CFM2 Antibody

What is CFM2 and why is it significant in research?

CFM2 is a reported synonym of the RFLNA gene, which encodes refilin A. This protein plays a crucial role in regulating the perinuclear actin network and nuclear shape through its interaction with filamins. The human version of CFM2 has a canonical length of 216 amino acid residues and a molecular mass of approximately 23.6 kilodaltons. Two distinct isoforms have been identified to date. CFM2 is primarily localized in the cytoplasm and is notably expressed in specific tissues including the testis, stomach, small intestine, and fallopian tube. It belongs to the Refilin protein family, which makes it an important target for studying cytoskeletal dynamics and nuclear morphology in various cellular contexts .

What are the key differences between Arabidopsis CFM2 and human CFM2?

While both are referred to as CFM2, the Arabidopsis and human variants represent distinct proteins with different functions. The commercially available antibodies featured in search results predominantly target Arabidopsis CFM2, as indicated by their reactivity specifications. Arabidopsis CFM2 is involved in plant-specific processes, whereas human CFM2 (refilin A) participates in cytoskeletal organization through filamin interactions. When designing experiments, researchers must carefully select antibodies specific to their target organism and validate cross-reactivity claims, particularly when working with conserved protein domains. Sequence homology analysis between plant and mammalian CFM2 variants can help predict potential cross-reactivity issues in experimental applications .

How should researchers select the appropriate anti-CFM2 antibody for their specific experimental needs?

When selecting an anti-CFM2 antibody, researchers should consider several critical factors: (1) Target species specificity - confirm the antibody has been validated against your species of interest, noting that available commercial antibodies show reactivity to Arabidopsis; (2) Application compatibility - verify the antibody has been validated for your specific applications (e.g., Western blot, ELISA); (3) Epitope location - consider whether the target epitope is accessible in your experimental conditions; (4) Clonality - monoclonal antibodies offer high specificity for a single epitope while polyclonal antibodies may provide broader detection across multiple epitopes; (5) Conjugation requirements - determine if you need an unconjugated antibody or one with a specific conjugate (e.g., biotin, fluorophore) .

Before committing to large-scale experiments, conduct preliminary validation studies with positive and negative controls. Review literature for previous successful applications with your chosen antibody, and consider performing small-scale pilot studies to confirm specificity and sensitivity in your specific experimental system .

What validation methods are essential before using a CFM2 antibody in critical experiments?

A comprehensive validation protocol for CFM2 antibodies should include:

• Western blot analysis: Confirm a single band of expected molecular weight (~23.6 kDa for human CFM2) in positive control samples and absence in negative controls.

• Knockout/knockdown controls: Test the antibody against samples where CFM2 expression has been eliminated or reduced through genetic approaches (CRISPR/Cas9, siRNA) to verify specificity.

• Peptide competition assay: Pre-incubate the antibody with purified CFM2 protein or immunizing peptide to demonstrate specific binding through signal abolishment.

• Cross-reactivity assessment: Test against related proteins, particularly other Refilin family members, to ensure specificity.

• Immunoprecipitation followed by mass spectrometry: Confirm the antibody pulls down authentic CFM2 protein.

Validation methods should be selected based on the techniques you intend to use in your research. For immunohistochemistry applications, include tissue-specific positive and negative controls. For quantitative applications, establish the linear detection range and limit of detection through dilution series experiments .

What are the optimal conditions for using CFM2 antibodies in Western blot applications?

For optimal Western blot results with CFM2 antibodies, follow these methodological guidelines:

• Sample preparation: Use RIPA buffer supplemented with protease inhibitors for extraction. For cytoskeletal proteins like CFM2/refilin A, include phosphatase inhibitors to preserve post-translational modifications.

• Gel selection: Use 12-15% polyacrylamide gels to effectively resolve the ~23.6 kDa CFM2 protein and its isoforms.

• Transfer conditions: Employ semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour using PVDF membranes (preferred over nitrocellulose for this protein).

• Blocking procedure: Block with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Antibody incubation: Dilute primary antibody (typically 1:1000, but verify manufacturer's recommendation) in blocking solution and incubate overnight at 4°C with gentle rocking.

• Washing and detection: Wash thoroughly (4-5 times for 5 minutes each) before adding appropriate HRP-conjugated secondary antibody at 1:5000 dilution for 1 hour at room temperature.

Include positive controls (tissues known to express CFM2 such as testis or small intestine extracts) and negative controls (tissues with minimal expression or CFM2-knockout samples). For challenging detection scenarios, consider enhanced chemiluminescence (ECL) substrates with extended exposure times .

How can CFM2 antibodies be effectively used in ELISA-based experiments?

For developing robust ELISA protocols with CFM2 antibodies:

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

• Blocking: Block with 1-3% BSA in PBS for 1-2 hours at room temperature to minimize background.

• Sample preparation: Create standard curves using recombinant CFM2 protein (0.1-1000 ng/ml). For cell lysates, use a concentration of 10-50 μg total protein per well.

• Detection antibody: Use a different anti-CFM2 antibody targeting a separate epitope (sandwich ELISA) or directly conjugated primary antibody for direct ELISA.

• Optimization steps:

  • Test multiple antibody concentrations to determine optimal signal-to-noise ratio

  • Validate assay linearity across a wide range of target concentrations

  • Determine the lower limit of detection and quantification

• Controls: Include full standard curve, blank wells, and samples with known concentrations of CFM2.

When developing ELISAs for detecting Arabidopsis CFM2, be aware that plant proteins may require specialized extraction buffers to prevent interference from plant secondary metabolites and polyphenols .

How can researchers investigate CFM2 interactions with filamin proteins using immunoprecipitation techniques?

To effectively study CFM2-filamin interactions using immunoprecipitation (IP):

• Lysis buffer selection: Use a gentle non-denaturing lysis buffer (e.g., 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% Triton X-100) supplemented with protease and phosphatase inhibitors to preserve native protein complexes.

• Pre-clearing step: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• IP protocol:

  • Incubate 500-1000 μg of pre-cleared lysate with 2-5 μg of anti-CFM2 antibody overnight at 4°C with gentle rotation

  • Add protein A/G beads for 2-4 hours at 4°C

  • Wash extensively (4-5 times) with cold lysis buffer

  • Elute complexes by boiling in SDS sample buffer

• Co-IP analysis: Analyze eluates by Western blotting using antibodies against filamin A or other suspected interaction partners.

• Controls: Include:

  • IgG control IP to identify non-specific interactions

  • Input samples (5-10% of starting material)

  • Reverse co-IP using anti-filamin antibodies to confirm bidirectional interaction

• Validation approaches: Confirm interactions through alternative methods such as proximity ligation assay (PLA) or fluorescence resonance energy transfer (FRET) in intact cells.

This methodology allows researchers to investigate how CFM2/refilin A regulates the perinuclear actin network through its interactions with filamin proteins under various cellular conditions .

What approaches can be used to investigate the subcellular localization of CFM2 in different cell types?

For comprehensive subcellular localization studies of CFM2:

• Immunofluorescence optimization:

  • Fixation: Compare paraformaldehyde (4%, 10-15 min) with methanol fixation (-20°C, 10 min) to determine optimal epitope preservation

  • Permeabilization: Test different agents (0.1-0.5% Triton X-100, 0.1-0.5% Saponin) and durations (5-15 min)

  • Blocking: Use 5% normal serum from the same species as the secondary antibody

  • Primary antibody dilution: Typically 1:100-1:500, optimized through titration

  • Co-staining: Include markers for cytoskeletal structures (phalloidin for F-actin), nuclear membrane (lamin antibodies), and other subcellular compartments

• Live-cell imaging:

  • Generate CFM2-GFP fusion constructs with careful consideration of tag position to avoid interference with protein function

  • Create stable cell lines with inducible expression systems for long-term studies

  • Apply lattice light-sheet or spinning disk confocal microscopy for dynamic localization studies

• Subcellular fractionation:

  • Separate cytoplasmic, nuclear, membrane, and cytoskeletal fractions

  • Analyze each fraction by Western blotting with CFM2 antibody

  • Include fraction-specific markers (GAPDH, Lamin A/C, Na+/K+ ATPase, β-actin) to confirm fraction purity

• Tissue-specific expression:

  • Compare localization patterns across tissues known to express CFM2 (testis, stomach, small intestine, fallopian tube)

  • Analyze how subcellular distribution changes under different physiological conditions or stressors

This multifaceted approach provides comprehensive insights into the spatial distribution of CFM2 and can reveal tissue-specific localization patterns relevant to its function .

How should researchers address non-specific binding issues when working with CFM2 antibodies?

When encountering non-specific binding with CFM2 antibodies, implement the following systematic troubleshooting approach:

• Optimize blocking conditions:

  • Test alternative blocking agents (5% BSA, 5% normal serum, commercial blocking solutions)

  • Extend blocking time (2-3 hours at room temperature or overnight at 4°C)

  • Add 0.1-0.3% Tween-20 to blocking and antibody diluent solutions

• Improve antibody specificity:

  • Increase washing stringency (more wash steps, higher salt concentration in wash buffer)

  • Perform antibody pre-adsorption against tissues/lysates from CFM2-negative samples

  • Titrate antibody concentration to find optimal signal-to-noise ratio

  • Consider using a different clone targeting a unique epitope

• Sample preparation refinements:

  • Ensure complete protein denaturation for Western blots (heat samples at 95°C for 5 minutes in reducing conditions)

  • Filter lysates to remove particulates that can cause non-specific binding

  • Use freshly prepared samples to minimize protein degradation

• Controls for validation:

  • Include peptide competition controls to confirm binding specificity

  • Use lysates from CFM2 knockout or knockdown models as negative controls

  • Test the antibody against recombinant CFM2 protein as a positive control

• Detection system adjustments:

  • Switch to more specific detection systems (e.g., from conventional HRP to polymer-based detection)

  • Reduce exposure time in chemiluminescence detection

  • Consider fluorescent secondary antibodies with precise quantification capabilities

Document all optimization steps systematically to develop a reproducible protocol tailored to your specific experimental system .

What are the key considerations when interpreting CFM2 expression data across different experimental systems?

When interpreting CFM2 expression data across diverse experimental systems, consider these critical factors:

• Species-specific variation:

  • Human CFM2/refilin A (23.6 kDa) differs from Arabidopsis CFM2 in structure and function

  • Antibody cross-reactivity between species should be experimentally verified, not assumed

  • Evolutionary conservation analysis helps contextualize observed differences

• Isoform detection:

  • Confirm which CFM2 isoforms are recognized by your antibody

  • The presence of multiple bands may indicate isoforms, post-translational modifications, or degradation products

  • Verify band identity through mass spectrometry or isoform-specific antibodies

• Tissue-specific expression patterns:

  • CFM2/refilin A shows variable expression across tissues (highest in testis, stomach, small intestine, fallopian tube)

  • Normalize expression to tissue-specific housekeeping genes rather than using universal normalizers

  • Consider cell type heterogeneity within tissue samples when interpreting results

• Quantification methodology:

  • Western blot: Use standardized loading controls and calibration curves for accurate quantification

  • ELISA: Validate linearity across the working range and determine lower limits of detection

  • Immunohistochemistry: Apply digital pathology approaches for objective quantification

• Experimental variables affecting expression:

  • Cell cycle stage can influence CFM2 expression and localization

  • Cell density and culture conditions may alter cytoskeletal organization and CFM2 levels

  • Stress conditions might trigger expression changes in CFM2 and related proteins

By addressing these considerations, researchers can generate more reliable and contextually meaningful data on CFM2 expression patterns across different experimental systems .

How can researchers effectively study the functional relationship between CFM2 and nuclear morphology?

To investigate the functional relationship between CFM2 and nuclear morphology:

• Genetic manipulation approaches:

  • Generate CFM2 knockout/knockdown models using CRISPR/Cas9 or RNAi

  • Create CFM2 overexpression systems with inducible promoters

  • Develop domain-specific mutants to identify regions critical for nuclear shape regulation

• Advanced imaging techniques:

  • Implement live-cell imaging with nuclear membrane markers (e.g., lamin-GFP fusions)

  • Apply super-resolution microscopy (STORM, PALM, SIM) to visualize perinuclear actin networks

  • Use atomic force microscopy to measure nuclear stiffness in CFM2-modified cells

• Nuclear morphometry analysis:

  • Develop automated image analysis pipelines to quantify nuclear parameters (area, perimeter, circularity, aspect ratio)

  • Track dynamic changes in nuclear shape over time in response to stimuli

  • Correlate CFM2 expression/localization with nuclear morphology metrics

• Biochemical interaction studies:

  • Map the binding interfaces between CFM2 and filamin proteins

  • Identify additional nuclear envelope proteins that interact with CFM2

  • Investigate post-translational modifications of CFM2 that regulate its function

• Mechanical stress experiments:

  • Apply controlled mechanical forces to cells while monitoring nuclear responses

  • Compare wild-type and CFM2-deficient cells under various mechanical stress conditions

  • Evaluate nuclear rupture frequency and repair efficiency in relation to CFM2 status

This multidisciplinary approach provides mechanistic insights into how CFM2 contributes to nuclear shape regulation through its interaction with the perinuclear actin cytoskeleton .

What methodologies can be applied to resolve contradictory data regarding CFM2 function in different experimental models?

When facing contradictory results about CFM2 function across different experimental models, implement the following resolution strategies:

• Standardized experimental framework:

  • Develop a consensus protocol that normalizes key variables (cell density, passage number, sample processing)

  • Create a repository of validated reagents (antibodies, expression constructs, cell lines) for cross-laboratory testing

  • Implement blinded analysis to minimize expectation bias

• Multi-modal validation approach:

  • Verify findings using orthogonal techniques (e.g., if Western blot and immunofluorescence show discrepancies, add mass spectrometry)

  • Apply genetic approaches (CRISPR/Cas9, RNAi) alongside antibody-based methods

  • Combine in vitro and in vivo models to establish biological relevance

• Context-dependent function analysis:

  • Systematically vary experimental conditions (cell type, differentiation state, tissue origin)

  • Test function under different stressors (mechanical stress, oxidative stress, nutrient deprivation)

  • Map condition-specific interaction networks using techniques like BioID or APEX2 proximity labeling

• Quantitative systems biology:

  • Develop mathematical models that integrate conflicting datasets and propose testable hypotheses

  • Use Bayesian approaches to update models based on new evidence

  • Identify parameters that most strongly influence experimental outcomes

• Collaborative verification:

  • Organize multi-laboratory testing of key findings with standardized protocols

  • Establish minimum reporting standards for CFM2 experimental methods

  • Create open databases of raw data for community reanalysis

By systematically addressing experimental variables and implementing rigorous validation approaches, researchers can resolve contradictions and develop a more nuanced understanding of context-dependent CFM2 functions .