CFL1 Antibody

What is CFL1 and why is it significant in cellular research?

CFL1 (Cofilin-1) is a critical actin-binding protein belonging to the ADF/cofilin family with significant roles in cytoskeletal dynamics. In humans, CFL1 is a 166 amino acid protein with a molecular mass of approximately 18.5 kDa . As a ubiquitous actin-binding factor, CFL1 is essential for reorganizing actin filaments through its pH-sensitive F-actin depolymerizing activity .

Methodologically, CFL1's significance stems from its central role in several cellular processes:

• It severs actin filaments by creating more positive ends on filament fragments

• It causes depolymerization at the minus end of filaments, preventing reassembly

• It recycles older ADP-F-actin, helping cells maintain ATP-G-actin pools required for sustained motility

CFL1 function is regulated by various factors including pH, phosphorylation, and phosphoinositides, making it a critical node in cellular signaling networks .

What applications are CFL1 antibodies most commonly used for in research?

CFL1 antibodies are versatile research tools employed across multiple experimental applications. Based on citation analysis, there are over 980 publications describing the use of CFL1 antibodies in research . The methodological applications include:

When selecting an application, researchers should consider that CFL1 localizes to both cytoplasm and nucleus and may show different distribution patterns depending on cell activation state and tissue type .

How can I validate the specificity of a CFL1 antibody for my research?

Methodological validation of CFL1 antibody specificity is crucial for generating reliable research data. A comprehensive validation approach should include:

• Positive control testing: Use cells/tissues known to express CFL1 (such as MCF-7 human breast cancer cell line) .

• Knockout/knockdown validation: Compare antibody signals between WT and CFL1-depleted samples. Studies have utilized:

  • Genetic knockout models (although complete CFL1 KO is embryonic lethal)

  • siRNA knockdown of CFL1 in cell lines

  • Inducible knockouts using systems like K14CreER or Ah-Cre transgenes

• Cross-reactivity assessment: Test against related proteins, particularly ADF (70% sequence homology to CFL1) .

• Rescue experiments: Restore protein expression via retroviral infection with WT CFL1 after depletion and confirm antibody reactivity returns .

• Multiple antibody comparison: Use antibodies from different vendors or those targeting different epitopes of CFL1.

Remember that CFL1 is widely expressed across tissues, making it essential to include appropriate positive and negative controls when validating antibody specificity .

How should I optimize immunofluorescence protocols for detecting both active and inactive forms of CFL1?

Optimizing immunofluorescence protocols for CFL1 requires careful consideration of its phosphorylation state, which dictates activity. Inactive CFL1 is phosphorylated at Serine-3 (pCFL1-S3), while the non-phosphorylated form is active .

Methodological approach:

• Fixation optimization:

  • For total CFL1: Standard 4% paraformaldehyde fixation works well

  • For phospho-CFL1: Quick fixation (2-5 minutes) with phosphatase inhibitors is crucial to preserve phosphorylation state

• Antigen retrieval: For formalin-fixed paraffin sections, boil sections in 10mM citrate buffer (pH 6.0) for 20 minutes .

• Dual immunostaining protocol:

  • Use antibodies against both total CFL1 and phospho-CFL1 (pCFL1-S3)

  • Select antibodies from different host species (e.g., rabbit anti-CFL1 and mouse anti-pCFL1)

  • Use appropriate secondary antibodies with non-overlapping fluorophores

• Co-localization controls:

  • Include F-actin staining (phalloidin) to visualize CFL1 activity on actin filaments

  • Include nuclear counterstaining as CFL1 can shuttle between cytoplasm and nucleus

• Activity modulation controls:

  • Treatment with phosphatase inhibitors (increases pCFL1)

  • Treatment with LIMK inhibitors (decreases pCFL1)

For optimal visualization, CFL1 typically shows diffuse cytoplasmic and nuclear staining, while activated CFL1 often localizes to the leading edge of migrating cells and areas of active actin turnover .

What are the critical considerations when using CFL1 antibodies in cancer research?

Cancer research applications of CFL1 antibodies require specific methodological considerations due to CFL1's altered expression and regulation in malignancies. Studies have demonstrated higher expression and activation of CFL1 in chronic lymphocytic leukemia (CLL) compared to control groups , and CFL1 autoantibodies have emerged as potential biomarkers in ovarian cancer .

Critical methodological considerations include:

• Expression level assessment:

  • Quantitative methods (Western blot, ELISA) with validated loading controls are essential

  • Compare matched normal-tumor pairs when possible

  • Consider tissue microarrays for high-throughput screening

• Activity state discrimination:

  • Use phospho-specific antibodies (pCFL1-S3) to distinguish inactive from active CFL1

  • Calculate the ratio of active/total CFL1 as a metric of pathway activation

• Cellular localization patterns:

  • Nuclear versus cytoplasmic distribution often changes in cancer

  • Co-staining with markers of invasive structures (invadopodia, lamellipodia)

• Functional validation approaches:

  • Knockdown studies using siRNA followed by phenotypic assays

  • Rescue experiments with WT versus mutant CFL1

• Biomarker potential evaluation:

  • For autoantibody studies, use a panel approach as demonstrated in ovarian cancer research combining anti-CFL1, anti-EZR, and anti-CYPA for optimal diagnostic value

A comprehensive cancer study might employ the diagnostic panel approach as shown in this table from ovarian cancer research:

This panel demonstrated significantly improved diagnostic capability over single markers alone .

How can I effectively distinguish between ADF and CFL1 in experimental systems?

• Antibody selection strategies:

  • Choose antibodies raised against regions with lowest sequence homology

  • Validate antibody specificity using knockout/knockdown systems for each protein

  • Consider using epitope-tagged versions in overexpression studies

• Expression pattern analysis:

  • ADF and CFL1 show tissue-specific expression patterns; CFL1 is more ubiquitous

  • RT-qPCR can quantify relative expression (studies show CFL1 is often 7-8 fold higher than ADF in some cell types)

• Functional discrimination approaches:

  • Single and double knockdown/knockout experiments reveal distinct phenotypes

  • CFL1 knockout is embryonically lethal while ADF knockout mice are viable

  • Double knockout of ADF/CFL1 in adult tissues causes catastrophic cytoskeletal defects not seen in single knockouts

• Biochemical activity differences:

  • ADF has stronger F-actin depolymerizing activity

  • CFL1 shows enhanced severing activity

  • pH sensitivity differs slightly between the proteins

For conclusive experiments, implementing genetic approaches like the CreER-loxP system used in studies to generate ADF⁻/⁻CFL1⁻/⁻ cells provides the clearest distinction between these highly homologous proteins .

What are common technical challenges when using CFL1 antibodies for Western blotting?

Western blotting for CFL1 presents several methodological challenges that researchers should anticipate and address:

• Size discrimination issues:

  • CFL1's small size (18.5 kDa) requires careful gel percentage selection (15% or gradient gels)

  • Close molecular weight to ADF may cause band overlap

  • Use positive controls with known CFL1 expression (MCF-7 cells work well)

• Phosphorylation state detection:

  • Phospho-CFL1 and total CFL1 run similarly on gels

  • Use phosphatase inhibitors during lysate preparation

  • Consider Phos-tag™ gels for enhanced separation of phosphorylated forms

• Antibody specificity concerns:

  • Multiple proteins in the ADF/cofilin family may cross-react

  • Validate antibody specificity with recombinant CFL1 and/or knockdown samples

  • Consider using multiple antibodies that recognize different epitopes

• Quantification challenges:

  • Loading control selection is critical (avoid actin as CFL1 affects actin dynamics)

  • Use total protein staining methods (Ponceau S, REVERT) for normalization

  • When studying CFL1 phosphorylation, always normalize phospho-CFL1 to total CFL1

• Extraction method optimization:

  • CFL1 distribution between cytosolic and cytoskeletal fractions varies with activation

  • Two-step lysis with phalloidin-mediated F-actin stabilization can help assess G-/F-actin ratios

  • Detergent selection affects CFL1 extraction (RIPA buffer works well for total extraction)

A methodological approach to studying CFL1's association with actin can be performed using a G-/F-actin fractionation protocol as described in published studies, which revealed a large increase in F- compared to G-actin in ADF/CFL1-depleted cells .

How do I troubleshoot inconsistent CFL1 staining patterns in immunohistochemistry?

Inconsistent CFL1 staining patterns in immunohistochemistry (IHC) can arise from several methodological factors. Systematic troubleshooting should address:

• Fixation-related issues:

  • Overfixation can mask CFL1 epitopes—optimize fixation time

  • Different fixatives affect CFL1 staining (formalin, methanol, acetone)

  • For FFPE tissues, proper antigen retrieval is critical (boil sections in 10mM citrate buffer, pH 6.0, for 20 minutes)

• Antibody selection considerations:

  • Not all CFL1 antibodies work equally well for IHC

  • Monoclonal antibodies may be more consistent but recognize fewer epitopes

  • Polyclonal antibodies offer more robust signal but may have batch-to-batch variation

  • Clone AT1C1 has been validated for IHC applications

• Signal-to-noise optimization:

  • Blocking protocol optimization (BSA vs. serum vs. commercial blockers)

  • Secondary antibody selection (polymer-based detection systems offer enhanced sensitivity)

  • Endogenous peroxidase quenching (if using HRP-based detection)

  • Autofluorescence reduction for IF (sodium borohydride treatment)

• Biological variability sources:

  • CFL1 expression varies by tissue type and physiological state

  • Phosphorylation status affects epitope accessibility

  • Nuclear vs. cytoplasmic localization changes with cell state

  • Tissue-specific post-translational modifications

• Controls implementation:

  • Include known positive controls (epithelium, lymphocytes)

  • Use knockout/knockdown tissues when available

  • Include isotype controls to assess non-specific binding

  • Peptide competition assays for validating signal specificity

For methodological consistency, maintain a detailed protocol log tracking all variables between experiments, and consider using automated IHC platforms for improved reproducibility when available.

How can CFL1 antibodies be utilized to study cytoskeletal dynamics in neurodegenerative diseases?

CFL1 antibodies provide powerful tools for investigating cytoskeletal abnormalities in neurodegenerative conditions through several methodological approaches:

• Neuronal morphology analysis:

  • CFL1 is crucial for neural tube morphogenesis and neural crest cell migration

  • Immunostaining protocols should examine:

    • Dendritic spine morphology (co-stain with phalloidin)

    • Growth cone dynamics (co-stain with phospho-CFL1)

    • Axonal transport (co-localization with vesicle markers)

• Stress response visualization:

  • CFL1 forms rod-like inclusions during cellular stress

  • Methodological approach to rod detection:

    • Fix rapidly to preserve transient structures

    • Use detergent-resistant fraction isolation

    • Co-stain for ADF and actin to confirm rod composition

• Activity-dependent reorganization studies:

  • Track CFL1 phosphorylation state changes following stimulation

  • Use phospho-specific antibodies to monitor LIMK-CFL1 pathway activation

  • Implement live-cell imaging with tagged CFL1 constructs to complement antibody-based fixed cell analysis

• Disease-specific aggregation investigation:

  • Examine co-localization of CFL1 with disease-specific proteins (tau, α-synuclein, huntingtin)

  • Apply proximity ligation assays to detect interaction between CFL1 and disease proteins

  • Study CFL1 in patient-derived samples using standardized IHC protocols

• Therapeutic intervention monitoring:

  • Use CFL1 antibodies to assess cytoskeletal restoration following treatment

  • Quantify CFL1 phosphorylation state as a marker of pathway normalization

  • Track subcellular localization changes as indicators of functional recovery

These approaches have revealed that nuclear integrity depends on proper actin dynamics regulated by ADF/CFL1, suggesting that cytoskeletal dysregulation may contribute to nuclear pathologies observed in various neurodegenerative conditions .

What is the significance of detecting anti-CFL1 autoantibodies in cancer diagnostics?

The detection of anti-CFL1 autoantibodies represents an emerging methodological approach in cancer diagnostics, particularly for ovarian cancer (OC). Recent research demonstrates several significant aspects to this approach:

• Diagnostic panel development:

  • Anti-CFL1 autoantibodies alone show limited diagnostic value, but when combined in panels their utility increases significantly

  • A panel containing anti-CFL1, anti-EZR, and anti-CYPA demonstrated superior performance with:

    • AUC: 0.762

    • Sensitivity: 55.56%

    • Specificity: 81.31%

• Early detection potential:

  • Anti-CFL1 autoantibodies may appear before clinical manifestations

  • Methodological approach for early detection studies:

    • Longitudinal sampling from high-risk populations

    • Correlation with conventional markers (CA125, HE4)

    • Assessment in combination with ROMA (Risk of Ovarian Malignancy Algorithm)

• Differential diagnosis capabilities:

  • Anti-CFL1 autoantibodies show significantly higher levels in OC compared to both benign ovarian disease (BD) and normal controls

  • No significant difference was observed between BD and normal controls

  • This suggests specificity for malignant transformation

• Implementation considerations:

  • ELISA-based detection protocols provide quantitative measurements

  • Standardization of cut-off values is critical

  • Machine learning approaches can optimize diagnostic algorithms

• Screening protocol development:

  • The predicted probability model:
    P(OC) = 1/(1 + EXP(−(−3.399 + 4.512 × anti-CFL1 + 3.997 × anti-EZR + 6.71 × anti-CYPA)))

  • This mathematical model integrates multiple autoantibody markers

  • Validation across diverse patient cohorts is essential for clinical implementation

This research demonstrates that while anti-CFL1 autoantibodies alone have limited diagnostic value, they contribute significantly to multi-marker panels with potential clinical utility in ovarian cancer management .

How does CFL1 function relate to therapeutic resistance in chronic lymphocytic leukemia?

Recent research has illuminated CFL1's role in chronic lymphocytic leukemia (CLL) therapy resistance, offering methodological approaches to study this relationship:

• Expression and activation assessment:

  • CLL patients show significantly higher expression and activation of CFL1 compared to healthy controls

  • Methodological detection via:

    • RT-qPCR for mRNA quantification

    • Western blot for protein levels and phosphorylation state

    • Flow cytometry for single-cell analysis

• Functional investigation approaches:

  • CFL1 gene silencing experiments reveal:

    • Decreased chemotaxis and migration capabilities

    • Increased apoptosis correlating with G2/M phase arrest

    • Compromised prosurvival signaling pathways

• Therapeutic target assessment:

  • CFL1 inhibition affects viability of CLL cells

  • Experimental approaches include:

    • siRNA-mediated knockdown

    • Small molecule inhibitors targeting CFL1-actin interaction

    • Upstream pathway modulation (LIMK inhibitors)

• Resistance mechanism characterization:

  • CFL1 maintains prosurvival signaling in CLL cells

  • Investigation methodology:

    • Track CFL1 phosphorylation state before and after therapy

    • Monitor cytoskeletal reorganization in resistant vs. sensitive cells

    • Examine CFL1-dependent cell adhesion-mediated drug resistance

• Biomarker potential evaluation:

  • CFL1 expression/activity as predictive markers of treatment response

  • Longitudinal sampling before, during, and after therapy

  • Correlation with established prognostic markers and clinical outcomes

This research indicates that CFL1 is not merely a passenger in CLL but plays an active role in maintaining malignant cell viability and influencing therapeutic outcomes. The significantly higher expression and activation of CFL1 in CLL suggests its potential as both a biomarker and therapeutic target .

What emerging techniques are enhancing CFL1 antibody applications in single-cell analysis?

Emerging methodological approaches are revolutionizing CFL1 antibody applications at the single-cell level:

• Mass cytometry (CyTOF) integration:

  • Metal-conjugated CFL1 antibodies enable multiplexed protein detection

  • Allows simultaneous assessment of CFL1 with dozens of other proteins

  • Enables correlation of CFL1 status with cell signaling networks and phenotypic markers

  • Methodological considerations include careful antibody panel design and validation

• Super-resolution microscopy approaches:

  • Advanced techniques reveal nanoscale organization of CFL1-actin interactions

  • STORM/PALM microscopy can visualize individual CFL1 molecules and their clustering

  • Expansion microscopy physically enlarges samples for enhanced resolution

  • These approaches have revealed CFL1's precise localization within cytoskeletal structures

• Live-cell imaging combined with FRET sensors:

  • Enables real-time visualization of CFL1 activity state changes

  • Genetically-encoded sensors can be validated with fixed-cell antibody approaches

  • Allows correlation of CFL1 activity with cellular behaviors like migration and division

• Spatial transcriptomics and proteomics correlation:

  • Antibody-based imaging paired with RNA sequencing

  • Maps CFL1 protein distribution in relation to its transcriptional regulation

  • Reveals tissue microenvironmental influences on CFL1 expression and function

• CRISPR-based genomic tagging:

  • Endogenous tagging of CFL1 for native-level expression studies

  • Tagged CFL1 can be validated with antibody detection

  • Enables longitudinal tracking of CFL1 dynamics in living cells

These techniques are particularly valuable for understanding CFL1's context-dependent functions in complex tissues and heterogeneous cell populations, where traditional bulk analysis methods may obscure important single-cell behaviors and regulatory mechanisms.

How can phosphorylation-specific CFL1 antibodies contribute to understanding cytoskeletal regulation in disease states?

Phosphorylation-specific CFL1 antibodies provide crucial methodological tools for dissecting cytoskeletal dysregulation in pathological conditions:

• Signal pathway activation mapping:

  • Phospho-CFL1 (Ser3) antibodies detect the inactive form

  • Enables quantitative assessment of LIMK-CFL1 pathway activation

  • Methodological approach:

    • Ratio analysis of phospho-CFL1/total CFL1 as activation metric

    • Spatial mapping of phosphorylation status across tissues

    • Temporal dynamics of phosphorylation in response to stimuli

• Drug response biomarker development:

  • CFL1 phosphorylation state changes rapidly with cytoskeletal-targeting therapies

  • Monitoring protocol:

    • Baseline phospho-CFL1 assessment before treatment

    • Time-course evaluation during therapy

    • Correlation with phenotypic responses (migration, invasion, proliferation)

• Disease mechanism elucidation:

  • Aberrant CFL1 phosphorylation contributes to various pathologies

  • Investigation approaches:

    • Compare phospho-CFL1 patterns in healthy vs. diseased tissues

    • Correlate with disease-specific markers

    • Manipulate phosphorylation state to assess causality

• Organelle dynamics investigation:

  • Nuclear shape depends on proper actin dynamics regulated by ADF/CFL1

  • Nuclear deformation in ADF/CFL1 knockout models suggests critical role in nuclear integrity

  • Methods to study this relationship:

    • Co-staining of phospho-CFL1 with nuclear envelope markers

    • Live-cell imaging of nuclear morphology during CFL1 activity modulation

    • Correlation of phospho-CFL1 patterns with chromatin organization

• Therapeutic strategy development:

  • CFL1 activity manipulation as potential intervention

  • Target discovery framework:

    • Screen for compounds that normalize aberrant CFL1 phosphorylation

    • Validate hits with functional assays

    • Assess specificity using phospho-specific antibodies

These approaches have revealed that aberrant CFL1 phosphorylation contributes to pathologies ranging from cancer to neurodegeneration, making phospho-specific antibodies invaluable tools for both basic research and translational medicine.

What is the potential for CFL1 antibodies in developing actin cytoskeleton-targeted therapeutics?

CFL1 antibodies offer unique methodological approaches for developing therapeutics targeting the actin cytoskeleton:

• High-throughput screening platforms:

  • CFL1 antibodies enable image-based screens for compounds affecting:

    • CFL1 phosphorylation state

    • CFL1-actin binding

    • CFL1 subcellular localization

  • Methodological approach:

    • Automated immunofluorescence in multi-well format

    • Quantitative image analysis for phenotypic profiling

    • Correlation with functional readouts (migration, division)

• Target validation methodologies:

  • Genetic approaches have demonstrated catastrophic effects of ADF/CFL1 co-depletion

  • Translational research strategies:

    • Titrated CFL1 inhibition to find therapeutic window

    • Tissue-specific targeting approaches

    • Conditional and inducible systems for temporal control

• Combination therapy rationales:

  • CFL1 inhibition sensitizes cells to cytoskeletal-targeting drugs

  • Experimental designs:

    • Systematic combination screens with CFL1 antibodies as readouts

    • Mechanistic studies of synergistic interactions

    • In vivo validation with appropriate biomarkers

• Delivery system optimization:

  • Antibody fragments or intrabodies targeting CFL1

  • Development considerations:

    • Cell-penetrating peptide conjugation

    • Nanoparticle encapsulation strategies

    • Tissue-specific targeting moieties

• Therapeutic biomarker development:

  • CFL1 status as predictor of response to actin-targeting therapies

  • Implementation approach:

    • Standardized IHC or ELISA protocols

    • Quantitative cutoff determination

    • Correlation with clinical outcomes