibody signal

  • Co-localization of signals confirms antibody specificity

• Cross-species reactivity assessment:

  • If working with non-human samples, compare epitope conservation across species

  • Test antibody in samples from species with known epitope conservation

  • Validate using the approaches above in each species of interest

Antibody validation should be performed for each application (Western blot, immunofluorescence, etc.) and each experimental system, as antibody performance can vary significantly between applications and biological contexts.

How can ZNF624 antibodies contribute to understanding the role of zinc finger proteins in transcriptional regulation?

ZNF624 antibodies can serve as valuable tools for elucidating the broader functions of zinc finger proteins in transcriptional regulation, particularly through these research approaches:

• Chromatin Immunoprecipitation (ChIP) studies:

  • ZNF624 antibodies could be used to identify genomic binding sites through ChIP-seq

  • This would reveal the specific DNA sequences recognized by ZNF624

  • Comparison with binding profiles of other zinc finger proteins could reveal common and unique regulatory targets

  • Integration with transcriptomic data would connect DNA binding with gene expression outcomes

• Protein complex identification:

  • Immunoprecipitation followed by mass spectrometry can identify ZNF624-associated proteins

  • This approach could reveal whether ZNF624, like other zinc finger proteins such as EZI, interacts with transcription factors (e.g., STAT3)

  • Understanding these interactions would help place ZNF624 within specific transcriptional regulatory networks

• Subcellular localization dynamics:

  • Immunofluorescence with ZNF624 antibodies can track the protein's localization under different conditions

  • Similar to EZI, which enhances STAT3 nuclear localization , ZNF624 might influence the nuclear retention of other transcription factors

  • Live-cell imaging with labeled antibody fragments could reveal dynamic changes in localization

• Domain-specific functions:

  • Using antibodies targeting different regions of ZNF624 in functional assays

  • This approach could help determine which domains are essential for nuclear localization, DNA binding, and protein-protein interactions

  • Comparisons with other zinc finger proteins could reveal conserved functional principles

• Epigenetic regulation:

  • Combined ChIP-seq for ZNF624 and histone modifications

  • This would reveal relationships between ZNF624 binding and chromatin state

  • Similar analyses for other zinc finger proteins would establish general principles about how this protein family influences epigenetic landscapes

• Developmental and disease contexts:

  • Tracking ZNF624 expression and localization across developmental stages or disease progression

  • This could reveal temporal and context-specific functions

  • Comparing results with other zinc finger proteins would identify family-wide versus member-specific roles

By conducting these studies across multiple zinc finger proteins, including ZNF624, researchers can develop a more comprehensive understanding of how this large protein family contributes to transcriptional regulation in diverse biological contexts.

What emerging techniques could enhance the utility of ZNF624 antibodies in functional genomics research?

Several emerging techniques could significantly enhance the utility of ZNF624 antibodies in functional genomics research:

• Proximity-dependent labeling combined with antibody validation:

  • BioID or APEX2 fusion with ZNF624 to identify proximity interaction partners

  • ZNF624 antibodies can validate these interactions through co-immunoprecipitation

  • This combination provides both discovery and validation approaches for protein interaction networks

• CUT&RUN and CUT&TAG technologies:

  • These techniques offer advantages over traditional ChIP-seq by requiring fewer cells and providing higher resolution

  • ZNF624 antibodies could be employed in these methods to map genomic binding sites with greater precision

  • Particularly valuable for rare cell populations or when studying dynamic transcriptional events

• Single-cell protein analysis:

  • Emerging single-cell proteomics techniques combined with ZNF624 antibodies

  • This would reveal cell-to-cell variation in ZNF624 expression and localization

  • Integration with single-cell transcriptomics could connect ZNF624 levels with transcriptional outcomes at single-cell resolution

• CRISPR screens with antibody-based readouts:

  • CRISPR perturbation of potential ZNF624 interacting partners or regulatory pathways

  • Antibody-based detection of changes in ZNF624 localization, modification, or expression

  • This approach could systematically identify factors that regulate ZNF624 function

• Liquid-phase separation studies:

  • Recent research suggests many transcription factors function within phase-separated condensates

  • ZNF624 antibodies could help determine if this zinc finger protein participates in such condensates

  • Immunofluorescence studies under various cellular conditions could reveal dynamic association with nuclear condensates

• Spatial transcriptomics integration:

  • Combining ZNF624 immunofluorescence with spatial transcriptomics

  • This would connect the spatial distribution of ZNF624 protein with localized transcriptional outputs

  • Particularly valuable for understanding tissue-specific functions in development or disease

• Cryo-electron tomography with antibody labeling:

  • Gold-conjugated ZNF624 antibodies for visualization in cellular ultrastructure

  • This approach could reveal the nanoscale organization of ZNF624-containing complexes

  • Particularly relevant for understanding how zinc finger proteins organize within chromatin structures

• Mass cytometry (CyTOF) applications:

  • Metal-conjugated ZNF624 antibodies for high-dimensional protein profiling

  • This enables simultaneous detection of ZNF624 along with dozens of other proteins

  • Valuable for understanding how ZNF624 expression correlates with cellular phenotypes and states

These emerging techniques, when combined with well-validated ZNF624 antibodies, have the potential to substantially advance our understanding of this zinc finger protein's function in complex biological systems.