ZNF774 Antibody

What is ZNF774 and what is its role in cellular processes?

ZNF774 is a member of the large family of zinc finger proteins with a molecular weight of approximately 55 kDa. It functions as a transcriptional regulator, modulating gene expression by interacting with DNA at promoter regions . The protein contains zinc finger domains that facilitate DNA binding, allowing it to recognize specific DNA sequences and regulate the expression of target genes. ZNF774 is encoded by the ZNF774 gene, identified with Entrez Gene ID 342132 and UniProt ID Q6NX45 . Although the specific genes regulated by ZNF774 are not fully characterized in the current literature, its structural features suggest it plays important roles in developmental processes and cellular homeostasis through transcriptional control. Understanding ZNF774 function contributes to our knowledge of gene regulation networks and may provide insights into disease mechanisms where transcriptional dysregulation occurs.

What types of ZNF774 antibodies are currently available for research applications?

Several types of ZNF774 antibodies are available for research purposes, primarily differing in their clonality, host species, and immunogen design. Monoclonal antibodies like PCRP-ZNF774-3F7 are produced by the Developmental Studies Hybridoma Bank (DSHB) using mouse as the host species and full-length recombinant human ZNF774 protein as the immunogen . These provide high specificity and consistency between batches. Polyclonal antibodies are also available, such as Abcam's rabbit polyclonal antibody (ab122137) generated against a recombinant fragment within the first 100 amino acids of human ZNF774 . Another polyclonal option from Abbexa is produced in rabbit using a recombinant fragment corresponding to amino acids 142-422 of human ZNF774 . Each antibody type offers distinct advantages, with monoclonals providing high specificity for a single epitope and polyclonals offering broader detection capabilities by recognizing multiple epitopes on the target protein. Researchers should select the appropriate antibody based on their specific experimental requirements, target species, and intended applications.

What are the recommended applications for ZNF774 antibodies?

ZNF774 antibodies have been validated for multiple research applications, with varying recommended dilutions depending on the specific antibody and application. Western blotting (WB) is a common application, with recommended dilutions ranging from 1:500 to 1:2000 for polyclonal antibodies . When using WB, ZNF774 typically appears as a band at approximately 50-55 kDa . Immunohistochemistry on paraffin-embedded tissues (IHC-P) is another validated application, with dilutions typically in the range of 1:100 to 1:200 . Abcam's antibody has been confirmed for immunocytochemistry/immunofluorescence (ICC/IF) applications, where it has demonstrated positive staining in the cytoplasm and cytoskeleton (intermediate filaments) of human cell lines like A-431 . ELISA is also a supported application for some ZNF774 antibodies, with recommended dilutions ranging from 1:20,000 to 1:80,000 . When designing experiments, researchers should consider that the optimal working dilution may need to be determined empirically for each specific experimental condition and sample type to achieve the best signal-to-noise ratio.

How can I validate the specificity of a ZNF774 antibody for my research?

Validating antibody specificity is essential for generating reliable research data with ZNF774 antibodies. A comprehensive validation approach should employ multiple complementary techniques. Begin with Western blot analysis using positive control samples like RT4 or U251MG cell lysates, where ZNF774 antibodies have demonstrated specific binding at the expected molecular weight of approximately 55 kDa . Include negative controls such as lysates from cells where ZNF774 is not expressed or has been knocked down using siRNA or CRISPR. Peptide competition assays provide another validation method, where pre-incubating the antibody with excess synthetic ZNF774 peptide (corresponding to the immunogen) should abolish specific signals in your application. For immunohistochemistry applications, compare staining patterns with documented ZNF774 expression in tissues like colon, where positive staining has been reported . Orthogonal validation can be performed by correlating protein detection with mRNA expression data from qPCR or RNA-seq experiments. For researchers with access to mass spectrometry facilities, immunoprecipitation followed by mass spectrometry analysis can provide definitive confirmation of antibody specificity by identifying the captured proteins.

What are the best practices for optimizing ZNF774 antibody use in Western blot experiments?

Optimizing Western blot protocols for ZNF774 detection requires attention to several key parameters. Sample preparation should include appropriate protease inhibitors to prevent degradation of ZNF774 protein, and optimization of lysis buffer conditions (detergent type and concentration) may be necessary depending on the cellular localization of your target. When separating proteins by SDS-PAGE, use a 10-12% gel to achieve optimal resolution around the 55 kDa range where ZNF774 is expected . Transfer conditions should be optimized for proteins in this molecular weight range, typically using PVDF membranes and a transfer time of 60-90 minutes at 100V or overnight at lower voltage. For blocking, 5% non-fat milk or BSA in TBST is generally effective, but empirical testing may be needed to determine which blocking agent provides the best signal-to-noise ratio. Primary antibody incubation should be performed using manufacturer-recommended dilutions (typically 1:500 to 1:2000 for ZNF774 antibodies) , with overnight incubation at 4°C often yielding the best results. After washing, use an appropriate HRP-conjugated secondary antibody matched to the host species of your primary antibody (anti-mouse for PCRP-ZNF774-3F7 or anti-rabbit for polyclonal antibodies) . For detection, enhanced chemiluminescence (ECL) systems are suitable, with exposure times optimized to prevent over-saturation while maintaining sensitivity.

What considerations should be made when designing immunofluorescence experiments with ZNF774 antibodies?

Designing effective immunofluorescence experiments with ZNF774 antibodies requires careful consideration of sample preparation, antibody selection, and imaging parameters. Begin by selecting an appropriate fixation method, considering that paraformaldehyde fixation (PFA) combined with Triton X-100 permeabilization has been successfully used with ZNF774 antibodies in cell lines like A-431 . Cell type selection is critical, as ZNF774 expression varies across tissues and cell lines. Sample adherence should be optimized to prevent cell loss during processing steps. When choosing primary antibodies, consider that rabbit polyclonal antibodies like ab122137 have demonstrated positive immunofluorescent staining in the cytoplasm and cytoskeleton (intermediate filaments) . Titrate antibody concentrations to determine optimal working dilutions, with 1-4 μg/ml reported as effective for some ZNF774 antibodies . For co-localization studies, carefully select secondary antibodies with minimal spectral overlap and include appropriate controls for antibody cross-reactivity. When conducting the experiment, incorporate proper negative controls (secondary antibody only, isotype control) and positive controls (tissues or cells known to express ZNF774). During imaging, optimize exposure settings to prevent photobleaching while maintaining adequate signal, and collect z-stack images if three-dimensional information is required. Data analysis should include quantification methods appropriate for the subcellular distribution pattern observed for ZNF774.

How can I troubleshoot weak or no signal when using ZNF774 antibodies?

When encountering weak or absent signals with ZNF774 antibodies, several systematic troubleshooting steps can help identify and resolve the issue. First, confirm that your experimental system expresses ZNF774 by checking RNA expression databases or performing RT-PCR, as expression levels vary across tissues and cell lines. If expression is confirmed, examine antibody handling and storage conditions, as improper storage or multiple freeze-thaw cycles can reduce antibody activity . Verify antibody viability using positive control samples such as RT4 or U251MG cell lysates where ZNF774 detection has been validated . For Western blotting applications, increase protein loading amount, extend primary antibody incubation time (overnight at 4°C), or try more sensitive detection systems like enhanced chemiluminescence substrates. Consider optimizing antigen retrieval methods for immunohistochemistry applications, as some epitopes may be masked during fixation. If the antibody concentration appears insufficient, titrate to higher concentrations while monitoring background levels. For applications where protein conformation is important, try alternative sample preparation methods that might better preserve epitope accessibility. If a monoclonal antibody fails, the specific epitope might be inaccessible in your experimental context, and switching to a polyclonal antibody that recognizes multiple epitopes could resolve the issue .

What could cause unexpected bands or patterns when using ZNF774 antibodies in Western blot?

Unexpected bands or staining patterns when using ZNF774 antibodies may result from several factors that require systematic investigation. Post-translational modifications of ZNF774, such as phosphorylation, glycosylation, or ubiquitination, can cause the protein to migrate at molecular weights different from the predicted 55 kDa . Protein degradation during sample preparation may generate lower molecular weight fragments; adding fresh protease inhibitors to lysis buffers can address this issue. Alternatively, unexpected higher molecular weight bands might represent dimers or complexes that were incompletely denatured; increasing SDS concentration or boiling time in sample buffer may resolve these. Cross-reactivity with structurally similar proteins, particularly other zinc finger proteins, can occur especially with polyclonal antibodies. This can be assessed through peptide competition assays or using samples with ZNF774 knockdown. Different isoforms of ZNF774 may exist due to alternative splicing, potentially explaining bands of unexpected sizes. For non-specific background, optimize blocking conditions by testing alternative blocking agents (BSA vs. milk) or increasing blocking time. If using enhanced chemiluminescence detection, reducing exposure time can help distinguish specific from non-specific signals. Finally, secondary antibody cross-reactivity can be evaluated by performing a control blot with secondary antibody only.

How should I interpret differences in ZNF774 localization between immunofluorescence and fractionation experiments?

Discrepancies in ZNF774 localization between immunofluorescence microscopy and subcellular fractionation experiments require careful interpretation and validation. Such differences may reflect biological realities rather than technical artifacts. ZNF774, as a zinc finger protein potentially involved in transcriptional regulation, might shuttle between cellular compartments depending on cell cycle stage, signaling events, or physiological conditions . Immunofluorescence data showing ZNF774 in cytoplasm and cytoskeleton (intermediate filaments) with Abcam's antibody may capture this dynamic localization . Different antibodies targeting distinct epitopes might preferentially recognize ZNF774 in specific compartments or conformational states; comparing results using multiple antibodies can help resolve this. Fixation methods significantly impact protein localization in immunofluorescence; PFA fixation may preserve cytoplasmic pools while methanol fixation might better reveal nuclear antigens. For validation, perform immunofluorescence with careful co-staining using established markers for subcellular compartments (lamin for nuclear envelope, tubulin for cytoskeleton). In parallel, conduct subcellular fractionation with Western blotting for ZNF774 alongside compartment-specific markers to confirm separation quality. Additionally, consider super-resolution microscopy techniques for more precise localization. Orthogonal approaches like proximity ligation assay or immunoelectron microscopy can provide complementary data on protein localization. Finally, functional studies examining ZNF774's activity in different compartments may help reconcile discrepant observations.

How can ZNF774 antibodies be used in chromatin immunoprecipitation (ChIP) experiments to study DNA binding properties?

Chromatin immunoprecipitation (ChIP) using ZNF774 antibodies can provide valuable insights into the genomic binding sites and transcriptional regulatory functions of this zinc finger protein. When designing a ChIP experiment for ZNF774, antibody selection is critical, as the antibody must recognize the native, DNA-bound form of the protein. While specific ChIP validations are not explicitly mentioned in the available data for current ZNF774 antibodies, polyclonal antibodies that recognize multiple epitopes (such as those from Abcam or Abbexa) may offer better chances of success for initial ChIP experiments . Crosslinking conditions should be optimized for transcription factors, typically using 1% formaldehyde for 10-15 minutes at room temperature. Sonication parameters need careful optimization to generate DNA fragments of 200-500 bp, verified by gel electrophoresis. For immunoprecipitation, use 2-5 μg of antibody per chromatin sample from 1-2 million cells, and include appropriate controls (IgG negative control, histone mark positive control). After reversing crosslinks and purifying DNA, qPCR primers should target predicted ZNF774 binding sites based on consensus zinc finger binding motifs or regions containing the KRAB-associated protein 1 (KAP1) co-repressor. For comprehensive binding site identification, consider ChIP-seq analysis, which would require verification of antibody specificity through motif enrichment analysis and comparison with known zinc finger protein binding patterns.

What is known about ZNF774 expression patterns across different tissues and disease states?

Current information on ZNF774 expression patterns across tissues and disease states remains limited in the published literature, presenting opportunities for novel research contributions. Based on antibody validation data, ZNF774 protein has been detected in several human tissues and cell lines. Western blot analyses have identified ZNF774 expression in RT4 and U251MG cell lines, human plasma, liver tissue, and tonsil tissue . Immunohistochemistry studies have demonstrated ZNF774 presence in human colon tissue sections . The detection of ZNF774 in both epithelial cells (RT4, colon) and cells of neural origin (U251MG) suggests it may have broad tissue expression patterns rather than highly restricted expression. Public transcriptomic databases might provide additional insights into tissue-specific expression patterns at the mRNA level, which could guide further protein-level studies. Regarding disease associations, current literature is sparse, but the presence of ZNF774 in U251MG glioma cells suggests potential expression in neural tumors . As a transcriptional regulator, ZNF774 might have altered expression or function in diseases characterized by dysregulated gene expression. Researchers investigating ZNF774 in disease contexts should consider performing comparative expression analyses between normal and pathological tissues using techniques like tissue microarrays with immunohistochemistry or Western blot analyses of clinical samples, potentially revealing new biomarker applications.

How might novel antibody technologies enhance ZNF774 research in the future?

Emerging antibody technologies offer exciting possibilities for advancing ZNF774 research beyond current capabilities. Single-domain antibodies (nanobodies) derived from camelid immunoglobulins could provide superior access to sterically hindered epitopes within the zinc finger domains of ZNF774, potentially offering new insights into protein-DNA interactions . Recombinant antibody fragment technologies, highlighted in recent research, allow for precise engineering of binding properties and can be optimized for specific applications like super-resolution microscopy to visualize ZNF774 localization with unprecedented detail . Proximity-dependent labeling using antibody-enzyme fusions (such as APEX2 or TurboID fused to anti-ZNF774 antibodies) could reveal the protein interactome of ZNF774 in living cells, identifying transcriptional co-factors and other interaction partners. The application of structure-based antibody design approaches, potentially guided by AlphaFold2 predictions of ZNF774 structure, could yield antibodies with enhanced specificity for distinguishing between highly similar zinc finger proteins . Antibody-oligonucleotide conjugates would enable highly multiplexed imaging of ZNF774 alongside dozens of other transcription factors, providing comprehensive snapshots of transcriptional machinery. Additionally, emerging computational approaches for antibody design that leverage protein language models could accelerate the development of next-generation ZNF774 antibodies with improved developability profiles and reduced immunogenicity for in vivo applications . These technological advances promise to transform our understanding of ZNF774 biology and its role in transcriptional regulation.

What are the key considerations for selecting the appropriate ZNF774 antibody for specific research applications?

Selecting the optimal ZNF774 antibody requires systematic evaluation of several critical factors to ensure experimental success. First, consider the clonality that best suits your application: monoclonal antibodies like PCRP-ZNF774-3F7 offer high specificity and reproducibility ideal for quantitative applications, while polyclonal antibodies provide greater sensitivity through recognition of multiple epitopes, beneficial for detecting low-abundance targets or proteins in their native conformation . The host species of the antibody (mouse for PCRP-ZNF774-3F7, rabbit for others) should be compatible with your experimental design, particularly for multi-color immunofluorescence or when working with tissue from related species . The immunogen used to generate the antibody significantly impacts epitope recognition; antibodies raised against different regions of ZNF774 (N-terminal 1-100 amino acids versus 142-422 amino acids) may perform differently depending on protein folding or post-translational modifications in your experimental system . Validated applications should guide your selection—ensure the antibody has been specifically validated for your intended application, whether Western blot, immunohistochemistry, immunofluorescence, or ELISA . For species reactivity, verify that the antibody has been confirmed to react with your species of interest; while human reactivity is well-documented for available antibodies, cross-reactivity with mouse or rat ZNF774 may be predicted but requires validation . Finally, consider technical support availability from the supplier for troubleshooting assistance if needed.