ZNF75D Antibody

What is ZNF75D and why is it studied in molecular biology research?

ZNF75D (zinc finger protein 75D), also known as ZNF75 or ZNF82, is a 289 amino acid member of the Krueppel C2H2-type zinc finger protein family. It contains five C2H2-type zinc fingers and one KRAB domain through which it is thought to be involved in DNA-binding and transcriptional regulation . ZNF75D is primarily localized to the nucleus and is believed to function as a transcription factor . Most zinc finger proteins contain a Krueppel-type DNA binding domain and a KRAB domain that interacts with KAP1, thereby recruiting histone-modifying proteins . This makes ZNF75D an important subject for studying gene regulation mechanisms, transcriptional networks, and potential roles in disease pathways.

How do I select the most appropriate ZNF75D antibody for my research?

When selecting a ZNF75D antibody, consider these critical factors:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, ELISA, etc.)

• Species reactivity: Ensure the antibody recognizes ZNF75D in your species of interest (human, mouse, rat)

• Epitope location: Different antibodies recognize different regions of ZNF75D, which may affect detection depending on protein conformation or interactions

• Clonality: While most available ZNF75D antibodies are polyclonal, consider whether your experiment requires the specificity of a monoclonal antibody

• Validation data: Examine the manufacturer's validation data in contexts similar to your planned experiments

For most general applications studying human ZNF75D, a rabbit polyclonal antibody with validation data in Western blot applications provides a good starting point .

What is the expected molecular weight when detecting ZNF75D by Western blot?

While the calculated molecular weight of ZNF75D is approximately 59-60 kDa based on its amino acid sequence , researchers should note that the observed molecular weight in Western blot experiments is often around 35 kDa . This discrepancy may be due to:

• Post-translational modifications

• Protein processing or degradation

• Splice variants

• The detection of protein fragments

Always run appropriate positive controls to confirm the specific band pattern in your experimental system and be prepared to observe potential variations in molecular weight depending on cell/tissue type and experimental conditions .

What are the recommended dilutions and protocols for using ZNF75D antibodies in Western blot applications?

Based on multiple manufacturer recommendations, the following dilution ranges for Western blot applications should be considered:

For optimal Western blot results:

• Use fresh cell or tissue lysates with protease inhibitors

• Load 20-40 μg of total protein per lane

• Transfer to PVDF or nitrocellulose membrane

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

• Incubate with primary antibody overnight at 4°C

• Wash thoroughly before adding the appropriate secondary antibody

• Develop using your preferred detection method (ECL, fluorescence)

Antibody performance may be sample-dependent, so optimization for your specific experimental system is recommended .

How can I validate the specificity of a ZNF75D antibody in my experimental system?

To ensure your ZNF75D antibody is detecting the intended target with specificity:

• Positive controls: Include cell lines known to express ZNF75D (RAW 264.7 cells have been validated)

• Blocking peptide: Use a competitive binding assay with the immunizing peptide to confirm specificity

• siRNA knockdown: Compare signal between normal cells and those with ZNF75D knocked down

• Multiple antibodies: Use antibodies targeting different epitopes of ZNF75D

• Mass spectrometry: For definitive confirmation, isolate the detected band and perform mass spectrometry

• Immunoprecipitation followed by Western blot: Use for additional confirmation of specificity

A well-validated antibody should show consistent results across multiple validation methods and demonstrate absence of non-specific bands .

What applications beyond Western blot are supported by ZNF75D antibodies?

ZNF75D antibodies have been validated for multiple experimental applications:

• Immunohistochemistry (IHC): Typical dilutions range from 1:100-1:300

• ELISA: Highly sensitive detection with dilutions up to 1:10,000-1:312,500

• Immunofluorescence: For subcellular localization studies (nuclear localization expected)

• Immunoprecipitation: For studying protein-protein interactions

• ChIP assays: For investigating ZNF75D binding to DNA targets

Application-specific protocols may require optimization, as sensitivity and specificity can vary. Always validate the antibody for your specific application if the manufacturer hasn't provided validation data for that particular use .

What are common issues when working with ZNF75D antibodies and how can they be resolved?

For persistent issues, consider using alternative ZNF75D antibodies targeting different epitopes or validated for your specific application .

What are the optimal storage conditions for maintaining ZNF75D antibody performance?

To preserve antibody functionality and prevent degradation:

• Long-term storage: Store at -20°C as recommended by manufacturers

• Working aliquots: For frequent use, small aliquots can be kept at 4°C for up to one month

• Avoid freeze-thaw cycles: Multiple freeze-thaw cycles can significantly reduce antibody activity

• Storage buffer: Most ZNF75D antibodies are supplied in PBS with 50% glycerol, 0.02% sodium azide, and sometimes BSA (0.1-0.5%)

• Reconstitution: For lyophilized antibodies, reconstitute in distilled water to a final concentration of 1 mg/mL

Remember that antibody stability can vary between manufacturers and lots. Always check the specific storage recommendations for your antibody .

How can ZNF75D antibodies be used to investigate transcriptional regulatory networks?

For advanced transcriptional network studies:

• ChIP-seq experiments: Use ZNF75D antibodies to immunoprecipitate chromatin, followed by sequencing to identify genome-wide binding sites

• Sequential ChIP (Re-ChIP): Determine if ZNF75D co-occupies specific genomic regions with other transcription factors

• Co-immunoprecipitation: Identify protein partners that interact with ZNF75D to form transcriptional complexes

• Proximity ligation assay: Visualize and quantify protein interactions in situ

• CUT&RUN or CUT&Tag: Higher resolution alternatives to traditional ChIP for mapping ZNF75D binding sites

For these advanced applications, antibody specificity is critical. Validate using knockout/knockdown controls and ensure the antibody recognizes the native, folded protein rather than just denatured epitopes .

What insights can be gained from studying ZNF75D across different cellular contexts and disease models?

ZNF75D research across diverse biological contexts can reveal:

• Tissue-specific functions: Compare ZNF75D expression and localization patterns across tissues using immunohistochemistry

• Disease associations: Investigate altered ZNF75D expression or localization in disease states through immunohistochemical comparison of normal vs. pathological samples

• Stress responses: Examine how cellular stressors affect ZNF75D expression, modification, or localization

• Developmental regulation: Track ZNF75D expression during developmental processes

• Relationship to other zinc finger proteins: Comparative studies may reveal functional redundancy or specialization

For cross-tissue or cross-species studies, ensure your ZNF75D antibody has been validated for the specific context. The epitope recognized should be conserved across the species being studied .

How can post-translational modifications of ZNF75D be investigated using antibodies?

To study post-translational modifications (PTMs) of ZNF75D:

• Phospho-specific antibodies: If available, these can detect specific phosphorylated forms of ZNF75D

• Immunoprecipitation followed by PTM-specific detection: Use general ZNF75D antibodies for IP, then probe with antibodies against specific modifications (phospho, ubiquitin, SUMO, etc.)

• 2D gel electrophoresis: Separate ZNF75D isoforms by charge and mass, followed by Western blotting

• Mass spectrometry: After immunoprecipitation with ZNF75D antibodies, perform mass spectrometry to identify specific modifications

• In vitro kinase assays: Test whether specific kinases modify immunoprecipitated ZNF75D

Understanding PTMs may help explain the discrepancy between the calculated molecular weight (59-60 kDa) and observed weight (~35 kDa) in some experimental systems .

How might ZNF75D antibodies contribute to understanding epigenetic regulation mechanisms?

ZNF75D contains a KRAB domain that typically interacts with KAP1 (TRIM28), which recruits chromatin-modifying complexes. ZNF75D antibodies can be used to investigate these epigenetic connections through:

• Chromatin immunoprecipitation followed by sequencing (ChIP-seq): Map ZNF75D binding sites genome-wide and correlate with histone modification patterns

• Co-immunoprecipitation with chromatin modifiers: Identify interactions between ZNF75D and histone deacetylases, methyltransferases, or other epigenetic regulators

• Sequential ChIP: Determine co-occupancy of ZNF75D with specific histone modifications

• CUT&RUN or CUT&Tag with antibody combinations: Higher resolution mapping of ZNF75D in relation to chromatin states

• Immunofluorescence co-localization: Visualize ZNF75D in relation to heterochromatin or euchromatin markers

These approaches can help elucidate how ZNF75D contributes to gene silencing or activation through recruitment of specific chromatin-modifying complexes .

What considerations are important when designing experiments to study ZNF75D isoforms or splice variants?

When investigating potential ZNF75D isoforms or splice variants:

• Epitope mapping: Determine exactly which region(s) of ZNF75D your antibody recognizes

• Multiple antibodies approach: Use antibodies targeting different domains to identify potential isoforms

• RNA analysis correlation: Combine protein detection with RT-PCR or RNA-seq data to confirm expression of splice variants

• Isoform-specific knockdown: Design siRNAs targeting specific exons to selectively deplete certain isoforms

• Mass spectrometry validation: Confirm the identity of bands detected at unexpected molecular weights

Understanding which ZNF75D domain(s) your antibody targets is crucial, as some isoforms may lack specific domains and therefore might not be detected by certain antibodies. The antibody information should specify which region of ZNF75D was used as the immunogen .