ZNF785 Antibody

What is ZNF785 and what is its function in human cells?

ZNF785 (Zinc Finger Protein 785) is a transcription regulatory protein predicted to enable DNA-binding transcription repressor activity, RNA polymerase II-specific and RNA polymerase II transcription regulatory region sequence-specific DNA binding activity. It is predicted to be involved in negative regulation of transcription by RNA polymerase II and is primarily located in the nucleus . Recent studies have identified ZNF785 as one of the KRAB-zinc finger proteins that interact with TRIM28 (also known as KAP1), a master regulator of gene expression . This interaction suggests ZNF785 may play a role in epigenetic gene silencing mechanisms.

What are the general characteristics of commercially available ZNF785 antibodies?

Most commercially available ZNF785 antibodies are rabbit polyclonal antibodies designed for research applications. Key characteristics include:

These antibodies are typically supplied in liquid form with glycerol-based storage buffers and should be stored at -20°C to maintain stability .

What are the optimal dilutions for ZNF785 antibody in different experimental applications?

The optimal dilution of ZNF785 antibody varies by application and specific product. Based on manufacturer recommendations:

For Western blot applications, most manufacturers recommend starting with a 1:1000 dilution and adjusting based on signal strength . It's always advisable to perform a dilution series to determine the optimal concentration for your specific experimental conditions and cell/tissue type. When using a new lot of antibody, validation at different dilutions is recommended to ensure consistent results.

How should ZNF785 antibody samples be prepared and stored for optimal performance?

For optimal performance and longevity of ZNF785 antibodies, follow these preparation and storage guidelines:

• Upon receipt, briefly centrifuge the antibody vial before opening to collect all material at the bottom.

• Aliquot the antibody into smaller volumes to avoid repeated freeze-thaw cycles, which can degrade antibody performance.

• Store aliquots at -20°C in the dark. Most ZNF785 antibodies are supplied in a buffer containing 50% glycerol, PBS, and 0.02% sodium azide or thimerosal, which helps maintain stability during freezing .

• Avoid storing diluted antibody solutions for extended periods; prepare fresh working dilutions when possible.

• Prior to use, thaw aliquots at room temperature or on ice rather than at higher temperatures.

• Do not vortex antibody solutions; instead, mix gently by inversion or light tapping.

Most manufacturers indicate that properly stored antibodies maintain stability for 12 months from the shipping date .

How does ZNF785 interact with TRIM28, and why is this relevant for epigenetic research?

Recent research has demonstrated that ZNF785 is one of several KRAB-zinc finger proteins (KRAB-ZNFs) that interact with TRIM28 (also known as KAP1), a key regulator of gene silencing. This interaction is relevant for epigenetic research for several reasons:

• The TRIM28 protein contains an RBCC (RING, B1 and B2 boxes, coiled-coil domain) motif that facilitates homodimerization and specifically interacts with the KRAB domain of zinc finger proteins like ZNF785 .

• Studies using immunoprecipitation followed by mass spectrometry have confirmed the interaction between TRIM28 and ZNF785, with verification by IP-Western blotting .

• When TRIM28 undergoes acetylation-mimic mutation (K304Q), its interaction with ZNF785 is attenuated, which affects gene expression in leukemic K562 cells .

• Knockdown experiments of related ZNFs have shown upregulation of specific targets, suggesting that ZNF785 may similarly participate in repression of target genes through its interaction with TRIM28 .

This interaction is particularly important for researchers studying epigenetic silencing mechanisms, transcriptional regulation, and the role of KRAB-zinc finger proteins in health and disease states.

What cell lines and tissues have been validated for ZNF785 antibody applications?

ZNF785 antibodies have been tested and validated in various cell lines and tissues, with consistent detection across multiple human cancer cell lines. The following table summarizes validated cell lines and detection methods:

For tissue expression analysis, the Human Protein Atlas provides mRNA expression data for ZNF785 across various tissues and cell types, including immune cells . When studying ZNF785 in new cell lines or tissues, preliminary validation experiments are recommended to confirm detection at the expected molecular weight.

How can researchers validate the specificity of ZNF785 antibodies in their experimental systems?

Validating antibody specificity is crucial for generating reliable research data. For ZNF785 antibodies, consider implementing these validation strategies:

• Knockout/Knockdown Controls: Use ZNF785 knockout (KO) cell lines or knockdown approaches (siRNA/shRNA) to verify that the signal disappears or is significantly reduced. This is considered the gold standard for antibody validation .

• Peptide Competition Assay: Preincubate the antibody with its immunizing peptide before application. A specific antibody will show reduced or absent signal when the epitope is blocked by the competing peptide. This approach has been used for validating some ZNF785 antibodies, as seen in Western blot analysis of HepG2 cell lysates .

• Molecular Weight Verification: Confirm that the detected band appears at the expected molecular weight (46-87 kDa for ZNF785). Multiple bands may indicate isoforms, post-translational modifications, or non-specific binding .

• Multiple Antibodies Approach: Use different antibodies targeting distinct epitopes of ZNF785 to confirm consistent detection patterns.

• Recombinant Expression: Overexpress tagged ZNF785 and confirm detection by both the ZNF785 antibody and an antibody against the tag.

The YCharOS initiative (mentioned in search result ) provides standardized antibody characterization and validation data, which may include information on ZNF785 antibodies to help researchers select validated reagents.

What are common technical challenges when using ZNF785 antibodies in Western blotting, and how can they be addressed?

When using ZNF785 antibodies for Western blotting, researchers may encounter several technical challenges. Here are common issues and their solutions:

• Background Signal/Non-specific Binding:

  • Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Use 5% BSA instead of milk for blocking and antibody dilution

  • Increase washing time and frequency (5-6 washes of 5-10 minutes each)

  • Optimize primary antibody dilution (try 1:1000-1:2000 range)

• Weak or No Signal:

  • Ensure adequate protein loading (25-50 μg total protein per lane)

  • Use fresh transfer buffers and optimize transfer conditions

  • Reduce antibody dilution (use more concentrated antibody)

  • Increase exposure time during detection

  • Try enhanced chemiluminescence (ECL) with extended exposure

• Multiple Bands:

  • Verify if additional bands represent isoforms, post-translational modifications, or degradation products

  • Adjust sample preparation (use fresh protease inhibitors)

  • Try different lysis buffers to maintain protein integrity

  • Run a peptide competition assay to identify specific vs. non-specific bands

• Inconsistent Results Between Experiments:

  • Standardize protein extraction protocols

  • Aliquot and minimize freeze-thaw cycles of antibody

  • Use consistent exposure times across experiments

  • Include positive control samples (HepG2 or HeLa cell lysates work well for ZNF785)

How can ZNF785 antibodies be used to study its role in transcriptional regulation networks?

ZNF785 antibodies can be powerful tools for investigating the protein's role in transcriptional regulation networks through several advanced applications:

• Chromatin Immunoprecipitation (ChIP):

  • While not explicitly validated in the search results, appropriately validated ZNF785 antibodies could potentially be used in ChIP assays to identify genomic binding sites of ZNF785

  • This would reveal direct target genes regulated by ZNF785 and allow analysis of binding motifs

• Co-Immunoprecipitation (Co-IP) Studies:

  • ZNF785 antibodies have been successfully used in immunoprecipitation studies to detect protein-protein interactions, particularly with TRIM28

  • This approach can be extended to identify other protein partners in transcriptional regulatory complexes

  • Example protocol: Use anti-ZNF785 antibody conjugated to protein A/G beads to pull down ZNF785 and associated proteins from cell lysates, followed by Western blot or mass spectrometry analysis

• Proximity Ligation Assay (PLA):

  • Combining ZNF785 antibodies with antibodies against suspected interaction partners for in situ detection of protein-protein interactions

  • This technique can provide spatial information about interactions within cellular compartments

• Gene Expression Correlation Studies:

  • After knockdown/knockout of ZNF785, use antibodies to verify protein depletion, then perform transcriptome analysis to identify affected genes

  • This has been successfully demonstrated with related zinc finger proteins where knockdown of ZNF558 resulted in upregulation of specific targets including ZNF568, ITGB3, and LINE1

By combining these approaches, researchers can build comprehensive models of ZNF785's role in transcriptional regulation networks and its contributions to gene silencing through interaction with TRIM28 and other epigenetic modifiers.

What is known about post-translational modifications of ZNF785 and how can antibodies help study these modifications?

While the search results don't specifically address post-translational modifications (PTMs) of ZNF785, studying PTMs is an important aspect of understanding protein function and regulation. Here's how researchers can approach this topic:

• Detection of Total vs. Modified ZNF785:

  • Current commercial antibodies appear to detect total ZNF785 rather than specific modified forms

  • Researchers interested in PTMs would need to:
    a) Use antibodies specific to particular modifications (if available)
    b) Use general PTM detection methods followed by ZNF785 immunoprecipitation
    c) Perform mass spectrometry analysis after immunoprecipitation with ZNF785 antibodies

• Potential PTMs Based on Related Proteins:

  • Many zinc finger proteins undergo phosphorylation, SUMOylation, and ubiquitination that regulate their activity

  • The interaction with TRIM28, which has E3 ligase activity through its RING domain, suggests ZNF785 might be subject to ubiquitination or SUMOylation

• Methodological Approach to Study ZNF785 PTMs:

  • Immunoprecipitate ZNF785 using validated antibodies

  • Analyze by mass spectrometry to identify PTMs

  • Validate findings using modification-specific antibodies in Western blotting

  • Perform functional studies using site-directed mutagenesis of modified residues

• PTM Analysis in Different Cellular Contexts:

  • Compare PTM patterns across different cell types where ZNF785 is expressed

  • Examine changes in PTMs under different cellular stresses or stimulations

  • Assess how PTMs affect interactions with TRIM28 and other partners

This represents an area of potential future research, as detailed information about ZNF785 PTMs appears limited in the current literature.