CFL1 Antibody

What is CFL1 and what are its key functions?

CFL1 (Cofilin 1) is an 18.5 kDa protein comprising 166 amino acid residues in humans. As a member of the Actin-binding proteins ADF family, CFL1 binds to F-actin and exhibits pH-sensitive F-actin depolymerizing activity, playing a crucial role in regulating actin cytoskeleton dynamics . CFL1 is widely expressed across various tissues and undergoes post-translational modifications, particularly phosphorylation. The protein is essential for normal mitotic progression and cytokinesis, and it regulates cell morphology and cytoskeletal organization . Additionally, CFL1 is required for the up-regulation of atypical chemokine receptor ACKR2 from endosomal compartments to the cell membrane, facilitating chemokine uptake and degradation. It also plays a critical role in neural tube morphogenesis and neural crest cell migration .

What types of CFL1 antibodies are available for research?

Research-grade CFL1 antibodies are available in both polyclonal and monoclonal formats with various host species options. Polyclonal antibodies, such as rabbit anti-CFL1, recognize multiple epitopes across the CFL1 protein, providing robust detection capabilities . Monoclonal antibodies, like mouse anti-human CFL1 (clone 1093310), offer highly specific detection with reduced batch-to-batch variability . Both unconjugated antibodies and those with fluorescent or enzymatic conjugates are available. Cross-reactivity profiles vary, with some antibodies specifically recognizing human CFL1, while others detect orthologs in multiple species including mouse and rat . The choice between these types depends on the specific research application, required specificity, and experimental design considerations.

What are the common applications for CFL1 antibodies?

CFL1 antibodies are employed across multiple research applications with varying protocols:

Over 980 citations in the literature describe the use of CFL1 antibodies, with Western Blot being the most widely utilized application . Researchers should optimize dilutions for their specific experimental conditions.

What is the subcellular localization of CFL1 and how does this impact antibody selection?

CFL1 exhibits dual localization in both the cytoplasm and nucleus of cells, which has been confirmed through immunofluorescence and immunohistochemistry studies using validated antibodies . This protein is involved in the translocation of the actin-cofilin complex from the cytoplasm to the nucleus, which is part of its functional role in cellular processes . When selecting antibodies for localization studies, researchers should choose those validated for both compartments, particularly for techniques that preserve cellular architecture such as immunofluorescence. Fixation and permeabilization methods significantly impact the detection of nuclear CFL1, with methanol fixation often providing better nuclear antigen accessibility than formaldehyde-based protocols. The visualization of CFL1 in specific subcellular compartments requires careful consideration of antibody penetration capabilities and epitope accessibility in different fixation conditions .

How should researchers validate CFL1 antibodies before use?

Validation of CFL1 antibodies is critical as studies indicate nearly half of commercially available antibodies may not function as advertised . A comprehensive validation protocol should include:

• Specificity Testing: Perform Western blot analysis using positive control samples (e.g., Jurkat, MCF-7 cell lysates) to confirm the detection of a single band at approximately 17-18 kDa .

• Knockout/Knockdown Controls: Compare antibody reactivity in wild-type cells versus CFL1 knockout or knockdown samples to confirm signal specificity.

• Cross-reactivity Assessment: If planning to use the antibody across species, verify reactivity with each target species (human, mouse, rat) independently .

• Application-specific Validation: For each intended application (WB, IF, IHC, etc.), perform preliminary experiments with positive controls and optimize protocols accordingly.

• Epitope Verification: If the exact epitope is known, consider how post-translational modifications might affect antibody binding, especially for phosphorylation-sensitive epitopes.

Proper validation minimizes false positives and ensures reproducible results across experiments .

What criteria should be considered when selecting a CFL1 antibody?

Selecting the appropriate CFL1 antibody requires evaluation of multiple technical specifications:

• Intended Application: Verify the antibody has been validated for your specific application (WB, IF, IHC, FACS, ELISA), as performance can vary significantly between applications .

• Species Reactivity: Ensure compatibility with your experimental model organism. Some antibodies detect only human CFL1, while others recognize orthologs in mouse, rat, and other species .

• Antibody Format: Choose between polyclonal and monoclonal based on your needs for specificity versus epitope coverage.

• Immunogen Information: Review the immunogen used to generate the antibody. Antibodies raised against recombinant full-length protein (e.g., amino acids 4-144) may provide better recognition than peptide-derived antibodies .

• Validation Data: Examine manufacturer-provided validation data specific to your application, including images of Western blots, IF/IHC staining patterns, and positive controls used .

• Citation Record: Consider antibodies with established publication records in peer-reviewed literature, as these have demonstrated reliability in actual research settings .

• Batch Consistency: For longitudinal studies, consider manufacturers with good quality control practices to ensure consistency between lots.

How do polyclonal and monoclonal CFL1 antibodies compare in specificity and applications?

Polyclonal and monoclonal CFL1 antibodies offer distinct advantages depending on the research context:

For experiments requiring differentiation between phosphorylated and non-phosphorylated CFL1, epitope-specific monoclonal antibodies are preferred. For general detection of total CFL1 protein, particularly in fixed tissues or Western blots, polyclonal antibodies often provide robust detection . Clone selection is critical for monoclonal antibodies, as demonstrated by the varying cross-reactivity profiles observed between different clones targeting related proteins .

What controls should be included when working with CFL1 antibodies?

Proper experimental controls are essential for interpreting CFL1 antibody results accurately:

• Positive Control: Include cell lines or tissues with confirmed CFL1 expression, such as Jurkat, MCF-7, or HeLa cells, which consistently show detectable CFL1 levels .

• Negative Control:

  • Primary antibody omission control to assess background from secondary antibody

  • Isotype control (matching IgG class) to evaluate non-specific binding

  • Ideally, CFL1 knockout/knockdown samples where available

• Loading Control: For Western blots, include housekeeping proteins (GAPDH, β-actin) for normalization, being mindful that CFL1 itself affects actin dynamics.

• Cross-Reactivity Control: If concerned about specificity, include samples containing related proteins (e.g., other ADF family members) to assess potential cross-reactivity.

• Peptide Competition: Pre-incubate the antibody with immunizing peptide to confirm binding specificity, especially for polyclonal antibodies.

• Signal Validation: For fluorescence applications, include an autofluorescence control and single-stained controls when performing multiplex imaging.

These controls help distinguish true CFL1 signal from technical artifacts and biological variability .

What are the optimal protocols for using CFL1 antibodies in Western blot analysis?

Successful Western blot detection of CFL1 requires attention to several key technical parameters:

• Sample Preparation:

  • Lyse cells in RIPA or NP-40 buffer containing protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylated CFL1

  • Load 15-20 μg total protein per lane for cell lines with typical CFL1 expression

• Electrophoresis Conditions:

  • Use 12-15% polyacrylamide gels for optimal resolution of the ~18 kDa CFL1 protein

  • Include molecular weight markers spanning 10-25 kDa range

• Transfer Parameters:

  • Semi-dry or wet transfer at 100V for 60-90 minutes

  • Use PVDF membrane for best results with CFL1 detection

• Blocking and Antibody Incubation:

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

  • Dilute primary CFL1 antibody 1:1,000-2,000 in blocking buffer

  • Incubate overnight at 4°C or 2 hours at room temperature

  • Use appropriate HRP-conjugated secondary antibody at 1:5,000-10,000

• Detection:

  • Develop with ECL substrate

  • Expected band size: 17-18 kDa

This protocol has been validated in multiple cell lines including Jurkat, MCF-7, NIH-3T3, and C6, consistently showing specific CFL1 detection .

How should CFL1 antibodies be used for immunofluorescence and immunohistochemistry?

For successful detection of CFL1 in cells and tissues:

Immunofluorescence Protocol:

• Grow cells on coverslips to 70-80% confluence

• Fix with 4% paraformaldehyde (10 min) or ice-cold methanol (5 min)

• Permeabilize PFA-fixed cells with 0.1% Triton X-100 (5 min)

• Block with 1-5% BSA or normal serum in PBS (1 hour)

• Incubate with primary CFL1 antibody at 5-10 μg/ml (or 1:100-1:200 dilution) for 2-3 hours at room temperature

• Wash 3x with PBS

• Incubate with fluorophore-conjugated secondary antibody (1:500-1:1000) for 1 hour

• Counterstain nucleus with DAPI

• Mount and visualize

Immunohistochemistry Protocol:

• Deparaffinize and rehydrate FFPE tissue sections

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Block endogenous peroxidase with 3% H₂O₂ (10 min)

• Block with 5% normal serum in PBS (1 hour)

• Incubate with primary CFL1 antibody at 5 μg/ml overnight at 4°C

• Wash 3x with PBS

• Apply HRP-polymer detection system

• Develop with DAB substrate

• Counterstain with hematoxylin

• Dehydrate, clear, and mount

Expected staining patterns include both nuclear and cytoplasmic localization, with intensity varying by cell type and physiological state .

What are the recommended fixation and permeabilization methods for CFL1 detection?

Fixation and permeabilization protocols significantly impact CFL1 detection quality:

For double immunostaining of CFL1 with other cytoskeletal proteins, PFA fixation followed by 0.1-0.2% Triton X-100 permeabilization often yields optimal results. When studying CFL1-actin interactions, phalloidin co-staining is compatible with methanol fixation but requires careful protocol optimization.

For tissue sections, heat-induced epitope retrieval in citrate buffer (pH 6.0) typically provides good antigen recovery for CFL1 detection with most antibodies . The choice of fixation method should be validated empirically for each specific CFL1 antibody and experimental system.

How can researchers troubleshoot common issues with CFL1 antibody staining?

When encountering problems with CFL1 antibody applications, consider these targeted troubleshooting approaches:

No Signal in Western Blot:

• Verify sample preparation includes protease inhibitors

• Confirm protein transfer by Ponceau S staining

• Test multiple antibody concentrations (1:500 to 1:5000)

• Try both reducing and non-reducing conditions

• Ensure detection system is functional with positive control antibody

Multiple Bands in Western Blot:

• Increase washing stringency (0.1% to 0.3% Tween-20)

• Try alternative blocking agents (milk vs. BSA)

• Consider whether bands represent isoforms, post-translational modifications, or degradation products

• Compare patterns with published literature for expected CFL1 modifications

Weak Immunofluorescence Signal:

• Test alternative fixation methods (PFA vs. methanol)

• Increase antibody concentration or incubation time

• Enhance permeabilization (0.1% to 0.5% Triton X-100)

• Try signal amplification systems (TSA, polymer detection)

• Optimize antigen retrieval conditions for tissue sections

High Background in Immunohistochemistry:

• Include longer blocking steps (2+ hours)

• Add 0.1-0.3% Triton X-100 to antibody diluent

• Pre-absorb antibody with tissue powder

• Include additional washing steps

• Use more dilute antibody with longer incubation times

When troubleshooting, always run appropriate positive and negative controls simultaneously to distinguish technical issues from biological variability .

How can CFL1 antibodies be used to study actin cytoskeleton dynamics?

CFL1 antibodies enable sophisticated analyses of actin dynamics through several advanced approaches:

• Co-localization Studies: Combine CFL1 antibodies with phalloidin staining to visualize the spatial relationship between CFL1 and F-actin filaments. This approach reveals areas of active actin depolymerization where CFL1 is recruited .

• Live-Cell Imaging: Using membrane-permeable fluorescently-tagged CFL1 antibody fragments allows tracking of endogenous CFL1 dynamics during cell migration, division, or response to stimuli.

• Proximity Ligation Assay (PLA): This technique can detect direct interactions between CFL1 and other actin-binding proteins or regulatory molecules with single-molecule sensitivity in situ.

• Phospho-specific Detection: Antibodies recognizing phosphorylated CFL1 (Ser3) can distinguish between active (non-phosphorylated) and inactive (phosphorylated) forms, revealing the regulation of actin-severing activity in response to cellular signals.

• Super-resolution Microscopy: CFL1 antibodies compatible with techniques like STORM or PALM enable visualization of CFL1-actin interactions at nanoscale resolution, revealing spatial organization beyond the diffraction limit.

These approaches provide mechanistic insights into how CFL1 mediates cytoskeletal remodeling during processes such as cell migration, division, and morphological changes .

What is known about CFL1 phosphorylation and how can it be studied using antibodies?

CFL1 activity is primarily regulated through phosphorylation at Serine 3, which inhibits its actin-binding and severing functions. Studying this dynamic regulation requires specialized approaches:

• Phosphorylation Detection Methods:

  • Phospho-specific antibodies that selectively recognize CFL1 phosphorylated at Ser3

  • Two-dimensional gel electrophoresis followed by Western blotting to separate phosphorylated from non-phosphorylated forms

  • Phos-tag™ SDS-PAGE to create mobility shifts based on phosphorylation state

• Kinase-Phosphatase Pathways:
  LIM kinases (LIMK1/2) and testicular protein kinases (TESK1/2) phosphorylate CFL1, while Slingshot phosphatases (SSH1/2/3) and Chronophin dephosphorylate it. Co-immunoprecipitation studies using CFL1 antibodies can identify these regulatory interactions.

• Stimulus-Response Analysis:
  Quantitative Western blotting with phospho-specific and total CFL1 antibodies can track the kinetics of CFL1 phosphorylation following stimuli such as growth factors, chemokines, or mechanical stress.

• Functional Correlation:
  Correlative microscopy combining phospho-CFL1 immunostaining with live-cell imaging of actin dynamics can link phosphorylation state to functional outcomes at the cellular level.

These methodologies have revealed that CFL1 phosphorylation states vary significantly across cell types and respond dynamically to extracellular signals, cytoskeletal perturbations, and pathological conditions .

How are CFL1 antibodies utilized in cancer research?

CFL1 antibodies have become valuable tools in cancer research due to growing evidence of CFL1's role in tumor progression:

• Diagnostic Applications:

  • Detection of anti-CFL1 autoantibodies as biomarkers for ovarian cancer, showing significant elevation compared to normal controls and benign disease

  • Part of a diagnostic panel with anti-EZR and anti-CYPA autoantibodies achieving 55.56% sensitivity and 81.31% specificity for ovarian cancer detection

• Metastasis Studies:

  • Immunohistochemical analysis of CFL1 expression in primary tumors versus metastatic lesions

  • Correlation of CFL1 levels with invasion capacity and metastatic potential

  • Identification of CFL1-dependent invadopodia formation in invasive cancer cells

• Therapeutic Target Validation:

  • Monitoring CFL1 expression changes in response to actin cytoskeleton-targeting drugs

  • Evaluating CFL1 involvement in chemoresistance mechanisms

  • Screening for compounds that modulate CFL1 activity or expression

• Mechanistic Investigations:

  • Analysis of CFL1 interactions with cancer-specific signaling pathways

  • Examination of CFL1's role in cancer cell metabolism and stress responses

  • Study of nuclear CFL1 functions in cancer cell transcriptional regulation

The correlation between CFL1 dysregulation and clinical outcomes suggests its potential as both a prognostic marker and therapeutic target in multiple cancer types .

What methods can be used to study CFL1 translocation between cytoplasm and nucleus?

CFL1 shuttles between cytoplasmic and nuclear compartments, carrying out distinct functions in each location. This dynamic process can be investigated through several approaches:

• Subcellular Fractionation:

  • Biochemical separation of nuclear and cytoplasmic fractions

  • Western blot analysis of each fraction using CFL1 antibodies

  • Quantification of the nuclear/cytoplasmic CFL1 ratio under various conditions

• Immunofluorescence Analysis:

  • Fixed-cell imaging with CFL1 antibodies and nuclear counterstains

  • High-content imaging platforms for quantitative assessment of nuclear/cytoplasmic signal intensity

  • Time-course studies following stimuli known to induce translocation

• Live-Cell Translocation Assays:

  • Fluorescently-tagged CFL1 antibody fragments for real-time tracking

  • Photoactivatable or photoconvertible CFL1 fusion proteins for pulse-chase experiments

  • FRAP (Fluorescence Recovery After Photobleaching) to measure kinetics of movement

• Molecular Interaction Studies:

  • Co-immunoprecipitation of CFL1 with importin/exportin machinery

  • Proximity ligation assays to detect interactions with nuclear transport factors

  • Analysis of post-translational modifications that regulate nuclear localization

Research has shown that CFL1's nuclear translocation increases during stress conditions and may be involved in regulating gene expression through its effects on nuclear actin. The actin-CFL1 complex translocation represents a significant mechanism by which cytoskeletal dynamics influence nuclear events .

How should researchers interpret varying CFL1 expression levels across different cell types?

Interpreting CFL1 expression patterns requires consideration of cell-type specific contexts:

This multilayered interpretation approach helps distinguish biologically meaningful CFL1 expression changes from technical variation or cell-specific baselines .

What are the known cross-reactivity issues with CFL1 antibodies?

Awareness of potential cross-reactivity is crucial for accurate data interpretation:

• Known Cross-Reactivity Targets:

  • ADF (Destrin): High homology to CFL1 (~70% sequence identity)

  • Cofilin-2 (CFL2): Muscle-specific isoform with ~80% sequence identity

  • Other actin-binding proteins with similar molecular weights

• Distinguishing Features in Detection:

  • CFL1: 18.5 kDa, widely expressed

  • CFL2: 18.7 kDa, predominantly in muscle tissue

  • ADF: 18.5 kDa, expression overlaps with CFL1

• Validation Approaches:
  Similar to the clone comparison shown for CD85d antibodies, where one clone showed high specificity while another exhibited cross-reactivity with related family members, CFL1 antibodies should be validated against recombinant CFL1, CFL2, and ADF proteins .

• Application-Specific Considerations:

  • Western blot: Cross-reactivity may appear as additional bands near 18-19 kDa

  • IHC/IF: May manifest as unexpected staining in tissues known to express related proteins

  • IP: Could co-precipitate family members, affecting downstream analyses

• Mitigation Strategies:

  • Use antibodies raised against unique regions of CFL1

  • Validate in tissues with differential expression of CFL family members

  • Consider genetic approaches (knockout/knockdown) for definitive target verification

These considerations are particularly important when studying tissues that express multiple cofilin family members, such as developing or regenerating muscles .

How can researchers quantify CFL1 levels accurately using antibody-based methods?

Accurate quantification of CFL1 requires rigorous methodological approaches:

• Western Blot Quantification:

  • Include recombinant CFL1 standards at known concentrations (5-100 ng range)

  • Use fluorescent secondary antibodies for wider linear detection range

  • Apply rolling ball background subtraction before densitometry

  • Normalize to total protein (Stain-Free or Ponceau S) rather than single housekeeping proteins

  • Report relative changes using biological replicates (n≥3)

• ELISA Approaches:

  • Sandwich ELISA using capture and detection antibodies targeting different CFL1 epitopes

  • Include standard curves with 7+ points and appropriate quality controls

  • Validate sample dilution linearity to confirm quantification within the linear range

  • Consider sample matrix effects when interpreting absolute concentrations

• Flow Cytometry Quantification:

  • Use calibration beads with known antibody binding capacity

  • Report data as molecules of equivalent soluble fluorochrome (MESF)

  • Include isotype controls and unstained samples for accurate background subtraction

  • Apply standardized gating strategies across experimental cohorts

• Image-Based Quantification:

  • Maintain consistent acquisition parameters (exposure, gain, offset)

  • Include fluorescence intensity calibration standards

  • Apply automated segmentation algorithms for unbiased measurement

  • Report integrated intensity rather than maximum pixel values

These approaches enable not only relative comparisons between experimental conditions but also more absolute quantification of CFL1 protein levels when properly calibrated .

What are the potential confounding factors in CFL1 antibody-based research?

Several experimental variables can confound CFL1 antibody data interpretation:

• Post-translational Modifications:

  • Phosphorylation at Ser3 may mask epitopes recognized by some antibodies

  • Oxidation of CFL1 during sample preparation can alter antibody recognition

  • Other modifications (acetylation, ubiquitination) may affect antibody binding

• Sample Preparation Variables:

  • Fixation artifacts: Overfixation can mask CFL1 epitopes, particularly in IHC/IF

  • Extraction efficiency: CFL1 bound to actin may extract differently than free CFL1

  • Protease activity during sample handling can generate fragments detected as multiple bands

• Cell State Dependencies:

  • Cell cycle phase affects CFL1 expression and localization

  • Cell density impacts CFL1 phosphorylation status

  • Mechanical stress during cell handling can rapidly alter CFL1 activity

• Technical Factors:

  • Antibody batch variation, especially for polyclonal antibodies

  • Detection system sensitivity limits for low-abundance forms

  • Non-specific binding in particular tissue types or fixation conditions

• Biological Complexity:

  • Coexpression of ADF or CFL2 in the same cells

  • Compensatory regulation when manipulating CFL1 levels

  • Feedback mechanisms affecting CFL1 in response to actin dynamics perturbations

Controlling for these variables through appropriate experimental design, including time-course analyses, multiple detection methods, and careful sample handling protocols, is essential for generating reliable CFL1 data .