HINFP Antibody

What is HINFP and why is it important in molecular research?

HINFP (histone H4 transcription factor) is a zinc-finger transcription factor with a calculated molecular weight of approximately 60 kDa that functions as a critical regulator of histone gene expression, particularly histone H4 genes . It plays essential roles in cell cycle progression, chromatin organization, and genome stability maintenance. HINFP is ubiquitously expressed in proliferating cells but downregulated in post-proliferative differentiated cells .

The importance of HINFP in research stems from its multiple functions:

• Master regulator of mammalian histone H4 gene transcription

• Cell cycle-dependent activator of histone genes at the G1/S phase transition

• Guardian of genome stability by repressing transposable elements

• Essential factor for embryonic development (homozygous null mutations cause embryonic lethality)

What types of HINFP antibodies are available and what are their characteristics?

Multiple types of HINFP antibodies are currently available for research applications:

Polyclonal antibodies:

• Typically rabbit-derived (e.g., 10066-2-AP)

• Recognize multiple epitopes of HINFP

• Show reactivity with human, mouse, and rat samples

• Purified through antigen affinity methods

Monoclonal antibodies:

• Include mouse-derived antibodies (e.g., PCRP-HINFP-1A1)

• Recognize specific epitopes of HINFP

• Often have more specific reactivity patterns (e.g., human-specific)

• May be produced using recombinant protein immunogens

Most commercially available HINFP antibodies detect the protein at 60-70 kDa observed molecular weight range in Western blot applications .

How should HINFP antibodies be stored and handled for optimal performance?

HINFP antibodies require specific storage and handling conditions to maintain their activity:

Storage recommendations:

• Short-term storage (up to two weeks): 4°C is acceptable

• Long-term storage: Divide into small aliquots (at least 20 μl) and store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles which can compromise antibody activity

Buffer composition:

• Most HINFP antibodies are supplied in PBS with preservatives (e.g., 0.02% sodium azide) and stabilizers (e.g., 50% glycerol, pH 7.3)

• Some smaller volume preparations may contain BSA (0.1%) as additional stabilizer

For optimal results, antibodies should be allowed to equilibrate to room temperature before use and centrifuged briefly to collect solution at the bottom of the vial.

What are the validated applications for HINFP antibodies and their recommended dilutions?

HINFP antibodies have been validated for multiple experimental applications with specific recommended dilutions:

It is advisable to titrate each antibody in your specific experimental system to determine optimal conditions, as performance can be sample-dependent .

How can HINFP antibodies be used to study histone regulation and cell cycle progression?

HINFP antibodies serve as valuable tools for investigating histone regulation and cell cycle progression:

Methodology for cell cycle studies:

• Chromatin immunoprecipitation (ChIP): HINFP antibodies can be used to identify binding of HINFP to histone H4
  gene promoters, particularly at the critical Site II element which mediates cell cycle-dependent activation .

• Co-immunoprecipitation: Can detect interactions between HINFP and p220 NPAT (nuclear protein ataxia-telangiectasia locus), which function together in the CDK2/cyclin E pathway to regulate histone gene expression at the G1/S phase transition .

• Immunofluorescence with cell cycle markers: Combining HINFP detection with markers of cell cycle phases can reveal how HINFP localization and abundance changes during progression through the cell cycle.

• Western blot analysis during synchronized cell cycle progression: Allows quantification of HINFP protein levels at different cell cycle stages to correlate with histone gene expression patterns .

Research has shown that HINFP operates as a nonredundant CDK2-responsive transcription factor that functions independently of the E2F/pRB pathway, making it a unique player in cell cycle regulation .

What control samples should be included when using HINFP antibodies in experiments?

Proper experimental controls are essential for validating HINFP antibody specificity and interpreting results accurately:

Positive controls:

• HepG2 cells, human liver tissue, mouse liver tissue, rat liver tissue, and L02 cells have been validated as positive controls for Western blot applications

• HeLa cells and L02 cells are suitable positive controls for immunofluorescence/ICC applications

Negative controls:

• Knockout/knockdown validation: Tissues or cells with HINFP gene knockout or knockdown provide the most stringent negative controls

• Secondary antibody-only controls: Essential for immunofluorescence and IHC to assess background staining

• Isotype controls: Using matched isotype (e.g., rabbit IgG for polyclonal or mouse IgG2b for monoclonal) at equivalent concentrations

Recombinant expression controls:

• Overexpression of tagged HINFP in cells can serve as a positive control while helping to validate antibody specificity

When validating antibody specificity, researchers should consider that HINFP expression varies across tissues and developmental stages, with highest expression typically in proliferating cells .

How can HINFP antibodies be used to study the role of HINFP in genome stability and transposable element repression?

Recent research has established HINFP as a guardian of the somatic genome by repressing transposable elements . Methodological approaches using HINFP antibodies include:

Chromatin immunoprecipitation sequencing (ChIP-seq):

• Cross-link protein-DNA complexes in target tissues/cells

• Immunoprecipitate with HINFP antibody

• Sequence pulled-down DNA to identify HINFP binding sites across the genome, particularly at transposable element regions

• Analyze data for enrichment patterns at specific transposon families

Co-immunoprecipitation with epigenetic modifiers:

• Prepare nuclear extracts from tissues/cells of interest

• Immunoprecipitate with HINFP antibody

• Analyze co-precipitated proteins to identify interactions with histone-modifying enzymes or other chromatin regulators

• Western blot for specific candidates or perform mass spectrometry for unbiased analysis

Combined immunofluorescence for DNA damage and HINFP:
Studies in Drosophila have shown that loss of HINFP increases DNA damage and γH2Av staining . Similar approaches can be used in mammalian systems:

• Perform immunofluorescence for HINFP and DNA damage markers (e.g., γH2AX)

• Analyze correlation between HINFP levels and DNA damage

• Quantify using high-content imaging systems for statistical validation

These approaches can reveal mechanisms by which HINFP maintains genome stability, particularly its relationship with Histone1 expression which is critical for transposable element repression in somatic tissues .

What methodologies can be used to study HINFP in the context of cancer research?

HINFP has emerging roles in cancer biology, with recent evidence showing its downregulation is associated with senescence in bladder cancer tissues . Several methodological approaches are valuable:

Tissue microarray analysis:

• Use HINFP antibodies for immunohistochemistry on cancer tissue microarrays

• Score HINFP expression levels and correlate with clinical parameters

• Analyze subcellular localization patterns in tumor vs. normal tissues

• Correlate with markers of senescence (e.g., p16, SA-β-gal) or proliferation (Ki67)

Cell-autonomous studies using genetic manipulation:

• Generate HINFP knockdown or knockout in cancer cell lines using RNAi or CRISPR/Cas9

• Perform immunoblotting with HINFP antibodies to confirm knockdown efficiency

• Assess phenotypic changes using clonogenic assays, senescence markers, and invasion assays

• Analyze downstream effects on histone gene expression and transposable element activation

Co-expression network analysis:

• Combine HINFP immunohistochemistry with other cancer biomarkers

• Use multiplex immunofluorescence to simultaneously detect HINFP and senescence-associated secretory phenotype (SASP) markers

• Quantify correlation patterns using digital pathology tools

• Perform spatial analysis to identify heterogeneous expression patterns within tumors

These approaches can help elucidate how HINFP downregulation contributes to cancer progression through senescence-associated mechanisms and potential therapeutic vulnerabilities .

How can HINFP antibodies be utilized in developmental biology research?

HINFP is essential for embryonic development, with homozygous null mutations causing embryonic lethality at the peri-implantation stage . Research methodologies include:

Lineage-specific knockout studies:

• Generate conditional knockout models using Cre-loxP systems

• Validate tissue-specific deletion using HINFP antibodies in immunohistochemistry or Western blot

• Assess phenotypic consequences in specific lineages or developmental stages

• Compare histone gene expression between wild-type and knockout tissues

Embryonic stem cell differentiation models:

• Use HINFP antibodies to track expression during differentiation of pluripotent stem cells

• Perform time-course analysis of HINFP expression by Western blot or immunofluorescence

• Correlate with histone expression patterns and cell cycle dynamics

• Implement HINFP knockdown at specific differentiation stages to determine temporal requirements

Mosaic analysis with cell marking:
Similar to techniques used in Drosophila studies , researchers can:

• Generate mosaic tissues with marked HINFP-null clones

• Use HINFP antibodies to confirm absence of protein in mutant cells

• Analyze cell-autonomous effects on development, proliferation, and differentiation

• Quantify clone size and distribution to assess growth advantages or disadvantages

These approaches can reveal the developmental contexts in which HINFP function is most critical and the molecular mechanisms underlying developmental defects in its absence.

What are common issues with HINFP antibody applications and how can they be resolved?

Researchers may encounter several challenges when working with HINFP antibodies:

High background in immunostaining:

• Cause: Insufficient blocking, excessive antibody concentration, or cross-reactivity

• Solution: Increase blocking time (2+ hours), optimize antibody dilution (try 1:100, 1:500, 1:1000), use alternative blocking agents (5% BSA, 5% normal serum), or add 0.1-0.3% Triton X-100 for better permeabilization

Multiple bands in Western blot:

• Cause: Potential isoforms, degradation products, or non-specific binding

• Solution: Use fresh samples with protease inhibitors, optimize antibody dilution (start with 1:1000-1:4000), increase washing time/stringency, or validate with knockout/knockdown controls

Weak or no signal in immunoprecipitation:

• Cause: Insufficient antibody amount, poor antigen exposure, or incompatible buffer conditions

• Solution: Increase antibody amount (try 2-4 μg), modify lysis conditions, pre-clear lysate, or extend incubation time (overnight at 4°C)

Variable results between experiments:

• Cause: Antibody degradation, inconsistent sample preparation, or cell cycle-dependent expression

• Solution: Aliquot antibody to avoid freeze-thaw cycles, standardize sample collection protocols, or synchronize cells when studying cell cycle-dependent processes

It's important to remember that HINFP expression is cell cycle-regulated and varies between proliferating and differentiated cells, which can contribute to experimental variability .

How can flow cytometry data for HINFP be properly analyzed and interpreted?

Flow cytometry is an increasingly used technique for protein expression analysis, but proper data interpretation requires specific approaches:

Proper gating strategy:

• Use forward/side scatter to identify viable cells

• Apply single-cell gating to exclude doublets

• Use isotype controls to set negative population boundaries

• Consider using logarithmic or logicle (bi-exponential) transformation for optimal visualization

Data transformation considerations:
As noted in research on flow cytometry data interpretation, logarithmic displays may hide populations with low or negative expression values . For HINFP analysis:

• Consider using logicle (bi-exponential) transformation which provides better visualization of populations near zero fluorescence

• This is particularly important when examining heterogeneous populations where HINFP expression varies widely, such as in cancer samples with senescent and non-senescent cells

Multi-parameter analysis:

• Combine HINFP staining with cell cycle markers (e.g., DNA content, cyclin antibodies)

• Include markers for proliferation or senescence when studying cancer contexts

• Use dimensionality reduction techniques (e.g., tSNE, UMAP) for complex datasets

• Apply appropriate compensation when using multiple fluorophores

These approaches help ensure accurate interpretation of HINFP expression patterns, particularly in heterogeneous cell populations where expression levels may vary significantly .

What strategies can be used to validate HINFP antibody specificity in different experimental contexts?

Establishing antibody specificity is critical for reliable research outcomes. For HINFP antibodies, consider these validation approaches:

Genetic validation:

• Knockout/knockdown controls: Use CRISPR/Cas9 knockout or siRNA knockdown of HINFP to generate negative controls

• Rescue experiments: Re-express HINFP in knockout cells to restore antibody signal

• Heterozygous models: Test for gene dosage effects in heterozygous (HINFP+/-) samples

Biochemical validation:

• Peptide competition: Pre-incubate antibody with immunizing peptide/protein before application

• Multiple antibodies: Use antibodies targeting different epitopes of HINFP and compare results

• Mass spectrometry: Confirm identity of immunoprecipitated proteins or Western blot bands

Domain-specific validation:

• Truncation constructs: Express specific domains of HINFP to map epitope recognition

• Tagged constructs: Use epitope-tagged HINFP to compare with antibody staining patterns

• Post-translational modification sensitivity: Test whether antibody recognition is affected by phosphorylation or other modifications

These validation strategies should be applied in a context-dependent manner, particularly when studying HINFP in new cell types or experimental conditions not previously validated .

How does HINFP function in the epigenetic regulation of the genome?

HINFP plays crucial roles in epigenetic regulation through multiple mechanisms:

Histone gene regulation:
HINFP directly regulates histone gene expression, particularly histone H4, which is fundamental to nucleosome assembly and chromatin structure . This function positions HINFP as an upstream regulator of global chromatin organization.

Connection to DNA methylation machinery:
HINFP (previously also known as MIZF) interacts with MBD2, a methyl-CpG-binding protein critical for DNA methylation-mediated transcriptional repression . Through this interaction, HINFP participates in:

• Recruitment of histone deacetylase complexes (HDACs) to methylated DNA

• Establishment of repressive chromatin environments

• Silencing of specific genomic regions, including transposable elements

Histone H1-dependent genome stability:
In Drosophila, HINFP maintains Histone H1 expression, which is essential for:

• Higher-order chromatin assembly

• Repression of most transposable elements in somatic tissues

• Prevention of DNA damage and genomic instability

Methodologically, these functions can be studied using:

• ChIP-seq to map HINFP binding sites genome-wide

• Co-immunoprecipitation to identify interacting chromatin modifiers

• RNA-seq following HINFP manipulation to assess global transcriptional effects

• DNA methylation analysis (e.g., bisulfite sequencing) in HINFP-depleted cells

Understanding these mechanisms provides insight into how HINFP coordinates epigenetic landscapes that maintain genome stability and regulate gene expression.

What is the relationship between HINFP, cellular senescence, and cancer progression?

Recent research has uncovered important connections between HINFP, cellular senescence, and cancer:

HINFP downregulation and senescence induction:
Studies in bladder cancer have shown that heterogeneous downregulation of HINFP is associated with senescence in tumor tissues . Mechanistically:

• HINFP knockout transcriptionally inhibits H1F0 and H1FX (histone H1 variants)

• This inhibition triggers DNA damage

• DNA damage consequently induces cell senescence

• Senescence represses proliferation and growth of cancer cells

Senescence-associated secretory phenotype (SASP) and metastasis:
Paradoxically, while senescence restricts cancer cell proliferation, it can promote invasion and metastasis through SASP:

• HINFP downregulation induces senescence with SASP characteristics

• Increased expression of matrix metalloproteinases (MMP1/3) enhances invasion capacity

• These factors promote the invasion and metastasis of neighboring non-senescent cancer cells

Therapeutic implications:
The HINFP-senescence axis presents potential therapeutic targets:

• Histone deacetylase inhibitors (HDACis) can efficiently eliminate senescent cells induced by HINFP knockout

• This elimination suppresses the invasion and metastasis of bladder cancer cells

• HDACis may benefit cancer patients with metastases induced by cell senescence

Methodologically, studying these relationships requires combining HINFP antibody-based detection with senescence markers, invasion assays, and in vivo metastasis models to fully elucidate the complex role of HINFP in cancer biology.

How does HINFP function differ between species and what are the evolutionary implications?

HINFP shows both conserved and divergent functions across species, with important evolutionary implications:

Conserved roles:
Across species from Drosophila to mammals, HINFP functions as:

• A transcriptional regulator binding specific DNA sequences

• A controller of histone gene expression

• A factor in maintaining genome stability

Species-specific targets:
Despite functional conservation, target specificity varies:

• Mammals: HINFP primarily regulates histone H4 gene expression and cell cycle progression

• Drosophila: HINFP maintains Histone H1 expression to repress transposable elements in somatic tissues

Methodological approaches to study evolutionary divergence:

• Comparative genomics: Analyze HINFP binding site motifs across species

• Cross-species complementation: Test if human HINFP can rescue Drosophila HINFP mutant phenotypes

• Domain swap experiments: Exchange functional domains between species to identify critical regions

• ChIP-seq in different organisms: Compare genome-wide binding patterns to identify conserved and divergent targets

Evolutionary significance:
The conservation of HINFP across species highlights its fundamental importance in:

• Cell cycle regulation and proliferation control

• Genome defense against transposable elements

• Chromatin organization and maintenance

These studies not only illuminate the evolutionary history of chromatin regulation but also help identify which HINFP functions are most central to cellular homeostasis and which have been adapted to species-specific requirements.