PHF11 Antibody

What is PHF11 and what cellular functions does it serve?

PHF11 (PHD finger protein 11) is a nuclear protein with a reported length of 331 amino acid residues and a molecular mass of approximately 37.6 kDa in humans. It contains a PHD (Plant Homeodomain) finger domain that typically mediates protein-protein interactions, particularly with chromatin. PHF11 functions primarily as a positive regulator of Th1-type cytokine gene expression, suggesting its importance in immune response modulation . It is highly expressed in immune cells, particularly T and B lymphocytes, natural killer cells, and mature dendritic cells, indicating its significant role in immune function regulation . The protein has been identified to possess up to two different isoforms, allowing for potential functional diversity across different cellular contexts. Its nuclear localization suggests involvement in transcriptional regulation and other nuclear processes that may influence gene expression patterns within immune cells .

What are the key research applications for PHF11 antibodies?

PHF11 antibodies serve multiple critical research applications in immunology and molecular biology. Western Blot (WB) represents the most widely used application, enabling precise detection and quantification of PHF11 protein expression across different experimental conditions and cell types . Immunohistochemistry (IHC) applications allow researchers to visualize PHF11 distribution in tissue sections, providing spatial context for understanding its function in complex tissue environments . Immunofluorescence (IF) and Immunocytochemistry (ICC) techniques facilitate cellular localization studies to determine PHF11's subcellular distribution and potential co-localization with other proteins of interest . Flow cytometry applications using PHF11 antibodies enable quantitative analysis of PHF11 expression in individual cells within heterogeneous populations. Additionally, Enzyme-Linked Immunosorbent Assay (ELISA) methods provide sensitive quantification of PHF11 in various sample types . These diverse applications collectively enable comprehensive investigation of PHF11's expression patterns, regulatory mechanisms, and potential roles in both normal physiology and disease states.

What is known about PHF11 expression patterns across different tissues and cell types?

PHF11 exhibits a distinctive expression pattern that is predominantly concentrated in immune system cells. Research has documented particularly high expression levels in T and B lymphocytes, which are central to adaptive immunity . Similarly elevated expression appears in natural killer (NK) cells, which play crucial roles in innate immune responses and immunosurveillance . Mature dendritic cells, which function as professional antigen-presenting cells bridging innate and adaptive immunity, also demonstrate significant PHF11 expression . This expression profile strongly indicates PHF11's involvement in immune regulation pathways. While the protein is primarily expressed in these immune cell populations, lower expression levels may occur in other tissue types. The consistent nuclear localization of PHF11 across expressing cell types suggests conservation of its molecular function involving nuclear processes such as transcriptional regulation . This distinct cellular distribution pattern provides important context for researchers designing experiments to investigate PHF11's role in immune function, inflammatory responses, or potential involvement in immunological disorders.

What are the optimal protocols for using PHF11 antibodies in Western blot applications?

When utilizing PHF11 antibodies for Western blot applications, researchers should implement a carefully optimized protocol to ensure specific and reproducible detection. Begin with protein extraction using RIPA buffer supplemented with protease inhibitors, followed by determination of protein concentration via Bradford or BCA assay. For optimal separation of PHF11 (37.6 kDa), load 20-30 μg of protein per lane on a 10-12% SDS-PAGE gel and run at 100-120V until adequate separation is achieved . Transfer proteins to a PVDF membrane (recommended over nitrocellulose for PHF11) at 100V for 1 hour or 30V overnight at 4°C. For blocking, use 5% non-fat dry milk in TBST for 1 hour at room temperature, though 3% BSA may provide lower background in some cases.

Primary antibody incubation should be performed with anti-PHF11 antibody at a 1:500 to 1:1000 dilution (optimize based on specific antibody) overnight at 4°C . Following thorough washing (3-5 times with TBST), incubate with HRP-conjugated secondary antibody at 1:5000 dilution for 1 hour at room temperature. After additional washing steps, develop using ECL reagent and image using appropriate detection system. Important controls include a positive control (lysate from immune cells known to express PHF11), a negative control (lysate from cells with PHF11 knockdown/knockout), and a loading control (β-actin or GAPDH) to normalize expression levels . This methodological approach ensures reliable detection of PHF11 protein while minimizing technical artifacts.

How can researchers effectively validate PHF11 antibody specificity for their experiments?

Comprehensive validation of PHF11 antibody specificity requires a multi-faceted approach to ensure reliable experimental results. Begin with genetic validation techniques using cells expressing PHF11 alongside PHF11 knockout/knockdown samples generated via CRISPR-Cas9 or siRNA technology . Western blot analysis should demonstrate absence or significant reduction of the expected 37.6 kDa band in knockout/knockdown samples. Peptide competition assays provide additional validation by pre-incubating the PHF11 antibody with the immunizing peptide, which should abolish specific signals if the antibody is indeed targeting PHF11 .

Cross-reactivity assessment is essential, particularly for researchers working with non-human models. Compare antibody reactivity across species by testing samples from human, mouse, and other relevant organisms, as PHF11 antibodies show variable cross-reactivity with orthologs . Orthogonal validation using multiple antibodies targeting different PHF11 epitopes can confirm consistent detection patterns. Additionally, recombinant protein testing should verify antibody recognition of purified PHF11 protein at the expected molecular weight .

For definitive validation in advanced research contexts, mass spectrometry analysis of immunoprecipitated proteins can confirm PHF11 identity. Finally, application-specific validation should verify antibody performance in each intended application (WB, IHC, IF), as antibodies may perform differently across techniques . This comprehensive validation strategy ensures high confidence in antibody specificity, particularly important when investigating PHF11's role in complex immunological processes.

What controls should be included when investigating PHF11 expression in different cell lines?

Robust experimental design for studying PHF11 expression requires implementation of several critical controls to ensure data validity and interpretability. Positive controls should include immune cells with documented high PHF11 expression, such as T cells, B cells, NK cells, or mature dendritic cells, which serve as benchmarks for proper antibody function and expression level comparison . Negative controls must include cells with confirmed absence of PHF11 expression or cells treated with verified PHF11-targeting siRNA/shRNA to establish background signal levels and confirm signal specificity .

Loading controls are essential for normalizing PHF11 expression across different samples. While housekeeping proteins like β-actin, GAPDH, or tubulin are commonly used, researchers should select loading controls appropriate for the specific cellular compartment being analyzed (nuclear markers like Lamin B1 are preferable given PHF11's nuclear localization) . Isotype controls using non-specific antibodies of the same isotype as the PHF11 antibody help identify potential non-specific binding.

For comparative studies across cell lines, standardization of cell culture conditions, harvesting methods, and protein extraction protocols is crucial, as these factors can significantly influence PHF11 expression levels. Additionally, include technical replicates (minimum of three) and biological replicates (different passages of the same cell line) to account for technical variation and biological heterogeneity . These comprehensive controls enable accurate interpretation of PHF11 expression patterns while minimizing experimental artifacts and ensuring reproducibility in complex cellular systems.

How can PHF11 antibodies be utilized to investigate its role in antiviral immunity, particularly against foamy viruses?

PHF11 antibodies provide powerful tools for elucidating PHF11's newly discovered role in antiviral immunity, particularly its specific inhibition of spumavirus (foamy virus) replication. To investigate this function, researchers can implement several advanced methodological approaches. Viral replication analysis can be conducted by establishing cell lines with doxycycline-inducible PHF11 expression systems, followed by infection with GFP-encoding foamy viruses . Flow cytometry analysis of GFP expression in the presence versus absence of PHF11 induction will quantify the degree of viral inhibition. Western blot analysis using PHF11 antibodies can confirm proper expression timing and levels during these experiments .

To identify the specific stage of viral replication inhibited by PHF11, researchers should employ time-of-addition experiments where PHF11 expression is induced at different timepoints relative to viral infection. Integration assays using hygromycin-resistance reporters can distinguish between effects on integration versus post-integration steps . Immunoprecipitation studies using PHF11 antibodies can identify viral or host proteins that physically interact with PHF11 during infection, potentially revealing mechanistic insights.

For investigating promoter-specific effects, reporter assays with constructs containing different viral promoters (internal promoter versus LTR promoter) can be monitored in the presence/absence of PHF11 . Chromatin immunoprecipitation (ChIP) using PHF11 antibodies can determine whether PHF11 directly binds to viral promoters, providing mechanistic understanding of its inhibitory effects. These methodological approaches collectively enable comprehensive investigation of PHF11's specific role in antiviral immunity, particularly its unique ability to inhibit foamy virus gene expression by targeting transcription from the internal promoter.

What methodologies can be employed to investigate PHF11 domain-specific functions using antibodies?

Investigating domain-specific functions of PHF11 requires sophisticated experimental approaches utilizing domain-targeted antibodies. Begin by establishing a panel of antibodies targeting distinct epitopes within each functional domain: the N-terminal domain (NTD), PHD finger domain, and C-terminal domain (CTD) . This allows for domain-specific detection and functional analysis. For structural studies, domain-specific antibodies can be used in protein crystallography to stabilize specific conformations, potentially revealing mechanistic insights into domain interactions.

Domain-based protein-protein interaction studies can be conducted using co-immunoprecipitation with domain-specific antibodies, followed by mass spectrometry to identify domain-specific binding partners. To correlate specific domains with antiviral activity, researchers should employ chimeric protein approaches, replacing individual domains of PHF11 from species with antiviral activity (e.g., human) with corresponding domains from species lacking antiviral activity (e.g., feline) . The resulting chimeric proteins can be evaluated for antiviral function against foamy viruses.

For mechanistic insights, chromatin immunoprecipitation sequencing (ChIP-seq) using domain-specific antibodies can map genomic binding sites of PHF11, while RNA immunoprecipitation (RIP) can identify domain-specific RNA interactions. Domain-specific mutational analysis, where key residues within each domain are mutated and the resulting proteins analyzed using domain-specific antibodies, can pinpoint critical functional residues .

This comprehensive methodological approach enables researchers to dissect the specific contributions of each PHF11 domain to its various cellular functions, including immune regulation and antiviral activity, providing deeper mechanistic understanding of this multifunctional protein.

How can researchers use PHF11 antibodies to investigate its role in cytokine gene expression regulation?

Investigating PHF11's role in cytokine gene expression regulation requires sophisticated immunological techniques centered around appropriate antibody applications. Chromatin immunoprecipitation (ChIP) assays using validated PHF11 antibodies should be employed to identify direct binding of PHF11 to cytokine gene promoters or enhancer regions, particularly those involved in Th1-type responses . For comprehensive genome-wide binding analysis, researchers should implement ChIP-sequencing to map all potential PHF11 binding sites across the genome in relevant immune cell populations.

To determine functional consequences of PHF11 binding, coupling ChIP with reporter gene assays using constructs containing cytokine promoter regions can establish direct causality between PHF11 binding and transcriptional activation or repression. Co-immunoprecipitation studies using PHF11 antibodies followed by mass spectrometry can identify transcription factors, chromatin remodelers, or other regulatory proteins that form complexes with PHF11 at cytokine gene regulatory regions .

For investigating dynamic regulation, researchers should implement sequential ChIP (Re-ChIP) to determine if PHF11 co-occupies regulatory regions with other transcription factors during immune activation. Integration of PHF11 ChIP-seq data with RNA-seq and ATAC-seq from the same cell populations can correlate PHF11 binding with changes in chromatin accessibility and gene expression .

Cell-type specific analysis is crucial, comparing PHF11 binding patterns across different immune cell subsets (Th1, Th2, Th17, etc.) to identify cell-type-specific regulatory mechanisms. Additionally, stimulation time-course experiments analyzing PHF11 binding following immune activation with various stimuli can reveal dynamic regulatory patterns . These methodological approaches collectively enable comprehensive characterization of PHF11's role in cytokine gene regulation across diverse immunological contexts.

What are common technical challenges when using PHF11 antibodies and how can they be resolved?

Researchers frequently encounter several technical challenges when working with PHF11 antibodies that require systematic troubleshooting approaches. High background signal in Western blots often occurs due to non-specific binding. This can be resolved by implementing more stringent blocking conditions (5-10% milk or BSA instead of standard 3-5%), increasing washing duration and frequency (5-6 washes of 10 minutes each), optimizing antibody dilution through titration experiments (testing ranges from 1:250 to 1:2000), and using specialized blocking reagents containing both proteins and detergents to minimize non-specific interactions .

Weak or absent signal presents another common challenge, potentially resulting from insufficient protein, degraded antibody, or ineffective epitope accessibility. Researchers should increase protein loading (40-50μg for PHF11 detection), verify antibody viability through dot blot testing, implement antigen retrieval methods for fixed samples, and consider using alternative fixation methods that preserve PHF11 epitopes . Multiple bands in Western blots may indicate isoform detection, non-specific binding, or protein degradation. Verification through appropriate knockout controls, use of protease inhibitors during sample preparation, and comparison with predicted molecular weights of known isoforms (PHF11 has two reported isoforms) can distinguish between these possibilities .

Cross-reactivity issues between species can be addressed by selecting antibodies specifically validated for the study species or testing multiple antibodies targeting different epitopes. For immunoprecipitation difficulties, researchers should adjust lysis conditions to better preserve native protein conformation, pre-clear lysates thoroughly, and optimize antibody-to-bead ratios. Additionally, poor reproducibility can be mitigated through standardization of protocols, use of positive controls in each experiment, and implementation of automated systems where possible to reduce technical variation . These systematic troubleshooting approaches enable researchers to overcome common technical challenges and generate reliable data when working with PHF11 antibodies.

How should researchers interpret contradictory results when comparing PHF11 expression data across different antibodies?

When confronted with contradictory PHF11 expression results using different antibodies, researchers should implement a systematic analytical framework to resolve these discrepancies. Begin with comprehensive antibody validation assessment, examining the validation documentation for each antibody, including knockout/knockdown controls, peptide competition assays, and cross-reactivity profiles . Epitope mapping analysis is crucial - antibodies targeting different epitopes may yield different results if certain epitopes are masked by protein-protein interactions, subject to post-translational modifications, or inaccessible in specific conformational states.

Isoform specificity must be considered as PHF11 has two reported isoforms, and different antibodies may preferentially detect specific isoforms, leading to apparent expression discrepancies . Methodological differences in sample preparation, fixation techniques, antigen retrieval methods, or detection systems can significantly impact results and should be standardized across comparative experiments.

Cross-validation using orthogonal techniques provides critical verification - if Western blot results contradict immunofluorescence data, RNA analysis (RT-qPCR) or mass spectrometry can serve as independent verification methods. For definitive analysis, researchers should implement side-by-side comparison experiments using identical samples processed in parallel with different antibodies, followed by quantitative analysis of signal intensity and specificity .

The biological context of the samples may also explain discrepancies, as PHF11 expression and epitope accessibility may vary across cell types, activation states, or experimental conditions. When reporting such contradictory results, researchers should transparently acknowledge the discrepancies, clearly describe all methodological details that might influence interpretation, and present the full range of evidence while avoiding selective reporting of confirming results . This comprehensive approach enables accurate interpretation of complex antibody-based expression data for PHF11.

What quantitative methods should be used to accurately measure PHF11 protein levels in experimental samples?

Accurate quantification of PHF11 protein levels requires implementation of multiple complementary methodological approaches, each with specific technical considerations. Western blot densitometry represents a fundamental approach, requiring researchers to capture images within the linear detection range of their imaging system, normalize band intensity to appropriate loading controls (preferably nuclear proteins like Lamin B1 given PHF11's nuclear localization), and analyze using dedicated software that incorporates background correction . For more precise quantification, capillary electrophoresis-based Western analysis (e.g., Wes, Jess systems) provides higher reproducibility and broader dynamic range compared to traditional Western blotting.

ELISA-based quantification offers higher throughput capability, requiring validated PHF11-specific sandwich ELISA kits or development of custom assays using two antibodies targeting different PHF11 epitopes, along with recombinant PHF11 protein standards for absolute quantification . Flow cytometry enables single-cell level quantification, particularly valuable for heterogeneous cell populations, through careful optimization of fixation/permeabilization conditions (critical for nuclear proteins like PHF11), use of fluorescence standards for calibration, and comparison to isotype controls.

For the highest precision, mass spectrometry-based approaches such as selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) should be implemented using labeled reference peptides unique to PHF11 as internal standards. This approach enables absolute quantification independent of antibody binding efficiency .

Regardless of the method chosen, researchers should implement multiple technical replicates (minimum of three), appropriate statistical analysis accounting for the distribution characteristics of the data, and complementary approaches (e.g., combining Western blot with ELISA) for cross-validation. Additionally, researchers should report detailed methodological parameters, acknowledge technical limitations, and include all appropriate controls in publications to enable proper interpretation and reproducibility .

How can PHF11 antibodies be used to investigate its potential role in inflammatory and autoimmune diseases?

Investigation of PHF11's role in inflammatory and autoimmune diseases requires sophisticated implementation of antibody-based techniques across various experimental systems. For clinical sample analysis, immunohistochemistry and immunofluorescence using validated PHF11 antibodies can compare expression patterns in tissue samples from patients with various autoimmune conditions versus healthy controls, with particular attention to immune cell infiltrates . Flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) from patients versus controls can quantify PHF11 expression across specific immune cell subsets (Th1, Th17, Tregs) to identify disease-associated alterations.

In mechanistic studies, chromatin immunoprecipitation followed by sequencing (ChIP-seq) using PHF11 antibodies can map genome-wide binding patterns in disease-relevant cell populations, identifying potential dysregulated gene targets . Co-immunoprecipitation studies can elucidate PHF11's protein interaction network in normal versus disease states, potentially revealing altered interactions with transcriptional regulators.

For functional investigations, researchers should implement genome editing approaches (CRISPR-Cas9) to create cell lines or animal models with PHF11 mutations identified in patient populations, followed by comprehensive phenotypic characterization using PHF11 antibodies to confirm expression changes . Ex vivo stimulation assays with cells from patients versus controls, followed by PHF11 immunoblotting, can reveal potential differences in PHF11 expression dynamics or post-translational modifications in response to inflammatory triggers.

Additionally, PHF11 antibodies can be used in proximity ligation assays to detect and visualize protein-protein interactions in situ within tissue sections, potentially identifying disease-specific interaction patterns . Collectively, these methodological approaches enable comprehensive investigation of PHF11's potential contributions to inflammatory and autoimmune disease pathogenesis, potentially revealing new therapeutic targets or biomarkers.

What methods can researchers use to study post-translational modifications of PHF11 and their functional significance?

Investigating post-translational modifications (PTMs) of PHF11 requires specialized approaches centered around appropriate antibody applications and complementary techniques. Mass spectrometry-based PTM mapping represents the foundation of this research, where immunoprecipitation with PHF11 antibodies followed by LC-MS/MS analysis can identify specific modification sites and types (phosphorylation, acetylation, ubiquitination, SUMOylation, etc.) . For targeted PTM analysis, researchers should utilize modification-specific antibodies that recognize PHF11 only when modified at specific residues, enabling detection via Western blot, immunofluorescence, or flow cytometry.

To investigate dynamic regulation of PTMs, pulse-chase experiments with metabolic labeling followed by PHF11 immunoprecipitation can determine modification turnover rates. In parallel, stimulation time-course experiments examining PHF11 modifications following immune cell activation or other relevant stimuli can reveal context-dependent PTM patterns . Site-directed mutagenesis of identified modification sites, followed by functional assays, can establish causality between specific modifications and PHF11 functions.

For structural consequences of PTMs, hydrogen/deuterium exchange mass spectrometry of purified PHF11 (wild-type versus modified forms) can reveal how modifications alter protein conformation. Proximity ligation assays using PHF11 antibodies in combination with antibodies against specific modifying enzymes can visualize potential modification events in situ .

To investigate PTM crosstalk, researchers should implement sequential immunoprecipitation with antibodies against different modifications, potentially revealing combinatorial modification patterns. Complementary computational approaches can predict potential modification sites based on consensus motifs and structural accessibility . These comprehensive methodological approaches collectively enable detailed characterization of PHF11 post-translational modifications and their functional significance in various biological contexts, particularly in immune regulation and antiviral responses.

How might PHF11 antibodies contribute to understanding the evolutionary conservation of its antiviral function across species?

Investigating the evolutionary conservation of PHF11's antiviral function requires sophisticated comparative immunological approaches utilizing species-specific antibodies. Cross-species reactivity analysis should first be conducted to determine whether existing antibodies recognize PHF11 orthologs across diverse mammalian species (human, non-human primates, rodents, feline, bovine, etc.) . For orthologs not recognized by existing antibodies, researchers should develop species-specific antibodies targeting conserved epitopes to enable comparative studies.

Functional conservation assessment can be implemented through comparative viral inhibition assays, where PHF11 from different species is expressed in reporter cell lines and challenged with various retroviruses, particularly foamy viruses . Western blot analysis with species-specific antibodies confirms expression levels across experiments. For structural conservation studies, researchers should conduct immunoprecipitation of PHF11 orthologs from various species followed by mass spectrometry to identify interacting partners, revealing conservation or divergence in protein interaction networks .

Domain-specific functional analysis requires generation of chimeric proteins combining domains from PHF11 orthologs across species with differential antiviral activity, followed by functional assessment in viral inhibition assays . Immunofluorescence microscopy using species-specific antibodies can determine whether subcellular localization patterns are conserved across orthologs.

For regulatory conservation investigation, ChIP-seq studies using species-specific PHF11 antibodies can map genomic binding sites across orthologs, revealing conservation or divergence in regulatory targets . Additionally, phylogenetic analysis incorporating experimental data from antibody-based studies can correlate structural and functional evolution of PHF11 with host-pathogen co-evolution patterns . These comprehensive comparative approaches collectively enable detailed characterization of the evolutionary conservation and divergence of PHF11's antiviral function, potentially revealing important insights into host-virus co-evolution and species-specific immune mechanisms.