PFN4 Antibody

What is PFN4 and what biological processes is it involved in?

PFN4 (Profilin 4) is a member of the profilin protein family, but shares only approximately 30% homology with other profilin family members (PFN1-3). Notably, PFN4 does not encode for the actin and poly-L-proline binding sites that are present in PFN1-3, suggesting unique functions independent of actin dynamics regulation . PFN4 is predominantly expressed in testes and specifically localizes to the acrosome-acroplaxome-manchette complex during spermiogenesis . Its expression pattern indicates a specialized role in male reproductive biology, particularly in sperm head formation and acrosome development.

What are the typical applications of anti-PFN4 antibodies in research?

Anti-PFN4 antibodies are primarily utilized in several key research applications:

• Immunohistochemistry (IHC): To detect endogenous PFN4 expression in paraffin-embedded tissue sections, particularly in human brain and thyroid cancer tissues

• Western Blotting (WB): For protein expression analysis in tissue lysates

• Immunocytochemistry (ICC): For cellular localization studies

• Immunoprecipitation (IP): For protein-protein interaction studies

These applications enable researchers to investigate PFN4 expression patterns, subcellular localization, and potential interaction partners in various experimental contexts.

What are the key specifications researchers should know about commercially available PFN4 antibodies?

When selecting a PFN4 antibody for research, consider these specifications:

Researchers should verify antibody validation data, including positive controls in relevant tissues, and select appropriate secondary antibodies based on the host species .

How do PFN4-deficient mouse models contribute to our understanding of male fertility?

PFN4-deficient mouse models have provided critical insights into male fertility mechanisms:

These models demonstrate that PFN4 plays a crucial role in proper sperm development, particularly in manchette formation and acrosome biogenesis, which are essential for male fertility.

What methodological considerations should be taken when using PFN4 antibodies in immunohistochemistry?

When using PFN4 antibodies for IHC, researchers should consider:

• Tissue preparation: Use paraffin-embedded sections with appropriate antigen retrieval techniques

• Antibody dilution: Typical dilutions start at 1/20 for IHC applications based on validation data

• Positive controls: Human brain and thyroid cancer tissues have been validated as positive controls

• Detection systems: Compatible with standard secondary detection systems, particularly Goat Anti-Rabbit IgG conjugated with various reporters (AP, Biotin, FITC, HRP)

• Visualization: Appropriate counterstaining and imaging parameters should be established for optimal visualization of the acrosome-acroplaxome-manchette complex

• Cross-reactivity: Verify specificity against other profilin family members (PFN1-3) due to potential sequence homology

How does PFN4 contribute to manchette development and acrosome biogenesis at the molecular level?

PFN4's role in manchette development and acrosome biogenesis involves several molecular mechanisms:

Manchette development:

• PFN4-deficient mice show disrupted manchette formation with only marginal α-tubulin staining, appearing punctate and dispersed

• Ultrastructural analysis reveals mislocalization (steps 8-9) and an angular shape (step 10) of the microtubular manchette in PFN4-deficient spermatids

• Complete loss of manchette and amorphous shape of elongated sperm heads observed in steps 12-16 of spermiogenesis

• PFN4 deletion does not interfere with perinuclear ring formation and initial HOOK1 localization, but impedes microtubular organization of the manchette

Acrosome biogenesis:

• PFN4 is localized to the acroplaxome, suggesting a direct role in acrosome formation

• Disrupted cis- and *trans-*Golgi networks in PFN4-deficient mice affect proacrosomal vesicle production

• Proteomic analysis shows altered abundance of proteins involved in Golgi membrane trafficking (ARF3, SPECC1L, FKBP1)

• Disruption of PI3K/AKT pathway and autophagy inhibition may explain failure in acrosome formation

What proteomic changes are observed in PFN4-deficient testes and what signaling pathways are affected?

Proteomic analysis of PFN4-deficient testes revealed significant alterations in protein abundance and signaling pathways:

These changes suggest that PFN4 deficiency leads to:

• Disruption of Golgi membrane trafficking pathways

• Hyperactivation of the PI3K/AKT/mTOR signaling axis

• Inhibition of autophagy (reduced AMPK activity)

• Blockage of autophagic flux, potentially explaining acrosome formation failure

How can researchers determine antibody specificity and validate anti-PFN4 antibodies for their experimental systems?

To validate anti-PFN4 antibodies:

• Western blot analysis: Confirm single band of expected molecular weight (approximately 14 kDa) in testicular tissue

• Positive and negative tissue controls: Compare expression in testicular tissue (high expression) versus non-reproductive tissues (low/absent expression)

• Peptide competition assay: Pre-incubate antibody with excess immunizing peptide to confirm signal specificity

• Knockout validation: Use PFN4-deficient mouse tissues as negative controls

• Cross-reactivity testing: Test against other profilin family members (PFN1-3) to ensure specificity

• Multiple antibody approach: Compare staining patterns using antibodies raised against different PFN4 epitopes

• Complementary techniques: Validate antibody results with mRNA expression data (qRT-PCR, in situ hybridization)

What are the implications of PFN4 research for understanding male infertility in humans?

PFN4 research has significant implications for human male infertility:

• Diagnostic potential: PFN4 antibodies could be used to identify abnormal PFN4 expression or localization in testicular biopsies from infertile men

• Genetic screening: Identification of PFN4 mutations in infertile men could provide genetic diagnosis

• Therapeutic approaches: In vitro fertilization research with PFN4-deficient sperm demonstrated capability of fertilizing zona-free oocytes, suggesting potential treatment options for PFN4-related human infertility

• Contraceptive development: Understanding PFN4's essential role in male fertility could lead to novel contraceptive approaches targeting this protein

• Broader reproductive biology insights: PFN4 research contributes to understanding the complex molecular machinery required for sperm development

What controls should be included when using PFN4 antibodies in research applications?

Proper experimental controls for PFN4 antibody applications include:

For Western Blotting:

• Positive control: Testicular tissue lysate

• Negative control: Tissues known not to express PFN4

• Loading control: Housekeeping protein (β-actin, GAPDH)

• Specificity control: PFN4-deficient tissue or peptide competition assay

For Immunohistochemistry:

• Positive tissue control: Human brain or thyroid cancer tissue sections

• Negative tissue control: Tissues known not to express PFN4

• Technical negative control: Primary antibody omission

• Isotype control: Non-specific IgG from same host species

• Dilution series: Optimization of signal-to-noise ratio

For Immunoprecipitation:

• Input control: Pre-IP sample

• Negative control: Non-specific IgG precipitation

• Validation by mass spectrometry: Confirm precipitated protein identity

How should researchers address potential data contradictions when using different anti-PFN4 antibodies?

When faced with contradictory results using different anti-PFN4 antibodies:

• Epitope mapping: Identify the specific epitopes recognized by each antibody to understand potential differences in detection

• Isoform specificity: Determine whether antibodies detect different PFN4 isoforms or post-translational modifications

• Validation in knockout models: Test antibodies in PFN4-deficient tissues to confirm specificity

• Cross-reactivity assessment: Evaluate potential cross-reactivity with other profilin family members

• Complementary approaches: Use non-antibody methods (mRNA analysis, mass spectrometry) to resolve contradictions

• Sensitivity comparison: Determine detection limits of different antibodies

• Protocol optimization: Adjust fixation, antigen retrieval, and detection methods for each antibody

• Lot-to-lot variation: Test different lots of the same antibody to assess consistency

What methodological approaches can be used to study PFN4 function beyond antibody-based techniques?

Alternative approaches to study PFN4 function include:

• Genetic manipulation:

  • CRISPR/Cas9-mediated gene editing to create knockout models

  • Conditional knockout systems to study tissue-specific effects

  • Site-directed mutagenesis to study specific domains/residues

• Transcriptomic analysis:

  • RNA-seq to analyze global expression changes in PFN4-deficient models

  • Single-cell RNA-seq to study cell-specific expression patterns

  • qRT-PCR for targeted expression analysis

• Proteomic approaches:

  • Mass spectrometry to identify interaction partners

  • Proximity labeling techniques (BioID, APEX) to identify proximal proteins

  • Phosphoproteomics to study signaling pathway alterations

• Structural biology:

  • X-ray crystallography or cryo-EM to determine PFN4 structure

  • NMR to study protein dynamics and interactions

• Functional assays:

  • In vitro fertilization with PFN4-deficient sperm

  • Sperm motility and morphology analyses

  • Ultrastructural analysis using transmission electron microscopy

What are the emerging questions in PFN4 antibody research?

Key emerging questions include:

• How do post-translational modifications affect PFN4 function during spermiogenesis?

• Are there tissue-specific PFN4 isoforms that require specific antibody epitopes for detection?

• What is the evolutionary conservation of PFN4 function across species?

• How does PFN4 interact with other manchette and acrosome-associated proteins?

• Are there non-reproductive tissues where PFN4 plays functional roles?

• How might single nucleotide polymorphisms in human PFN4 contribute to male infertility?

• What compensatory mechanisms exist in heterozygous PFN4-deficient males that maintain fertility?

• How might PFN4 antibodies be used in clinical diagnostics for male infertility?

How can researchers optimize IHC protocols for detecting low-abundance PFN4 in non-reproductive tissues?

Optimization strategies include:

• Signal amplification systems:

  • Tyramide signal amplification (TSA)

  • Polymer-based detection systems

  • Quantum dot-based detection

• Tissue preparation optimization:

  • Compare multiple fixatives (formalin, Bouin's, zinc-based)

  • Test various antigen retrieval methods (heat-induced vs. enzymatic)

  • Optimize section thickness (5-10 μm)

• Antibody enhancement:

  • Prolonged primary antibody incubation (overnight at 4°C)

  • Higher antibody concentration with reduced background (blocking optimization)

  • Cocktails of multiple antibodies against different PFN4 epitopes

• Reducing background:

  • Avidin/biotin blocking for biotin-based detection systems

  • Endogenous peroxidase quenching optimization

  • Fc receptor blocking in tissues with high immunoglobulin content

• Sensitive imaging:

  • Confocal microscopy for improved signal-to-noise ratio

  • Super-resolution microscopy for precise localization

  • Digital image analysis with specialized software for weak signal detection

What cutting-edge techniques might advance our understanding of PFN4 function in reproductive biology?

Cutting-edge approaches include:

• Single-cell spatial transcriptomics to map PFN4 expression patterns in the testis with unprecedented resolution

• CRISPR activation/interference (CRISPRa/CRISPRi) for precise temporal control of PFN4 expression

• Organoid models of testicular tissue to study PFN4 function in a controlled 3D environment

• Live cell imaging with tagged PFN4 to track its dynamics during spermiogenesis

• AlphaFold2 and other AI approaches to predict PFN4 structure and potential interaction partners

• Optogenetics to control PFN4 activity with light-sensitive domains

• Patient-derived induced pluripotent stem cells (iPSCs) differentiated into germ cells to study human-specific PFN4 function

• Multi-omics integration combining genomics, transcriptomics, proteomics, and metabolomics data to build comprehensive models of PFN4 function