ZNF627 Antibody

What is ZNF627 and why is it studied in research?

ZNF627 (zinc finger protein 627) is a human protein encoded by the ZNF627 gene located on chromosome 19 . It belongs to the zinc finger protein family, which typically functions as transcription factors involved in various biological processes including gene expression regulation. These proteins contain zinc finger domains that enable specific DNA binding capabilities. ZNF627 has attracted research interest due to its potential roles in gene regulation networks and possible implications in cellular functions. While not as extensively characterized as some other zinc finger proteins, understanding ZNF627's function may provide insights into transcriptional regulation mechanisms and potentially disease associations . The protein's UniProt ID is Q7L945, with secondary accession numbers including O14846, Q4KMP9, Q6NT81, and Q9BRG4 .

What are the key considerations when selecting a ZNF627 antibody for research?

When selecting a ZNF627 antibody for research applications, researchers should consider several critical factors. First, evaluate the antibody's validation data across different techniques (WB, IHC, IF, ELISA) relevant to your experimental needs . Examine species reactivity - most commercial ZNF627 antibodies target human proteins with variable cross-reactivity to mouse (44-52%) and rat (45%) orthologs . Consider whether you need a polyclonal antibody (offering multiple epitope recognition) or monoclonal (for higher specificity to a single epitope). Review the immunogen sequence information (common sequences include "TGEKPYENPNPNASVVPVLS") to understand the antibody's binding region. Additionally, assess whether you need a conjugated version (like HRP-conjugated) or unconjugated format based on your detection method. Finally, evaluate the antibody's published validation data, checking for documentation of specificity through knockout controls, orthogonal validation, or independent antibody validation approaches.

How should ZNF627 antibodies be stored to maintain optimal activity?

Proper storage of ZNF627 antibodies is critical for maintaining their specificity and sensitivity. Store antibodies at -20°C in small aliquots to minimize freeze-thaw cycles, which can degrade antibody quality . For HRP-conjugated ZNF627 antibodies, it's particularly important to avoid exposure to light during storage as this can diminish enzymatic activity . Most commercial ZNF627 antibodies are supplied in buffer solutions containing glycerol (typically 50%) which prevents freezing at -20°C and allows for immediate use without completely thawing . When handling, always keep antibodies cold using an ice bucket. For short-term storage (1-2 weeks), refrigeration at 2-8°C is acceptable, but avoid storing diluted working solutions for extended periods. Document the freeze-thaw cycles and test antibody performance if you suspect degradation. Following manufacturer-specific recommendations is essential, as buffer compositions may vary (typical formulation: 0.01 M PBS, pH 7.4, with preservatives like 0.03% Proclin-300) .

What are the validated applications for ZNF627 antibodies and their recommended protocols?

ZNF627 antibodies have been validated for several experimental applications with specific protocol recommendations. For Western Blotting (WB), use standard protein extraction protocols with RIPA or NP-40 buffer, load 20-30μg of total protein per lane, and typically use antibody dilutions between 1:500-1:2000 depending on the specific product . For Immunohistochemistry (IHC), antigen retrieval using citrate buffer (pH 6.0) is often required, with antibody dilutions ranging from 1:100-1:500 . Immunofluorescence (ICC-IF) applications typically require dilutions of 0.25-2 μg/mL , with overnight incubation at 4°C often yielding best results. For ELISA applications, optimal dilutions must be determined empirically by the end user, though starting with manufacturer recommendations is advisable . Always include appropriate positive and negative controls, particularly human tissue or cell lysates known to express ZNF627. When optimizing protocols, perform dilution series experiments to determine optimal concentration for your specific sample type and detection system .

How can researchers troubleshoot non-specific binding when using ZNF627 antibodies?

Non-specific binding is a common challenge when working with ZNF627 antibodies. To address this issue, implement a systematic troubleshooting approach. First, increase blocking stringency by using 5% BSA or 5% milk in TBS-T for 1-2 hours at room temperature. Consider adding 0.1-0.3% Triton X-100 to reduce hydrophobic interactions. If background persists, implement additional washing steps (5-6 washes for 5-10 minutes each) with TBS-T containing higher salt concentration (up to 500mM NaCl). For Western blotting applications, pre-adsorption of the antibody with the immunizing peptide can help determine if binding is specific. When using polyclonal ZNF627 antibodies, which may recognize multiple epitopes, titrating to lower concentrations often helps reduce non-specific binding while maintaining specific signal. Additionally, use freshly prepared samples and ensure complete antigen retrieval for IHC applications. If cross-reactivity with related zinc finger proteins is suspected, validate specificity using knockout/knockdown controls or peptide competition assays. These methodological refinements should significantly improve signal-to-noise ratio in ZNF627 antibody applications .

What are the optimal sample preparation methods for detecting ZNF627 in different cellular compartments?

Detecting ZNF627 in different cellular compartments requires specific sample preparation strategies. For nuclear fraction isolation, use gentle lysis buffers (10mM HEPES pH 7.9, 1.5mM MgCl₂, 10mM KCl, 0.5mM DTT with protease inhibitors) followed by nuclear extraction buffer (20mM HEPES pH 7.9, 25% glycerol, 420mM NaCl, 1.5mM MgCl₂, 0.2mM EDTA, 0.5mM DTT with protease inhibitors). For whole-cell lysates, RIPA buffer supplemented with protease inhibitors, phosphatase inhibitors, and 1-2mM PMSF is recommended. When preparing samples for immunofluorescence, use 4% paraformaldehyde fixation (10 minutes at room temperature) followed by permeabilization with 0.1-0.3% Triton X-100. Critical parameters include maintaining cold temperatures during extraction, using fresh inhibitors, and processing samples quickly to prevent protein degradation. For tissue samples, optimal fixation time varies by tissue type but typically ranges from 24-48 hours in 10% neutral-buffered formalin followed by careful dehydration and paraffin embedding. For challenging applications, consider using antigen retrieval methods (heat-induced epitope retrieval with citrate buffer pH 6.0 or EDTA buffer pH 9.0) to enhance ZNF627 detection in formalin-fixed tissues. These approaches maximize detection sensitivity while preserving the native subcellular localization of ZNF627 .

How can researchers validate the specificity of ZNF627 antibodies in their experimental systems?

Validating ZNF627 antibody specificity requires a multi-faceted approach. First, perform peptide competition assays by pre-incubating the antibody with excess immunizing peptide (TGEKPYENPNPNASVVPVLS) before application to your samples—specific signals should be substantially reduced. Implement genetic validation using CRISPR/Cas9 knockout or siRNA knockdown of ZNF627, comparing antibody staining patterns between wild-type and modified samples. Orthogonal validation comparing protein levels detected by the antibody with mRNA expression (via qPCR) provides additional confidence. For advanced validation, employ independent detection methods using antibodies targeting different epitopes of ZNF627—concordant results strongly support specificity. Mass spectrometry validation of immunoprecipitated products can definitively confirm target identity. When working with tissues, use known positive controls (tissues with documented ZNF627 expression) and negative controls (tissues with minimal expression). For cross-reactivity assessment, test the antibody against recombinant proteins with similar sequences, particularly other zinc finger family members. Document all validation results systematically, including positive and negative controls, to establish robust evidence of specificity in your particular experimental context .

What are the critical differences in ZNF627 antibody performance across species and how should cross-reactivity be managed?

Managing species cross-reactivity is crucial when working with ZNF627 antibodies. Most commercial ZNF627 antibodies are raised against human protein sequences with documented cross-reactivity to mouse orthologs at approximately 44-52% and rat orthologs at approximately 45% . This partial homology creates significant challenges for cross-species applications. When working with non-human samples, verify the sequence homology between your target species and the immunogen sequence used to generate the antibody. For mouse studies, expect potential differences in binding affinity and epitope recognition compared to human samples. To manage cross-reactivity, always validate antibody performance in your specific species using appropriate positive and negative controls before conducting full experiments. If cross-reactivity issues arise, consider using species-specific antibodies or custom antibody development against conserved epitopes. When interpreting results from non-human species, be aware that differences in staining patterns may reflect either biological differences in protein expression or technical limitations of antibody cross-reactivity. Western blotting validation showing the correct molecular weight in your species of interest provides crucial supporting evidence for antibody specificity across species boundaries .

How does the choice of immunogen affect ZNF627 antibody performance in different applications?

The immunogen used to generate ZNF627 antibodies significantly impacts their performance across applications. Commercial ZNF627 antibodies are typically raised against specific peptide sequences (e.g., "TGEKPYENPNPNASVVPVLS") or recombinant protein fragments (e.g., amino acids 1-300) . Antibodies generated against linear peptide epitopes often perform well in denatured applications like Western blotting but may show reduced efficacy in applications requiring recognition of native conformations. Conversely, antibodies raised against folded recombinant protein domains may better recognize native ZNF627 in immunoprecipitation or immunofluorescence applications. The location of the immunogen within the protein structure is equally important—antibodies targeting highly conserved domains may exhibit greater cross-reactivity with related zinc finger proteins, while those targeting unique regions offer higher specificity but potentially reduced cross-species reactivity. For applications requiring detection of specific post-translational modifications, ensure the immunogen contains the relevant modification. When troubleshooting application-specific issues, consider whether the epitope might be masked by protein-protein interactions, buried within protein complexes, or altered by fixation procedures. Understanding these relationships between immunogen characteristics and application performance enables researchers to select appropriate ZNF627 antibodies for specific experimental contexts .

How can researchers optimize ZNF627 antibody dilutions for maximum signal-to-noise ratio in different applications?

Optimizing ZNF627 antibody dilutions requires systematic titration experiments tailored to each application. For Western blotting, prepare a dilution series (typically 1:250, 1:500, 1:1000, 1:2000, and 1:5000) using identical samples and standardized detection conditions. For immunofluorescence, recommended starting concentrations are 0.25-2 μg/mL , but systematic testing from 0.1-5 μg/mL may be necessary. For immunohistochemistry, begin with dilutions between 1:100-1:500 and adjust based on results. The optimization process should evaluate both signal intensity and background levels across different dilutions. Quantify the signal-to-noise ratio by measuring specific signal intensity compared to background in control regions or samples. Remember that optimal dilutions may vary by tissue type, fixation method, and detection system—document these variables carefully. For HRP-conjugated ZNF627 antibodies , enzyme activity can vary between lots, necessitating re-optimization with each new lot. Additionally, extended incubation times (overnight at 4°C) with more dilute antibody often yields better results than shorter incubations with concentrated antibody. After determining optimal dilutions, validate reproducibility across multiple experiments and document the specific conditions that yielded successful results for future reference .

What are the known or predicted functions of ZNF627 and how can antibodies help elucidate its role in different biological contexts?

ZNF627, as a zinc finger protein family member, is predicted to function primarily as a DNA-binding transcription factor involved in gene expression regulation. While its specific gene targets and biological functions remain under investigation, researchers can use ZNF627 antibodies to elucidate its roles through several experimental approaches. Chromatin immunoprecipitation (ChIP) experiments using validated ZNF627 antibodies can identify genomic binding sites, providing insights into regulated genes and pathways. For this application, antibodies must be thoroughly validated for specificity and ChIP efficiency, typically starting with a 2-5μg antibody per ChIP reaction. Co-immunoprecipitation (Co-IP) studies help identify protein interaction partners that may suggest functional roles in transcriptional complexes or signaling pathways. Immunohistochemistry across tissues and developmental stages can reveal expression patterns that hint at tissue-specific functions. In disease contexts, comparing ZNF627 expression and localization between normal and pathological samples may uncover potential roles in disease mechanisms. For functional studies, coupling antibody-based protein detection with knockdown/knockout phenotype analysis provides powerful insights into biological relevance. When interpreting such data, researchers should consider that zinc finger proteins often function redundantly, and that ZNF627's role may be context-dependent, varying across cell types, developmental stages, or stress conditions .

What methodological approaches should be used when comparing data from different ZNF627 antibodies that show discrepant results?

When facing discrepant results between different ZNF627 antibodies, researchers should implement a systematic comparative analysis approach. First, document the exact epitopes targeted by each antibody—differences in immunogen sequence (e.g., "TGEKPYENPNPNASVVPVLS" versus recombinant protein fragments ) may explain discrepancies if antibodies recognize different protein domains or isoforms. Generate a standardized panel of positive and negative control samples to test all antibodies under identical conditions. For definitive validation, implement orthogonal methods such as mass spectrometry analysis of immunoprecipitated material to determine which antibody most accurately detects ZNF627. Consider performing siRNA/shRNA knockdown or CRISPR knockout experiments to verify the specificity of each antibody—signals that disappear after target depletion indicate higher specificity. Examine antibody lot-to-lot variation by requesting validation data from manufacturers. When possible, sequence-verify your experimental samples to check for polymorphisms or mutations that might affect epitope recognition. Document application-specific performance separately, as antibodies may perform differently across applications (Western blot versus immunofluorescence). When publishing results, clearly report which antibody was used and include validation data demonstrating specificity in your experimental system, allowing the scientific community to better interpret potential discrepancies in the literature .

What quality control parameters should researchers evaluate when validating ZNF627 antibodies for specific applications?

Thorough validation of ZNF627 antibodies requires assessment of multiple quality control parameters. Begin with specificity testing using Western blot analysis to confirm detection of a single band at the expected molecular weight (approximately 66 kDa for human ZNF627). Evaluate sensitivity by determining the minimum detectable amount of target protein, typically through serial dilutions of positive control samples. For reproducibility assessment, compare results across multiple experiments, different lots of the same antibody, and between different laboratory members. Cross-reactivity testing against related zinc finger proteins helps establish specificity within this large protein family. For application-specific validation, document performance across different techniques—an antibody performing well in Western blot may not necessarily work in immunohistochemistry. For immunohistochemistry applications, evaluate tissue-specific background and optimize antigen retrieval methods. Batch-to-batch consistency is crucial—request lot-specific validation data from manufacturers and conduct in-house comparisons when switching lots. Additional parameters include antibody stability over time (shelf-life testing) and performance in different buffer conditions. Document all validation results systematically, including images of positive and negative controls, to establish confidence in antibody performance for your specific experimental system .

How do fixation and antigen retrieval methods affect ZNF627 antibody performance in immunohistochemistry?

Fixation and antigen retrieval methods significantly impact ZNF627 antibody performance in immunohistochemistry. Formalin fixation, while preserving morphology, creates protein cross-links that can mask ZNF627 epitopes, particularly affecting antibodies targeting conformational epitopes. To optimize detection, systematic comparison of fixation protocols is recommended—test 10% neutral-buffered formalin fixation for varying durations (12, 24, and 48 hours) against alternatives like zinc-based fixatives or alcohol-based fixatives that cause less epitope masking. For antigen retrieval, heat-induced epitope retrieval (HIER) typically yields better results than proteolytic methods for nuclear proteins like ZNF627. Compare multiple HIER buffers: citrate buffer (pH 6.0), EDTA buffer (pH 8.0-9.0), and Tris-EDTA buffer (pH 9.0) at different temperatures and durations (95-100°C for 10-30 minutes). Document the impact of these variables on both signal intensity and background staining. Fresh frozen tissues may provide better epitope preservation but sacrifice morphological detail. When transitioning protocols between tissue types (cell blocks versus whole tissues), re-optimization is often necessary. For challenging samples, consider alternative approaches like tyramide signal amplification or polymer-based detection systems to enhance sensitivity while maintaining specificity .

What are the best practices for quantifying ZNF627 expression levels using antibody-based techniques?

Accurate quantification of ZNF627 expression using antibody-based techniques requires rigorous methodological approaches. For Western blot quantification, implement a standard curve using recombinant ZNF627 protein at known concentrations (typically 5-8 points ranging from 0.1-100 ng) on each blot to establish a linear detection range. Use fluorescent secondary antibodies rather than chemiluminescence when possible, as they provide wider linear dynamic range and better reproducibility. Normalize ZNF627 signals to validated loading controls appropriate for your experimental context (GAPDH for whole cell lysates, histone H3 for nuclear fractions). For immunohistochemistry quantification, define objective scoring methods—consider H-score methodology (intensity × percentage positive cells) or automated image analysis using defined parameters for nuclear staining intensity. For flow cytometry applications, include appropriate fluorescence-minus-one (FMO) controls and isotype controls to establish specificity, and report results as median fluorescence intensity rather than percentage positive cells when analyzing proteins with continuous expression patterns. When comparing expression across sample types or experimental conditions, process and analyze all samples simultaneously to minimize technical variation. For all quantification methods, perform technical replicates (minimum triplicate) and biological replicates (minimum n=3) to establish statistical validity. Document and report all quantification parameters, including exposure settings, analysis software, and statistical approaches, to ensure reproducibility .

How can ZNF627 antibodies be utilized in emerging single-cell analysis techniques?

ZNF627 antibodies can be strategically integrated into emerging single-cell analysis techniques through several innovative approaches. For single-cell proteomics, conjugate validated ZNF627 antibodies with metal isotopes for mass cytometry (CyTOF) analysis, enabling simultaneous detection of ZNF627 alongside dozens of other proteins at single-cell resolution. When developing such protocols, titrate antibody concentrations carefully (typically 0.5-2 μg/mL) and validate specificity using positive and negative control cell populations. For spatial transcriptomics applications, combine ZNF627 immunofluorescence with in situ hybridization to correlate protein localization with mRNA expression patterns at the single-cell level. This approach requires highly specific antibodies with minimal background in tissue sections. For single-cell Western blotting technologies, adapt standard ZNF627 Western blot protocols by optimizing lysis conditions for microfluidic platforms and adjusting antibody concentrations for reduced sample volumes. When combining ZNF627 antibody detection with single-cell RNA-seq data, careful validation of antibody specificity becomes even more critical, as discordance between protein and mRNA levels may represent either biological regulation or technical artifacts. As these technologies continue to evolve, researchers should document antibody performance metrics specifically for single-cell applications, as sensitivity requirements often exceed those of bulk analysis methods .

What are the potential applications of ZNF627 antibodies in studying gene regulation networks?

ZNF627 antibodies offer powerful tools for investigating gene regulation networks through multiple experimental approaches. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) using validated ZNF627 antibodies can map genome-wide binding sites, revealing direct target genes and DNA binding motifs. For successful ChIP-seq applications, optimize antibody amounts (typically 2-10 μg per ChIP), sonication conditions, and IP protocols specifically for ZNF627. Cut-and-Run or CUT&Tag techniques provide alternatives with improved signal-to-noise ratios and reduced cell input requirements. To understand ZNF627's role in transcriptional regulation machinery, use co-immunoprecipitation with ZNF627 antibodies followed by mass spectrometry to identify protein interaction partners within regulatory complexes. For monitoring dynamic changes in ZNF627 occupancy across different cellular states, time-course ChIP experiments combined with reporter assays can connect binding events to transcriptional outcomes. When investigating potential roles in chromatin modification, combine ZNF627 ChIP with sequential ChIP for histone modifications to determine correlation with activating or repressive chromatin states. For in vivo relevance, tissue-specific ChIP using ZNF627 antibodies can reveal context-dependent regulatory functions. These approaches collectively build a comprehensive understanding of ZNF627's position within broader gene regulatory networks, potentially uncovering novel regulatory mechanisms and pathway interactions .

How might post-translational modifications affect ZNF627 antibody recognition, and what methodological approaches can address this challenge?

Post-translational modifications (PTMs) of ZNF627 present significant challenges for antibody recognition that require specialized methodological approaches. Zinc finger proteins commonly undergo modifications including phosphorylation, SUMOylation, and ubiquitination, which can either mask epitopes or create conformational changes affecting antibody binding. To address these challenges, researchers should first identify potential modification sites through bioinformatic prediction tools and existing proteomic datasets. Then, compare antibody performance under conditions that preserve or remove modifications—phosphatase treatment before Western blotting can reveal whether phosphorylation affects epitope recognition. For comprehensive analysis, use modification-specific antibodies alongside general ZNF627 antibodies to distinguish modified forms. To detect potentially masked epitopes, implement native versus denaturing conditions in parallel experiments, as some modifications may only affect antibody binding in the protein's native conformation. Mass spectrometry validation of immunoprecipitated material can identify specific modifications present. When inconsistent results arise across experimental conditions, consider whether stress responses, cell cycle phases, or signaling events might induce modifications affecting epitope accessibility. For critical applications, generate or source multiple antibodies targeting different regions of ZNF627 to minimize the impact of modification-dependent epitope masking. Document all treatments affecting protein modification status when reporting antibody performance to facilitate accurate interpretation of results across different experimental contexts .

What are the key specifications of commercially available ZNF627 antibodies?

This comprehensive table provides researchers with key specifications for comparing commercially available ZNF627 antibodies. When selecting an antibody, consider the specific application requirements, species compatibility, and validated performance metrics documented by the manufacturer. Optimal antibody selection should be based on the intended experimental application and validated in your specific research context .

What are the recommended dilution ranges for ZNF627 antibodies in different applications?

These recommended dilution ranges serve as starting points for optimization in specific experimental systems. Researchers should perform systematic titration experiments to determine optimal concentrations for their specific samples and detection methods. Document successful conditions for reproducibility across experiments .

What control samples are recommended for validating ZNF627 antibody specificity?