PHT4;7 Antibody

What is PHF14 and what cellular functions does it regulate?

PHF14 (PHD finger protein 14), also known as KIAA0783, is an 888 amino acid nuclear protein containing two PHD-type zinc fingers. It localizes to the nucleus and is involved in transcriptional regulation. The protein has a calculated molecular weight of 100 kDa but is typically observed at 140-150 kDa in experimental settings . PHF14 has been implicated in several cellular processes through its zinc finger domains, which typically mediate protein-DNA and protein-protein interactions in chromatin-associated proteins.

What applications has the PHF14 antibody (24787-1-AP) been validated for?

The PHF14 antibody (24787-1-AP) has been validated for multiple applications:

The antibody has demonstrated reactivity with human samples and has cited reactivity with mouse samples .

What is the best way to store and handle the PHF14 antibody to maintain its activity?

The PHF14 antibody should be stored at -20°C where it remains stable for one year after shipment. The antibody is supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For optimal preservation:

• Avoid repeated freeze-thaw cycles which can denature the antibody

• If using the 20μl size, note that it contains 0.1% BSA as a stabilizer

• Aliquoting is unnecessary for -20°C storage with this specific antibody formulation

• When handling, use aseptic technique to prevent contamination

• During experiments, keep the antibody on ice when not in use

Following these storage guidelines helps maintain antibody binding capacity and specificity throughout the research project.

What are the optimal conditions for using PHF14 antibody in Western blotting?

For optimal Western blotting results with PHF14 antibody:

• Sample Preparation:

  • Use fresh cell lysates from HeLa, HEK-293, or MDA-MB-453s cells (validated positive controls)

  • Include protease inhibitors in lysis buffer to prevent degradation

• Gel Electrophoresis:

  • Use 8-10% SDS-PAGE gels to properly resolve the 140-150 kDa protein

  • Load 20-50 μg of total protein per lane

• Transfer and Blocking:

  • Transfer proteins to PVDF membrane (recommended over nitrocellulose for high MW proteins)

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Antibody Incubation:

  • Primary antibody dilution: 1:500-1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

  • Secondary antibody: Anti-rabbit HRP at 1:3000-1:5000 dilution

• Detection:

  • Use enhanced chemiluminescence (ECL) detection

  • Expected band size: 140-150 kDa (note: calculated MW is 100 kDa but observed size is larger)

Following these guidelines will help obtain specific detection of PHF14 while minimizing background and non-specific binding.

How should immunohistochemistry protocols be optimized for PHF14 antibody?

For successful immunohistochemistry with PHF14 antibody (24787-1-AP):

• Tissue Processing:

  • Use formalin-fixed, paraffin-embedded (FFPE) human tissue sections

  • Human kidney tissue has been validated as a positive control

• Antigen Retrieval (critical step):

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • Heat-induced epitope retrieval using pressure cooker or microwave

• Blocking and Antibody Application:

  • Block with 10% normal serum from the same species as the secondary antibody

  • Apply PHF14 antibody at 1:20-1:200 dilution

  • Incubate in a humidified chamber overnight at 4°C

• Detection System:

  • Use polymer-based detection systems for increased sensitivity

  • DAB (3,3'-diaminobenzidine) is recommended as the chromogen

  • Counterstain with hematoxylin for nuclear visualization

• Controls:

  • Include negative controls (primary antibody omitted)

  • Include isotype controls to confirm specificity

  • Consider using tissues with known knockdown/knockout status for PHF14

Careful optimization of each step, particularly antibody concentration and antigen retrieval, is essential for specific immunolocalization of PHF14 in tissue sections.

What approaches should be used to validate antibody specificity for PHF14?

Comprehensive validation of PHF14 antibody specificity should include:

• Genetic Approaches:

  • Knockdown/knockout validation: Compare staining in PHF14 KD/KO vs. wild-type samples

  • Four publications have already utilized KD/KO approaches to validate this antibody

  • Overexpression: Compare staining intensity between overexpressed and endogenous levels

• Biochemical Approaches:

  • Immunoprecipitation followed by mass spectrometry

  • Peptide blocking experiments using the immunogen (PHF14 fusion protein Ag20542)

  • Pre-adsorption with the antigen to demonstrate signal reduction

• Orthogonal Methods:

  • Comparison with results using alternative antibodies against PHF14

  • Correlation with mRNA expression data

  • Use of tagged recombinant PHF14 protein as positive control

• Tissue/Cell Selection:

  • Use validated positive controls (HeLa, HEK-293, MDA-MB-453s cells)

  • Include known negative controls based on expression databases

As noted in guidelines on antibody use in physiology research, "The responsibility for antibody validation is a shared one, with investigators also needing to contribute" to ensure experimental reproducibility.

How can researchers troubleshoot inconsistent results with PHF14 antibody?

When encountering inconsistent results with PHF14 antibody:

• Antibody-Related Factors:

  • Verify antibody integrity: Check for precipitation, contamination, or degradation

  • Test new antibody lot against previous lots for consistency

  • Optimize concentration: Perform titration experiments (1:20 to 1:2000)

• Sample-Related Factors:

  • Ensure proper sample preparation and storage

  • Check for proteolytic degradation by adding fresh protease inhibitors

  • Verify protein expression in your specific sample type

• Protocol-Related Factors:

  • For IHC: Adjust antigen retrieval method (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • For WB: Modify blocking conditions, incubation times, washing stringency

  • For IP: Adjust antibody amount (0.5-4.0 μg for 1.0-3.0 mg of total protein)

• Experimental Controls:

  • Run positive controls (HEK-293 cells for IP; HeLa, HEK-293, MDA-MB-453s for WB)

  • Include loading controls for WB

  • Use isotype controls to assess non-specific binding

• Technical Verification:

  • Confirm results using an alternative detection method

  • Consider advanced multiplexing to verify colocalization with expected markers

  • Use orthogonal techniques to support antibody-based findings

Systematic troubleshooting helps identify the source of inconsistency and improves experimental reproducibility.

How can PHF14 antibody be effectively utilized in chromatin immunoprecipitation (ChIP) experiments?

For successful ChIP experiments using PHF14 antibody:

• Experimental Design:

  • Cross-linking: Use 1% formaldehyde for 10 minutes at room temperature

  • Chromatin shearing: Optimize sonication to achieve 200-500 bp fragments

  • Input: Reserve 5-10% of chromatin as input control

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Use 5-10 μg PHF14 antibody per ChIP reaction

  • Include IgG control from same species (rabbit)

  • Incubate overnight at 4°C with rotation

• Washing and Elution:

  • Use stringent washing conditions to reduce background

  • Elute DNA-protein complexes and reverse cross-links

  • Purify DNA using column-based methods

• Analysis:

  • qPCR for known or predicted target sites

  • ChIP-seq for genome-wide binding analysis

  • Bioinformatic analysis to identify enriched motifs

• Validation:

  • Confirm enrichment by comparing to IgG control

  • Verify results with re-ChIP or alternative PHF14 antibodies

  • Correlate with expression data of potential target genes

This approach has been validated in at least three published studies using the PHF14 antibody , making it a reliable method for investigating PHF14's role in chromatin regulation and transcriptional control.

What are the considerations for using PHF14 antibody in co-immunoprecipitation to study protein-protein interactions?

When performing co-immunoprecipitation (Co-IP) with PHF14 antibody:

• Lysis Conditions:

  • Use mild non-denaturing lysis buffers to preserve protein-protein interactions

  • Consider buffer compositions with different salt concentrations (150-300 mM NaCl)

  • Include protease and phosphatase inhibitors

• Pre-Clearing:

  • Pre-clear lysates with appropriate beads to reduce non-specific binding

  • Use 0.5-4.0 μg of PHF14 antibody per 1.0-3.0 mg of total protein lysate

  • HEK-293 cells have been validated for IP with this antibody

• Controls and Validation:

  • Include negative controls: IgG from same species, irrelevant antibody

  • Reverse Co-IP: Use antibodies against suspected interacting partners

  • Input control: Load 5-10% of pre-IP lysate

• Detection Methods:

  • Western blot analysis with antibodies against potential interacting partners

  • Mass spectrometry for unbiased identification of binding partners

  • Functional assays to verify biological relevance of identified interactions

• Technical Considerations:

  • Cross-linking may be necessary for transient interactions

  • Consider native vs. denatured IP depending on research question

  • For protein complexes, optimize antibody:lysate ratio

This approach has been validated in two published studies and can provide insights into PHF14's involvement in protein complexes related to transcriptional regulation and chromatin modification .

How can PHF14 antibody be used in multiplex immunofluorescence studies?

For multiplex immunofluorescence studies with PHF14 antibody:

• Panel Design:

  • Select compatible antibodies raised in different species to avoid cross-reactivity

  • Choose fluorophores with minimal spectral overlap

  • Include markers for subcellular compartments (nuclear, cytoplasmic)

• Sequential Staining Protocol:

  • Option 1: Sequential antibody application with intermediate blocking steps

  • Option 2: Simultaneous application of primary antibodies followed by species-specific secondaries

  • For PHF14 (rabbit polyclonal): Use anti-rabbit secondary conjugated to spectrally distinct fluorophore

• Controls:

  • Single-stain controls to assess bleed-through

  • Isotype controls for each primary antibody

  • Absorption controls using the immunizing peptide

• Imaging and Analysis:

  • Use confocal microscopy for subcellular localization

  • Employ spectral unmixing for closely overlapping fluorophores

  • Quantify colocalization using appropriate statistical methods

• Advanced Applications:

  • Combine with proximity ligation assay (PLA) to verify protein-protein interactions

  • Integrate with FRET analysis for proteins in close proximity

  • Consider expansion microscopy for super-resolution imaging of nuclear proteins

Multiplex immunofluorescence can reveal PHF14's spatial relationships with other nuclear proteins and chromatin-associated factors, providing insights into its functional interactions within the nuclear environment.

How should researchers interpret unexpected molecular weight observations for PHF14?

The PHF14 antibody (24787-1-AP) detects a protein at 140-150 kDa, despite a calculated molecular weight of 100 kDa . This discrepancy requires careful interpretation:

• Potential Explanations:

  • Post-translational modifications (phosphorylation, glycosylation, SUMOylation)

  • Alternative splicing yielding larger isoforms

  • Highly charged regions affecting protein migration

  • Incomplete denaturation maintaining tertiary structure

• Validation Approaches:

  • Mass spectrometry to confirm protein identity

  • Phosphatase treatment to assess contribution of phosphorylation

  • Comparison with recombinant PHF14 lacking post-translational modifications

  • Use of different gel systems (gradient gels, different acrylamide percentages)

• Experimental Considerations:

  • Always include molecular weight markers covering 100-170 kDa range

  • Use positive control lysates (HeLa, HEK-293, MDA-MB-453s)

  • Consider alternative denaturation conditions

• Literature Comparison:

  • Review published literature reporting PHF14 molecular weight

  • Note that the observed 140-150 kDa is consistent across multiple studies

  • Check for documented PHF14 modifications in proteomic databases

Understanding the basis for this molecular weight difference is important for proper data interpretation and can provide insights into PHF14 biology and post-translational regulation.

What are the best approaches for quantifying PHF14 expression levels in immunohistochemistry?

For accurate quantification of PHF14 expression in IHC:

• Sample Preparation Standardization:

  • Use consistent fixation protocols for all samples

  • Process all samples simultaneously when possible

  • Include calibration standards or reference tissues

• Staining Controls:

  • Include positive and negative tissue controls

  • Use internal controls within the same tissue section

  • Perform technical replicates to assess staining variability

• Quantification Methods:

  Method	Advantages	Limitations	Best For
  H-score	Accounts for both intensity and percentage	Subjective assessment	Semi-quantitative analysis
  Digital image analysis	Objective, reproducible	Requires specialized software	Large-scale studies
  Automated tissue cytometry	Single-cell resolution	Complex setup	Heterogeneous tissues
  Machine learning approaches	Pattern recognition	Requires training data	Complex expression patterns

• Statistical Analysis:

  • Use appropriate statistical tests for comparing expression levels

  • Account for multiple comparisons when necessary

  • Consider correlation with clinical parameters or other biomarkers

• Reporting Standards:

  • Clearly describe quantification methodology

  • Report both raw data and processed results

  • Include representative images of different expression levels

For PHF14 specifically, the recommended human kidney tissue can serve as a reference for standardizing quantification across experiments .

How do results from PHF14 polyclonal antibodies compare with monoclonal alternatives?

When comparing polyclonal PHF14 antibodies (like 24787-1-AP) with monoclonal alternatives:

• Epitope Recognition:

  • Polyclonal: Recognizes multiple epitopes, potentially providing signal amplification

  • Monoclonal: Targets single epitope, offering higher specificity but potentially lower sensitivity

  • For PHF14 with its multiple domains, epitope selection is critical for functionality studies

• Reproducibility Factors:

  • Polyclonal: Batch-to-batch variability may occur

  • Monoclonal: Higher consistency between production lots

  • Solution: Validate each new lot against previous results

• Application Suitability:

  Application	Polyclonal Advantage	Monoclonal Advantage
  Western Blot	Better for denatured proteins	Superior for specific isoforms
  IHC-FFPE	Often better epitope recognition after fixation	More consistent staining patterns
  IP	Higher avidity may improve pull-down	Less background in complex samples
  ChIP	Multiple epitope recognition beneficial	More precise binding site localization

• Cross-Reactivity Considerations:

  • Polyclonal: Higher risk of cross-reactivity with related proteins

  • Monoclonal: Lower cross-reactivity but may miss target if epitope is altered

  • Validation using knockout/knockdown remains essential for both types

• Selection Strategy:

  • Use polyclonals (like 24787-1-AP) for initial characterization and applications requiring high sensitivity

  • Consider monoclonals for highly specific applications or when distinguishing closely related proteins

  • For critical experiments, confirm findings using antibodies recognizing different epitopes

Understanding these differences helps researchers select the most appropriate antibody for their specific research question about PHF14.

What advantages do recombinant antibody technologies offer over traditional PHF14 antibodies?

Recombinant antibody technologies provide several advantages compared to traditional PHF14 antibodies:

• Production Consistency:

  • Recombinant: Defined genetic sequence ensures consistent production

  • Traditional: Animal-derived antibodies (like the rabbit polyclonal 24787-1-AP) have batch variation

  • Impact: Improved experimental reproducibility across studies

• Engineering Capabilities:

  • Antibody fragments (Fab, scFv) can access restricted epitopes

  • Fusion with reporters or affinity tags for specialized applications

  • Humanization for therapeutic potential

  • Site-specific modifications for controlled conjugation

• Performance Comparison:

  Feature	Traditional PHF14 Antibody	Recombinant Alternative
  Specificity	Good, but batch-dependent	Highly consistent
  Reproducibility	Variable between lots	Highly reproducible
  Customization	Limited	Extensive engineering options
  Production scalability	Limited by immunization	Unlimited once developed
  Application range	Well-established	Similar plus additional capabilities

• Emerging Applications:

  • Anti-idiotype development (similar to approach in )

  • Intrabodies for live-cell tracking of PHF14

  • Proximity-dependent labeling for protein interaction studies

  • Conditional activation for temporal control of binding

• Practical Considerations:

  • Higher initial development cost but greater long-term consistency

  • Potential for intellectual property protection of novel constructs

  • Increased data reproducibility aligns with current research standards

While the traditional PHF14 antibody (24787-1-AP) has proven utility with validation in multiple applications , recombinant technologies represent the future direction for even more reliable and versatile research tools.

How can researchers effectively combine PHF14 antibody-based assays with genomic and transcriptomic approaches?

For comprehensive understanding of PHF14 function, integrating antibody-based techniques with genomic/transcriptomic approaches offers powerful insights:

• ChIP-Seq Integration:

  • Use PHF14 antibody for ChIP followed by next-generation sequencing

  • Correlate binding sites with transcriptional start sites

  • Integrate with histone modification data to understand chromatin context

  • This approach has been validated in 3 published studies with this antibody

• Multi-omics Experimental Design:

  Technique Combination	Research Question	Analytical Approach
  ChIP-seq + RNA-seq	Direct transcriptional targets	Correlate binding with expression changes
  PHF14 IP-MS + RNA-seq	Protein complex influence on transcription	Identify co-regulated gene sets
  PHF14 IHC + spatial transcriptomics	Tissue-specific function	Correlate protein localization with gene expression patterns

• Functional Validation Framework:

  • PHF14 knockdown/knockout + antibody-based detection of downstream effects

  • Rescue experiments with mutant PHF14 constructs

  • Time-course analyses combining protein levels and transcriptional changes

• Data Integration Strategies:

  • Use computational approaches to integrate protein binding and expression data

  • Apply network analysis to identify PHF14-centered regulatory networks

  • Utilize machine learning to predict functional outcomes of PHF14 binding

• Visualization and Reporting:

  • Create integrative genome browser tracks showing PHF14 binding and expression

  • Develop network visualizations showing PHF14 protein interactions and regulated genes

  • Use dimensionality reduction techniques to identify patterns across multiple data types

This integrative approach leverages the specificity of the PHF14 antibody while providing broader functional context through complementary genomic and transcriptomic data.

What considerations are important when using PHF14 antibody in different cell and tissue types?

When applying PHF14 antibody across diverse biological samples:

• Expression Level Variations:

  • PHF14 expression may vary significantly between tissues and cell types

  • Adjust antibody concentration based on expected expression levels

  • For Western blotting, validated positive controls include HeLa, HEK-293, and MDA-MB-453s cells

  • For IHC, human kidney tissue serves as a validated positive control

• Protocol Modifications by Sample Type:

  Sample Type	Critical Adjustments	Validation Method
  FFPE tissues	Antigen retrieval optimization (TE buffer pH 9.0 or citrate buffer pH 6.0)	Comparison with fresh frozen samples
  Primary cells	Gentle fixation procedures	Correlation with mRNA expression
  Cell lines	Verify endogenous expression levels	Western blot quantification
  Tissue microarrays	Include known positive controls	Compare with whole-section staining

• Background Reduction Strategies:

  • For high autofluorescence tissues: Consider spectral unmixing or alternative detection methods

  • For tissues with high endogenous peroxidase: Additional blocking steps

  • For highly vascularized tissues: Optimize washing procedures

• Subcellular Localization Differences:

  • PHF14 is primarily nuclear but may show differential subnuclear patterns

  • Use counterstains (DAPI, Hoechst) to confirm nuclear localization

  • Consider co-staining with compartment markers (nucleoli, nuclear speckles)

• Cross-Species Applications:

  • While primarily validated in human samples, the antibody has cited reactivity with mouse samples

  • When expanding to new species, perform additional validation

  • Consider sequence homology when interpreting cross-species reactivity

Understanding these considerations ensures appropriate application of PHF14 antibody across diverse experimental systems while maintaining data reliability and reproducibility.