PFN1 Antibody Pair

What is PFN1 and why is it important in cellular research?

PFN1 is a ubiquitously expressed 15 kDa protein that functions as a key regulator of actin polymerization in cells. It maintains the speed of actin polymerization during filament elongation by controlling the exchange of ATP and ADP binding. Beyond cytoskeletal regulation, PFN1 engages in multiple cellular processes including cell migration, cytokinesis, membrane trafficking, and endocytosis across various cell types . More recently, PFN1 has been identified as having significant roles in DNA damage response and repair machinery, making it a multifunctional protein of interest in diverse research fields .

What types of PFN1 antibody pairs are available for research applications?

PFN1 antibody pairs typically consist of a capture antibody and a detection antibody designed for sandwich ELISA applications. The most common configuration includes:

These antibody pairs demonstrate broad species reactivity, including human, mouse, guinea pig, cow, chicken, rabbit, goat, and sheep samples . Both components are typically IgG isotype antibodies preserved in PBS buffer (0.01 M, pH 7.4) with 50% glycerol .

What are the main applications for PFN1 antibody pairs in research?

While sandwich ELISA represents the primary application for commercial PFN1 antibody pairs, individual PFN1 antibodies are employed across multiple techniques including:

• Western blotting (WB) for protein expression quantification

• Immunohistochemistry (IHC) for tissue localization studies

• Immunocytochemistry (ICC) for subcellular localization

• Immunoprecipitation (IP) for protein-protein interaction studies

• Flow cytometry (FCM) for quantitative cellular analysis

The versatility of these antibodies allows researchers to investigate PFN1's role in cytoskeletal dynamics, disease pathogenesis, and cellular regulatory mechanisms .

What is the optimal protocol for sandwich ELISA using PFN1 antibody pairs?

For optimal sandwich ELISA using PFN1 antibody pairs, follow this methodology:

• Plate Preparation: Coat a high-binding 96-well microplate with capture antibody (typically 2-5 μg/ml in carbonate buffer, pH 9.6) overnight at 4°C.

• Blocking: Block remaining binding sites with 1-2% BSA in PBS for 1-2 hours at room temperature.

• Sample Addition: Add standards and samples diluted in sample diluent with 0.05% Tween-20 for 2 hours at room temperature.

• Detection Antibody: Add biotinylated detection antibody (0.5-2 μg/ml) for 1-2 hours.

• Signal Development: Apply streptavidin-HRP conjugate followed by TMB substrate.

• Measurement: Stop reaction with 2N H₂SO₄ and read absorbance at 450nm with 570nm reference.

Critical parameters include antibody concentration optimization, thorough washing between steps (at least 3-5 washes), and inclusion of appropriate positive and negative controls . For accurate quantification, prepare a standard curve using recombinant human PFN1 protein.

How can researchers validate the specificity of PFN1 antibodies in their experimental systems?

Validating PFN1 antibody specificity requires a multi-faceted approach:

• Positive Control: Use recombinant PFN1 protein or cell lysates known to express PFN1 (most mammalian cell lines express detectable levels).

• Knockdown Validation: Compare antibody signal between wild-type cells and those with PFN1 knockdown using siRNA or shRNA. For example, researchers have validated PFN1 antibodies by examining tenfold reductions in PFN1 expression at both mRNA and protein levels after shPFN1 transduction .

• Peptide Competition: Pre-incubate the antibody with the immunizing peptide prior to application. For N-terminal directed antibodies, the peptide sequence "AGWNAYIDNL MADGTCQDAA IVGYKDSPSV WAAVPGKTFV NITPAEVGVL" can be used .

• Multiple Antibody Comparison: Compare staining patterns using antibodies targeting different epitopes of PFN1 (N-terminal versus C-terminal).

• Cross-reactivity Assessment: Test antibody against samples from multiple species to confirm predicted reactivity patterns .

What methodological approaches are recommended for studying PFN1's subcellular localization?

To effectively study PFN1's subcellular localization, researchers should employ complementary approaches:

• Co-immunofluorescence (co-IF): Combine PFN1 antibody staining with markers for specific cellular compartments. For cytoskeletal studies, co-stain with phalloidin to visualize filamentous actin, as demonstrated in studies examining PFN1 depletion effects on actin reorganization .

• Subcellular Fractionation: Separate nuclear and cytoplasmic fractions before western blotting to quantify PFN1 distribution. This approach is particularly valuable when studying PFN1's nuclear translocation during DNA damage response .

• Live Cell Imaging: For dynamic studies, express fluorescently-tagged PFN1 (GFP-PFN1) and monitor its localization in response to stimuli such as EGF treatment .

• Immunoelectron Microscopy: For high-resolution localization studies, especially when examining PFN1's association with membrane structures and cytoskeletal elements.

• Proximity Ligation Assay (PLA): To confirm PFN1's interactions with binding partners in specific subcellular compartments.

Research has shown that PFN1 can change its predominant cytoplasmic localization to nuclear upon DNA damage and subsequently restore cytoplasmic compartmentalization during recovery .

How does PFN1 contribute to DNA damage response and repair, and what methodologies are best to study this function?

PFN1 plays a previously underappreciated role in DNA damage response and repair mechanisms. Research demonstrates that PFN1 depletion accelerates DNA damage-mediated apoptosis and disrupts PTEN-AKT signaling cascade . To investigate this function:

• DNA Damage Induction Protocols:

  • Treat cells with genotoxic agents (e.g., UV radiation, etoposide)

  • Monitor PFN1 nuclear translocation via immunofluorescence at different timepoints

  • Compare wild-type versus PFN1-depleted cells

• DNA Repair Factor Recruitment Analysis:

  • Examine recruitment of repair factors like γH2AX to double-strand breaks

  • PFN1-deficient cells show failure to recruit DNA repair factors while control cells demonstrate significant increases in these genes

• Cell Cycle Analysis:

  • Use propidium iodide (PI) staining and flow cytometry to assess cell cycle stage

  • Analyze using appropriate software (e.g., CytExpert)

  • PFN1 depletion affects CHK1-mediated cell cycle arrest recovery following DNA damage

• PTEN-AKT Pathway Assessment:

  • Monitor phosphorylation status of PTEN and AKT by western blotting

  • Correlate PFN1 expression levels with pathway activation/inhibition

• Sphere Formation Assay:

  • Plate cells in appropriate conditions (e.g., 5×10^5 cells/well with shaking at 90 rpm)

  • Measure sphere size after 7 days to assess cellular integration capacity

What is the relationship between PFN1 and phosphoinositide metabolism, and how can researchers investigate this interaction?

PFN1 has emerged as an important molecular determinant of cellular PI(4,5)P₂ levels with significant implications for phosphoinositide signaling cascades. To investigate this relationship:

• PIP₂ Quantification Methodologies:

  • Immunostaining using specific anti-PIP₂ antibodies

  • Lipid dot-blot assays to measure total PIP₂ content

  • Studies have shown that silencing PFN1 expression leads to significant decreases in PIP₂ content across multiple cell lines (MDA-MB-231, BT-474, HeLa)

• PIP₃ and PI(3,4)P₂ Dynamics Assessment:

  • Monitor EGF-induced PM accumulation of PIP₃ in cells with normal or altered PFN1 expression

  • PFN1 knockdown enhances EGF-induced PM accumulation of PIP₃

• Lipid Phosphatase Interaction Studies:

  • Investigate PFN1's interaction with lipid phosphatases like SHIP2

  • Evidence suggests PFN1 binding may restrict PM recruitment of SHIP2

• Inositol Phosphate (IP₁) Accumulation Assay:

  • Measure IP₁ levels following EGF stimulation to assess PLC-mediated PIP₂ hydrolysis

  • Research shows PFN1 does not significantly modulate this pathway

• Membrane Association Analysis:

  • Challenge the conventional model of PFN1's direct binding to plasma membrane PPIs

  • Current data suggest PFN1's affinity to PPIs and plasma membrane presence in cells may be negligible

What methodological approaches can be used to investigate PFN1 mutations associated with amyotrophic lateral sclerosis (ALS)?

To investigate PFN1 mutations associated with ALS, researchers can employ these methodological approaches:

• Genetic Screening:

  • Screen ALS and related disorder patients (e.g., FTLD-TDP) for mutations in coding regions of PFN1 using Sanger sequencing

  • Use TaqMan genotyping assays to investigate identified variants in control subjects

• Aggregate Formation Analysis:

  • Express wild-type and mutant PFN1 in cell models (e.g., N2A cells)

  • Assess aggregate formation with and without proteasome inhibitors (e.g., MG132)

  • The p.E117G variant has been shown to form aggregates in N2A cells and exhibits raised insoluble protein levels upon proteasome inhibition

• Actin Binding Assessment:

  • Compare actin binding capacity between wild-type and mutant PFN1

  • Unlike other PFN1 mutants, p.E117G did not show reduced actin binding

• Immunohistochemistry on Autopsy Material:

  • Compare PFN1 and TDP-43 staining patterns between patients with and without specific PFN1 variants

  • Research has shown that PFN1 and TDP-43 staining did not differ between patients with and without the p.E117G variant

• Genotype-Phenotype Correlation Studies:

  • Compare mutation frequency between patients and control subjects

  • Analyze clinical presentations in patients with specific PFN1 mutations

  • The p.E117G variant was found in 0.4% of ALS/FTLD-TDP patients and 0.2% of control subjects

What are common issues with PFN1 western blotting and how can they be resolved?

When performing western blotting for PFN1 (15 kDa protein), researchers may encounter several challenges:

• Poor Signal Detection:

  • Problem: Low or absent PFN1 band despite proper sample preparation

  • Solution: Optimize antibody concentration (typically 0.5-2 μg/ml); increase protein loading (20-40 μg total protein); use PVDF membranes for better protein retention; extend primary antibody incubation to overnight at 4°C

• Multiple Bands/Non-specific Binding:

  • Problem: Detection of multiple bands beyond the expected 15 kDa

  • Solution: Increase blocking stringency (5% BSA or milk for 2 hours); use affinity-purified antibodies ; include 0.1% Tween-20 in washing buffers; optimize antibody dilution; consider using monoclonal antibodies for higher specificity

• Inconsistent Loading Control Issues:

  • Problem: Difficulties normalizing PFN1 expression due to its potential involvement in cellular processes

  • Solution: Use multiple housekeeping proteins; consider total protein normalization methods (e.g., stain-free technology); validate loading controls in your experimental system

• Detection of Post-translational Modifications:

  • Problem: Challenges in distinguishing phosphorylated forms of PFN1

  • Solution: Use phospho-specific antibodies when available; employ Phos-tag gels for mobility shift detection; combine with immunoprecipitation to enrich for modified forms

• Species Cross-reactivity Issues:

  • Problem: Antibody fails to detect PFN1 in certain species despite predicted reactivity

  • Solution: Verify species reactivity claims; select antibodies with demonstrated cross-reactivity to your species of interest ; consider using species-specific positive controls

How can researchers optimize immunohistochemistry protocols for PFN1 detection in tissue samples?

For optimal PFN1 detection in tissue samples through immunohistochemistry:

• Fixation Optimization:

  • Protocol Refinement: Compare 4% paraformaldehyde (PFA) versus formalin fixation

  • Timing Considerations: Limit fixation to 24-48 hours to prevent epitope masking

  • Post-fixation Processing: Process tissues promptly after fixation

• Antigen Retrieval Methods Comparison:

  • Heat-induced Epitope Retrieval (HIER): Test citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0)

  • Enzymatic Retrieval: For certain tissues, try proteinase K or trypsin-based retrieval

  • Optimization Strategy: Compare different methods on serial sections from the same sample

• Background Reduction Techniques:

  • Endogenous Peroxidase Blocking: Use 3% H₂O₂ in methanol for 10-15 minutes

  • Protein Blocking: Implement dual blocking with 2-5% normal serum from the same species as the secondary antibody, followed by 1% BSA

  • Autofluorescence Reduction: For fluorescent detection, use Sudan Black B (0.1-0.3%) treatment after antibody incubation

• Signal Amplification Methods:

  • Polymer-based Detection: Use HRP-polymer systems for enhanced sensitivity

  • Tyramide Signal Amplification: Consider for low-abundance detection

  • Multiple Antibody Approach: Validate findings using antibodies targeting different PFN1 epitopes

• Validation Controls Implementation:

  • Positive Control Selection: Include tissues known to express high PFN1 levels

  • Negative Controls: Perform parallel staining omitting primary antibody and using isotype controls

  • Competing Peptide Controls: Pre-incubate antibody with immunizing peptide to confirm specificity

How should researchers interpret changes in PFN1 expression in different disease contexts?

Interpreting changes in PFN1 expression across disease contexts requires careful consideration of several factors:

• Cancer Research Interpretation:

  • Downregulation Pattern: PFN1 is downregulated in clinical pancreatic tumor tissues and bladder cancer

  • Functional Significance: PFN1 overexpression can impair malignant phenotypes of tumor cells and increase sensitivity of breast cancer cells to camptothecin-induced apoptosis

  • Methodological Approach: Compare expression between matched tumor and adjacent normal tissues; correlate with clinical parameters and survival data

• Neurodegenerative Disease Context:

  • Mutation Analysis: Assess frequency of specific mutations (e.g., p.E117G in ALS patients versus controls)

  • Protein Aggregation: Evaluate PFN1 inclusion formation in patient tissues

  • Statistical Consideration: A 0.4% mutation frequency in patients versus 0.2% in controls may not reach statistical significance (p-value = 0.36)

• Cell-Type Specific Considerations:

  • Growth Pattern Analysis: PFN1-depleted HaCaT cells display independent growth versus colony formation in control cells

  • Sphere Formation: Measure changes in cellular integration capacity

  • Cytoskeletal Organization: Assess actin reorganization through co-IF staining of PFN1 with phalloidin

• Subcellular Localization Shifts:

  • Nuclear Translocation: PFN1 changes from predominant cytoplasmic localization to nuclear upon DNA damage

  • Recovery Dynamics: Monitor restoration of cytoplasmic compartmentalization during recovery periods

  • Interpretation Framework: Consider these shifts in context of cellular stress response mechanisms

• Expression Level Quantification:

  • Relative versus Absolute Changes: Adult pfn1ko/wt mice show 50% reduction in PFN1 expression without apparent functional impairment, but embryos exhibit developmental consequences

  • Normalization Strategy: Carefully select reference genes that remain stable across experimental conditions

What experimental approach should be used to determine if changes in cellular phenotype are directly attributable to PFN1 function?

To establish causality between PFN1 function and observed cellular phenotypes:

• Gene Manipulation with Rescue Experiments:

  • Knockdown-Rescue Methodology: Silence endogenous PFN1 and reintroduce either wild-type or mutant variants

  • Application Example: EGF-induced PM accumulation of PIP₃ is enhanced when PFN1 expression is silenced but reversed upon PFN1 re-expression

  • Controls: Include vector-only controls and expression of unrelated proteins

• Dose-Dependent Response Analysis:

  • Expression Level Modulation: Create cell lines with varying PFN1 expression levels

  • Quantitative Assessment: Correlate PFN1 expression levels with phenotypic changes

  • Example Approach: Transfect cells with myc-Pfn1 (coexpressed with GFP reporter) and assess PM PIP₂ staining intensity in GFP-positive cells compared to controls

• Structure-Function Analysis:

  • Domain-Specific Mutants: Generate PFN1 variants with mutations in actin-binding, PLP-binding, or PPI-binding domains

  • Binding Partner Disruption: Assess phenotypic consequences of disrupting specific interactions

  • Interpretation Framework: Determine which molecular interaction is critical for specific cellular functions

• Temporal Control Systems:

  • Inducible Expression/Knockdown: Use Tet-On/Off systems to control timing of PFN1 manipulation

  • Acute versus Chronic Effects: Distinguish between immediate responses and adaptive changes

  • Recovery Assessment: Monitor phenotype reversal upon restoration of normal PFN1 levels

• Correlation with Endogenous Regulators:

  • Pathway Manipulation: Alter upstream regulators of PFN1 expression or activity

  • Modifier Screens: Identify genes that enhance or suppress PFN1-associated phenotypes

  • Integrative Analysis: Combine with pharmacological interventions targeting specific pathways

This comprehensive approach ensures that observed phenotypes can be confidently attributed to PFN1 function rather than secondary effects or experimental artifacts.