DNM2 Antibody, FITC conjugated

What is DNM2 and why is it an important research target?

DNM2 (Dynamin 2) is a ubiquitously expressed large GTPase that plays critical roles in multiple cellular processes. It catalyzes the hydrolysis of GTP and utilizes this energy to mediate vesicle scission at plasma membranes during endocytosis and filament remodeling during organization of the actin cytoskeleton . DNM2 is essential for clathrin-mediated endocytosis (CME), exocytic processes, and clathrin-coated vesicle formation from the trans-Golgi network . During vesicular trafficking, DNM2 associates with membranes through lipid binding and self-assembles into ring-like structures through oligomerization, forming helical polymers around vesicle membranes that facilitate membrane scission . Its involvement in fundamental cellular processes makes it an important target for investigating membrane dynamics, cytoskeletal organization, and various disease mechanisms.

What are the key differences between DNM2 antibodies and how do FITC-conjugated versions enhance research capabilities?

DNM2 antibodies are available in various formats including rabbit polyclonal (e.g., ab3457) and mouse monoclonal (e.g., 68209-1-Ig) versions . While unconjugated antibodies require secondary detection steps, FITC-conjugated DNM2 antibodies offer direct visualization capabilities. This direct fluorescent labeling eliminates potential cross-reactivity issues associated with secondary antibodies and allows for:

• Single-step immunofluorescence protocols with reduced background

• Multi-color flow cytometry with minimal compensation requirements

• Live-cell imaging of DNM2 dynamics in real-time

• Reduced protocol time and complexity in fluorescence microscopy applications

The typical observed molecular weight of DNM2 is approximately 100 kDa, with a calculated molecular weight of 98 kDa, which is important to verify when validating antibody specificity .

What are the recommended applications for FITC-conjugated DNM2 antibodies?

Based on validation data for DNM2 antibodies, FITC-conjugated versions are optimally suited for:

It is strongly recommended to titrate the antibody in each specific testing system to obtain optimal results, as sample types can significantly influence performance .

How should I design validation experiments for a new FITC-conjugated DNM2 antibody?

A comprehensive validation strategy should include:

• Specificity testing: Compare staining patterns between wild-type cells and DNM2 knockdown/knockout models. Western blot with unconjugated antibody from the same clone can verify the target molecular weight (approximately 100 kDa) .

• Cross-reactivity assessment: Test the antibody across multiple species if cross-species reactivity is claimed. Current DNM2 antibodies show reactivity with human, mouse, rat, pig, and rabbit samples .

• Co-localization studies: Perform dual labeling with established markers of endocytic structures (clathrin, caveolin) to confirm proper localization.

• Functional validation: Assess DNM2 localization during GTPase-dependent processes using dynamin inhibitors (e.g., dynasore) to confirm functional specificity .

• Signal-to-noise optimization: Determine optimal fixation and permeabilization methods, as membrane-associated proteins like DNM2 can be sensitive to different fixation protocols.

What are the optimal sample preparation methods for immunofluorescence with FITC-conjugated DNM2 antibodies?

For optimal results with FITC-conjugated DNM2 antibodies in immunofluorescence applications:

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature preserves DNM2 structure while maintaining fluorophore activity. Avoid methanol fixation as it can disrupt membrane-associated protein localization.

• Permeabilization: Gentle permeabilization with 0.1% Triton X-100 for 5 minutes is sufficient for accessing intracellular DNM2. For membrane-associated DNM2 pools, consider milder permeabilization with 0.01% saponin.

• Blocking: Use 5% BSA or 10% normal serum from the same species as the secondary antibody (if using additional primary antibodies) for 1 hour to minimize non-specific binding.

• Antibody dilution: Prepare antibody in blocking buffer at 1:200-1:800 dilution, starting with manufacturer recommendations and optimizing as needed .

• Counterstaining considerations: When imaging DNM2 at plasma membrane or endocytic structures, consider membrane markers (WGA) or endosomal markers (EEA1) for co-localization studies.

• Anti-photobleaching: Mount with anti-fade reagents specifically optimized for FITC to extend imaging time and preserve signal strength.

How can I effectively use FITC-conjugated DNM2 antibodies to study endocytosis in live cells?

Live cell imaging of endocytosis with FITC-conjugated DNM2 antibodies requires careful preparation:

• Antibody loading: Use gentle permeabilization techniques (e.g., 0.01% digitonin) or microinjection to introduce antibodies into cells while maintaining cellular integrity.

• Concentration optimization: Begin with 1:400 dilution and adjust based on signal-to-noise ratio and cellular toxicity assessments.

• Imaging parameters: Use minimal laser power and exposure times to reduce photobleaching and phototoxicity. Consider using resonant scanners or spinning disk confocal for faster acquisition rates.

• Temperature control: Maintain cells at physiological temperature (37°C) as endocytosis rates are temperature-dependent; DNM2 GTPase activity changes significantly at sub-physiological temperatures.

• Temporal resolution: Acquire images at ≤1 second intervals to capture the rapid dynamics of DNM2 recruitment and vesicle scission events, which typically occur within 5-20 seconds.

• Co-labeling strategy: Combine with lipophilic membrane dyes (DiD, FM4-64) to simultaneously visualize membrane deformation during endocytosis.

What are common issues when using FITC-conjugated DNM2 antibodies and how can they be resolved?

For DNM2 specificity verification, compare staining patterns with known DNM2 distribution patterns at plasma membrane, endocytic vesicles, and trans-Golgi network locations.

How can I optimize FITC-conjugated DNM2 antibody performance for super-resolution microscopy?

Super-resolution microscopy with FITC-conjugated DNM2 antibodies requires special considerations:

• Sample preparation: Use thinner sections (80-100 nm for STORM/PALM) or #1.5H high-precision coverslips for SIM and STED.

• Fixation optimization: Implement 4% PFA with 0.2% glutaraldehyde to improve structural preservation of DNM2 ring-like assemblies around vesicle necks.

• Buffer composition: For STORM imaging, use oxygen scavenging buffers containing glucose oxidase/catalase system with cysteamine (MEA) to enhance FITC photoswitching.

• Labeling density: Adjust antibody concentration to achieve optimal labeling density (higher for SIM, lower for STORM/PALM) to match the specific super-resolution technique.

• Drift correction: Incorporate fiducial markers (TetraSpeck beads) for accurate drift correction during extended acquisition sessions.

• Multi-color considerations: When combining with other fluorophores, select those with minimal spectral overlap with FITC (e.g., Cy5, Alexa 647) to reduce chromatic aberration.

How can FITC-conjugated DNM2 antibodies be used to investigate the relationship between DNM2 and cytoskeletal dynamics?

DNM2 plays a crucial role in remodeling actin filaments in a GTPase-dependent manner and orchestrating the global actomyosin cytoskeleton . To investigate this relationship:

• Co-immunostaining protocol: Apply FITC-conjugated DNM2 antibody (1:200) alongside phalloidin-TRITC (1:1000) to visualize DNM2-actin associations. Include cortactin (CTTN) labeling, as this interaction stabilizes DNM2-actin filament binding and stimulates GTPase activity .

• Live imaging approach: In cells expressing Lifeact-RFP, introduce FITC-conjugated DNM2 antibody to monitor dynamic interactions during cytoskeletal remodeling.

• Experimental manipulations:

  • Apply actin depolymerizing agents (Latrunculin B, Cytochalasin D) at low doses to partially disrupt actin structures

  • Use jasplakinolide to stabilize actin filaments

  • Apply dynamin inhibitors (Dynasore, Dyngo-4a) to block GTPase activity

• Quantitative analysis: Measure colocalization coefficients (Pearson's, Manders') between DNM2 and actin at different subcellular regions and under various experimental conditions.

• Super-resolution approach: Implement STED or SIM imaging to resolve DNM2 ring-like structures around actin bundles, particularly in podocytes where DNM2 mediates arrangement of stress fibers .

How can FITC-conjugated DNM2 antibodies be applied to study disease models associated with DNM2 mutations?

DNM2 mutations are associated with several diseases including Charcot-Marie-Tooth (CMT) disease and centronuclear myopathy (CNM) . FITC-conjugated DNM2 antibodies can be leveraged to study these conditions:

• Patient-derived cell models: In fibroblasts or iPSC-derived cells from patients with DNM2 mutations, assess:

  • DNM2 localization patterns compared to healthy controls

  • Altered association with membranes and cytoskeletal structures

  • Changes in oligomerization using proximity-based assays

• Mouse model investigations: In DNM2 mouse models like the R369W/+ Dnm2 model for moderate CNM :

  • Quantify DNM2 protein levels in muscle tissues using flow cytometry

  • Assess DNM2 distribution in muscle fibers via immunofluorescence

  • Monitor changes in DNM2 expression following therapeutic interventions

• DNM2 and neutropenia assessment: In female mice with BM heterozygous Dnm2 haploinsufficiency that develop age-dependent neutropenia :

  • Analyze neutrophil CXCR4 surface expression following dynamin inhibition

  • Examine migration patterns of DNM2-deficient neutrophils

  • Correlate DNM2 expression levels with neutrophil counts and functionality

• Therapeutic monitoring: Following DNM2 downregulation therapy with antisense oligonucleotides or shRNA :

  • Track changes in DNM2 protein levels

  • Assess normalization of cellular phenotypes

What approaches can be used to simultaneously detect DNM2 isoforms using FITC-conjugated antibodies?

DNM2 has different isoforms with tissue-specific expression patterns, including those containing exon 10a/b in mutually exclusive splicing or the alternatively spliced exons 12b and 13b . For isoform-specific detection:

• Isoform-specific antibody approach: Use FITC-conjugated antibodies raised against unique epitopes in specific exons (e.g., exon 12b for muscle-specific isoforms).

• Combined immunoprecipitation-immunofluorescence strategy:

  • Perform isoform-specific immunoprecipitation

  • Label precipitated complexes with FITC-conjugated pan-DNM2 antibody

  • Quantify relative abundances of different isoforms

• Differential expression analysis: Compare DNM2 staining patterns in tissues known to express different isoform ratios (e.g., adult muscle expresses muscle-specific and ubiquitous DNM2 isoforms equally) .

• Developmental studies: Track changes in DNM2 isoform expression during muscle development, as exon 12b inclusion increases during this process .

• Post-immunostaining RNA scope: Combine FITC-conjugated DNM2 antibody staining with RNA scope for specific exons to correlate protein expression with specific mRNA variants.

How should researchers interpret differences in DNM2 subcellular localization patterns?

DNM2 exhibits distinct subcellular localization patterns that reflect its diverse functions:

• Plasma membrane association: Punctate staining at the cell periphery indicates involvement in clathrin-mediated endocytosis. Quantify by measuring fluorescence intensity along membrane regions and comparing to cytoplasmic signal.

• Endosomal localization: Co-localization with early endosomal markers (EEA1) indicates role in vesicular trafficking. Calculate Manders' overlap coefficient to determine the fraction of DNM2 associated with endosomes.

• Trans-Golgi network (TGN) association: Perinuclear DNM2 staining that overlaps with TGN markers reflects involvement in vesicle formation from the TGN .

• Cytoskeletal association: Linear patterns of DNM2 staining that align with actin filaments indicate cytoskeletal regulatory functions .

• Altered localization in disease states: In Charcot-Marie-Tooth disease or centronuclear myopathy, DNM2 may show:

  • Increased aggregation in specific compartments

  • Altered membrane association

  • Differential distribution between cytoskeletal and membrane structures

The interpretation should consider cell type-specific patterns, as DNM2 functions vary across tissues.

What controls and validation steps are essential when quantifying DNM2 expression levels using FITC-conjugated antibodies?

Rigorous controls and validation are critical for accurate quantification of DNM2 expression:

• Essential controls:

  • Isotype control (FITC-conjugated IgG of same species) to establish background fluorescence

  • DNM2 knockdown/knockout samples to confirm specificity

  • Positive control samples with known DNM2 expression levels

  • Unstained controls to determine autofluorescence levels

• Validation steps:

  • Correlation with Western blot data using unconjugated antibody from same clone

  • Comparison with mRNA expression data

  • Dose-response curves with recombinant DNM2 protein to establish linearity of detection

• Technical considerations:

  • Standard curve using calibration beads with known fluorophore quantities

  • Photobleaching correction by including reference standards

  • Background subtraction methods appropriate to imaging modality

• Data normalization approaches:

  • Normalize to total protein content for Western blot correlation

  • Use housekeeping proteins as internal controls for immunofluorescence

  • For flow cytometry, normalize to cell size/complexity parameters

How can researchers accurately analyze DNM2 dynamics in relation to GTPase activity using FITC-conjugated antibodies?

Analyzing DNM2 dynamics in relation to its GTPase activity requires specialized approaches:

• Conformational state analysis:

  • Use conformation-specific FITC-conjugated antibodies that preferentially bind GTP-bound or GDP-bound DNM2

  • Compare staining patterns before and after GTP-γ-S (non-hydrolyzable GTP analog) treatment

• Inhibitor-based approaches:

  • Apply dynamin inhibitors like dynasore that prevent GTPase activity

  • Monitor changes in DNM2 localization and oligomerization states

  • Correlate with functional outcomes like endocytosis inhibition or CXCR4 surface expression

• FRAP (Fluorescence Recovery After Photobleaching) analysis:

  • Measure DNM2 dynamics at membranes under different nucleotide states

  • Compare recovery half-times between wild-type and GTPase-deficient mutants

  • Correlate recovery kinetics with functional activities

• Co-localization with activity markers:

  • Combined staining with phospho-specific antibodies that recognize active conformations

  • Correlation with membrane curvature sensors (BAR domain proteins)

• Advanced analysis techniques:

  • Single-particle tracking to follow individual DNM2-positive vesicles

  • Residence time analysis at membranes during different stages of endocytosis

  • Mean squared displacement calculations to distinguish directed vs. random movement