MYO18A Antibody, FITC conjugated

What is MYO18A and why is it significant for cellular biology research?

MYO18A (Myosin XVIIIA) is an unconventional myosin protein that plays diverse roles in cellular architecture and function. It exists in different isoforms, with Myo18a-alpha colocalizing with the ER-Golgi complex, membrane ruffles, and filopodia, while Myo18a-beta localizes diffusely in the cytoplasm . Its significance stems from multiple biological functions:

• Stabilization and organization of the actin cytoskeleton

• Linking Golgi membranes to the cytoskeleton

• Participating in tensile forces required for vesicle budding from the Golgi

• Modulating lamellar actomyosin retrograde flow critical for cell protrusion and migration

• Maintaining stromal cell architectures required for cell-to-cell contact

• Regulating trafficking, expression, and activation of innate immune receptors on macrophages

• Suppressing inflammatory responses via CD14 trafficking modulation

• Acting as CD245, a receptor for surfactant A involved in activating human NK lymphocytes

The MYO18A protein has a calculated molecular weight of 233 kDa but is typically observed at 230 kDa and 190 kDa in experimental settings .

How does FITC conjugation enhance the utility of MYO18A antibodies in research applications?

FITC (fluorescein isothiocyanate) conjugation of MYO18A antibodies provides significant advantages for immunofluorescence and flow cytometry applications. The FITC fluorophore exhibits peak excitation at 495 nm and emission at 519 nm, producing bright green fluorescence that enables direct visualization without secondary antibody requirements. This conjugation eliminates potential cross-reactivity issues associated with secondary antibodies and reduces experimental time.

For MYO18A research specifically, FITC conjugation allows:

• Direct visualization of MYO18A subcellular localization and co-localization studies with differentially labeled cellular structures

• Multi-color imaging when combined with other non-overlapping fluorophore-conjugated antibodies

• Quantitative analysis of MYO18A expression through flow cytometry

• Reduced background compared to two-step detection methods

When designing experiments, researchers should account for FITC's sensitivity to photobleaching by optimizing exposure times and considering anti-fade mounting media for microscopy applications .

What are the recommended validation methods to ensure specificity of MYO18A antibodies before experimental use?

Before utilizing MYO18A antibodies in critical experiments, validation of specificity is essential through multiple complementary approaches:

• Western blot validation: Confirm single bands at expected molecular weights (230 kDa and 190 kDa) in positive control cell lines such as Jurkat, HeLa, K-562, Neuro-2a, and NIH/3T3 cells .

• Knockdown/knockout validation: Compare antibody signal in wild-type versus MYO18A knockout/knockdown samples. Published literature includes at least 4 studies using this approach for MYO18A antibody validation .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (for example, the synthetic peptide corresponding to amino acids 958-1008 or 1849-2054 depending on the antibody clone) to confirm specific binding .

• Cross-reactivity testing: Verify expected reactivity with human, mouse, and rat samples as indicated by the manufacturer specifications .

• Immunoprecipitation: Perform IP followed by mass spectrometry to confirm target pull-down, with HeLa cells being a recommended positive control .

Each validation approach provides complementary evidence of specificity, and researchers should document these validation steps in their methods sections.

What are the optimal fixation and permeabilization protocols for immunofluorescence applications with FITC-conjugated MYO18A antibodies?

The selection of fixation and permeabilization methods significantly impacts FITC-MYO18A antibody staining outcomes in immunofluorescence applications. Based on research protocols:

Recommended fixation protocols:

• 4% paraformaldehyde (10-15 minutes at room temperature) preserves cellular architecture while maintaining MYO18A epitope accessibility

• For dual cytoskeletal studies, a combination of 2% paraformaldehyde with 0.1% glutaraldehyde improves actin preservation while maintaining MYO18A antigenicity

Permeabilization options:

• 0.1-0.2% Triton X-100 (5-10 minutes) for general applications

• 0.5% saponin (10 minutes) for gentler permeabilization when studying membrane-associated MYO18A

• Avoid methanol-based fixation/permeabilization as it may disrupt the MYO18A epitope structure

When studying MYO18A-alpha's co-localization with ER-Golgi complexes or membrane structures, sequential mild fixation followed by gentle permeabilization produces superior results. For studies focusing on cytoplasmic MYO18A-beta, standard PFA fixation with Triton X-100 permeabilization is sufficient .

How can researchers optimize Western blot protocols for detecting MYO18A with high sensitivity and specificity?

Detecting MYO18A via Western blot requires optimization due to its high molecular weight (230 kDa/190 kDa). Follow these research-validated protocols:

Sample preparation optimization:

• Use RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors

• Sonicate lysates briefly (3-5 pulses) to shear DNA and reduce viscosity

• Heat samples at 70°C (not 95°C) for 5 minutes to prevent aggregation of high molecular weight proteins

Gel electrophoresis considerations:

• Use 6-8% polyacrylamide gels or gradient gels (4-12%) to effectively resolve high molecular weight proteins

• Extend SDS-PAGE running time to ensure adequate separation

Transfer parameters:

• Use wet transfer systems rather than semi-dry for high molecular weight proteins

• Transfer at low voltage (30V) overnight at 4°C or utilize commercial high molecular weight transfer systems

• Consider adding 0.1% SDS to transfer buffer while reducing methanol concentration to 10%

Antibody incubation:

• Recommended dilution range: 1:1000-1:6000 for Western blot applications

• Extend primary antibody incubation to overnight at 4°C for improved sensitivity

• Use 5% BSA rather than milk for blocking and antibody dilution

This optimized protocol helps overcome common challenges in detecting high molecular weight proteins like MYO18A and enables consistent results across experiments .

What specific controls are essential when performing flow cytometry with FITC-conjugated MYO18A antibodies?

For rigorous flow cytometric analysis using FITC-conjugated MYO18A antibodies, implement these essential controls:

Essential negative controls:

• Unstained cells to establish autofluorescence baseline

• Isotype control (rabbit IgG-FITC) at identical concentration to assess non-specific binding

• FMO (Fluorescence Minus One) controls for multicolor panels

• MYO18A knockdown cells to confirm antibody specificity

Positive controls:

• Cell lines with documented MYO18A expression (Jurkat, HeLa, K-562 cells)

• Positive cell population treated with known MYO18A expression inducers

Technical controls:

• FITC single-stained compensation beads for multicolor experiments

• Viability dye to exclude dead cells that may bind antibodies non-specifically

• Fc receptor blocking reagent when analyzing immune cells

Additional considerations:

• Perform titration experiments to determine optimal antibody concentration

• Include controls for each experimental condition and timepoint

• Document voltage settings and instrument calibration parameters

Implementation of these controls ensures reliable flow cytometry data interpretation and facilitates troubleshooting of unexpected results when studying MYO18A expression patterns .

How can researchers effectively design co-localization studies to investigate MYO18A interactions with cytoskeletal and membrane components?

Designing effective co-localization studies for MYO18A requires careful consideration of the distinct localization patterns of its isoforms. Myo18a-alpha primarily associates with the ER-Golgi complex, membrane ruffles, and filopodia, while Myo18a-beta exhibits diffuse cytoplasmic localization .

Recommended experimental design:

• Selection of appropriate co-staining markers:

  • Golgi markers: GM130 (cis-Golgi), TGN46 (trans-Golgi network)

  • ER markers: Calnexin, PDI

  • Actin cytoskeleton: Phalloidin (different fluorophore than FITC)

  • Membrane ruffle markers: Cortactin, WAVE2

  • Filopodia markers: Fascin, Myo10

• Multi-channel confocal microscopy setup:

  • Use sequential scanning to prevent bleed-through

  • Maintain consistent pinhole settings across channels

  • Capture Z-stacks to assess 3D relationships

• Quantitative co-localization analysis:

  • Calculate Pearson's correlation coefficient and Mander's overlap coefficient

  • Perform distance analysis between structures

  • Use intensity profile analysis along defined cellular regions

• Dynamic interaction studies:

  • Consider FRAP (Fluorescence Recovery After Photobleaching) to assess mobility

  • Implement live cell imaging with fluorescently-tagged MYO18A constructs

• Perturbation experiments:

  • Assess co-localization changes following cytoskeletal disruption (cytochalasin D, nocodazole)

  • Evaluate Golgi fragmentation effects on MYO18A distribution

These approaches allow researchers to dissect the spatial relationships between MYO18A and various cellular components, providing insight into its functional roles within the cell .

What strategies can be employed for multiplexed immunofluorescence studies that include FITC-conjugated MYO18A antibodies?

Multiplexed immunofluorescence incorporating FITC-conjugated MYO18A antibodies requires strategic planning to maximize information while avoiding spectral overlap. Consider these advanced approaches:

Spectral compatibility planning:

• Pair FITC (excitation: 495nm, emission: 519nm) with fluorophores having minimal spectral overlap

• Recommended compatible fluorophores: DAPI (nuclei), Cy3/RRX (red), Cy5 (far-red)

• Avoid PE and Alexa Fluor 488 due to spectral similarity with FITC

Sequential staining strategies:

• For epitopes requiring identical species antibodies, implement tyramide signal amplification (TSA)

• Consider primary antibody direct labeling with different fluorophores

• Utilize zenon labeling technology for same-species antibodies

Advanced microscopy techniques:

• Implement spectral unmixing on confocal platforms to resolve overlapping signals

• Consider super-resolution microscopy (STED, STORM, SIM) for co-localization at sub-diffraction resolution

• Utilize linear unmixing algorithms for closely overlapping fluorophores

Panel design considerations:

• Reserve FITC-MYO18A for structures requiring high sensitivity detection

• Place antibodies to abundant targets on less bright fluorophores

• Consider antigen abundance when designing staining sequence

Controls for multiplexed experiments:

• Single-color controls for each fluorophore

• FMO controls to establish gating boundaries

• Absorption controls to verify elimination of primary antibodies in sequential staining

This comprehensive approach enables complex multi-parameter analysis of MYO18A in relation to multiple cellular components simultaneously .

How do different fixation methods affect epitope presentation and signal intensity when using MYO18A antibodies?

The choice of fixation method significantly impacts epitope preservation and accessibility for MYO18A antibodies, with effects varying based on the specific epitope region targeted. Research findings demonstrate:

Comparative analysis of fixation methods for MYO18A detection:

For antibodies targeting amino acids 1849-2054 (C-terminal region), PFA and acetone fixation provide optimal epitope accessibility, while antibodies targeting regions corresponding to amino acids 958-1008 show better performance with PFA fixation .

Researchers should conduct preliminary fixation comparison studies with their specific antibody clone to determine optimal conditions, especially when studying MYO18A in different subcellular compartments or cell types.

What are the most common sources of false-positive and false-negative results when using MYO18A antibodies, and how can they be mitigated?

Researchers frequently encounter both false-positive and false-negative results when using MYO18A antibodies. Understanding these pitfalls and implementing appropriate controls is essential for accurate data interpretation.

Common sources of false-positive results:

• Non-specific binding: High molecular weight proteins often produce background bands in Western blotting.

  • Mitigation: Use more stringent blocking (5% BSA instead of milk) and increase wash duration/frequency.

• Cross-reactivity with related myosin family members:

  • Mitigation: Validate specificity through knockout/knockdown controls and peptide competition assays.

• Dead cell binding in flow cytometry:

  • Mitigation: Include viability dye and strict gating strategies to exclude dead/dying cells.

• Autofluorescence interference with FITC signal:

  • Mitigation: Implement spectral compensation, use unstained and isotype controls, consider alternative fluorophores for highly autofluorescent samples.

Common sources of false-negative results:

• Epitope masking during fixation:

  • Mitigation: Optimize fixation conditions (as detailed in section 3.3) or implement antigen retrieval.

• Insufficient antibody concentration for high molecular weight target:

  • Mitigation: Titrate antibody concentrations and extend incubation times (overnight at 4°C).

• Inefficient transfer of high molecular weight proteins in Western blotting:

  • Mitigation: Implement specialized high-molecular-weight transfer protocols (see section 2.2).

• Protein degradation during sample preparation:

  • Mitigation: Use fresh tissue/cells, maintain cold temperatures during lysis, and include protease inhibitor cocktails.

Implementing rigorous validation protocols and appropriate technical controls addresses these challenges, ensuring reliable and reproducible results when working with MYO18A antibodies .

How can researchers troubleshoot weak or inconsistent signals in immunofluorescence studies using FITC-conjugated MYO18A antibodies?

When encountering weak or inconsistent signals with FITC-conjugated MYO18A antibodies in immunofluorescence applications, implement this systematic troubleshooting approach:

Signal optimization flowchart:

• Antibody-related factors:

  • Verify antibody activity via dot blot or Western blot control

  • Increase concentration (consider testing 2-5× recommended dilution)

  • Extend incubation time (overnight at 4°C)

  • Check for FITC photobleaching (prepare fresh dilutions, protect from light)

• Sample preparation factors:

  • Optimize fixation protocol (test PFA vs. acetone fixation)

  • Adjust permeabilization (try 0.1%, 0.2%, and 0.5% Triton X-100)

  • Implement antigen retrieval (citrate buffer pH 6.0 or TE buffer pH 9.0)

  • Reduce background with longer/additional washing steps

• Target-related factors:

  • Verify target expression in sample (check literature for positive control cells)

  • Consider cell-cycle dependent expression patterns

  • Evaluate epitope accessibility (different fixation methods affect epitope exposure)

• Technical factors:

  • Optimize microscope settings (exposure time, gain, offset)

  • Use appropriate filters optimized for FITC

  • Consider signal amplification techniques (TSA)

  • Prepare fresh mounting media with anti-fade compounds

• Advanced solutions:

  • Try different antibody clones targeting alternative epitopes

  • Consider unconjugated primary followed by ultra-sensitive secondary detection

  • Implement automated image acquisition for consistency

  • Use computational image enhancement (deconvolution)

This structured approach helps identify and address specific factors contributing to suboptimal staining results, leading to improved detection of MYO18A in immunofluorescence applications .

What considerations are important when selecting between different MYO18A antibody clones that target distinct epitope regions?

The selection of MYO18A antibody clones targeting different epitope regions requires careful consideration of experimental goals and biological context. Research indicates that antibody performance varies significantly based on the targeted region, affecting detection sensitivity and specificity in different applications.

Epitope-specific performance characteristics:

Selection strategy based on research goals:

• For studies of protein-protein interactions: Select antibodies targeting regions away from known interaction domains (N-terminal region recommended)

• For subcellular localization studies: Choose antibodies with demonstrated performance in immunofluorescence (C-terminal AA 1849-2054 region recommended)

• For quantitative expression analysis: Select antibodies with linear signal response in Western blot (Middle region AA 958-1008 recommended)

• For isoform-specific detection: Choose antibodies targeting unique regions of specific isoforms

• For cross-species studies: Verify sequence conservation in the epitope region across target species

This strategic approach to antibody selection ensures optimal detection of MYO18A based on specific experimental requirements and biological questions .

How can FITC-conjugated MYO18A antibodies be utilized in studying the protein's role in immune cell regulation and inflammatory responses?

MYO18A's identification as CD245 (a receptor for surfactant A) and its role in regulating immune cell functions makes FITC-conjugated MYO18A antibodies valuable tools for immunology research. These antibodies enable sophisticated methodological approaches for investigating MYO18A's immunoregulatory functions:

Flow cytometry applications:

• Quantifying MYO18A expression levels on different immune cell populations (NK cells, macrophages)

• Tracking expression changes during activation/differentiation

• Correlating MYO18A expression with functional readouts (cytokine production, phagocytosis)

• Sorting MYO18A-high versus MYO18A-low populations for functional assays

Live-cell imaging approaches:

• Monitoring MYO18A dynamics during immune synapse formation

• Tracking receptor clustering during activation events

• Visualizing interactions with surfactant proteins in real-time

• Assessing co-localization with inflammatory signaling components

Functional blocking studies:

• Using non-conjugated antibodies to block MYO18A function followed by FITC-conjugated detection

• Assessing surfactant A binding after antibody-mediated perturbation

• Comparing effects of different epitope-targeting antibodies on NK cell activation

Translational research applications:

• Correlating MYO18A expression patterns with inflammatory disease states

• Developing flow cytometry panels for clinical immunophenotyping

• Investigating MYO18A as a potential therapeutic target for inflammatory conditions

These approaches leverage the direct visualization capabilities of FITC-conjugated antibodies while providing mechanistic insights into MYO18A's role in suppressing inflammatory responsiveness of macrophages and modulating CD14 trafficking .

What methodological approaches can be used to study MYO18A's association with the Golgi apparatus and its role in membrane trafficking?

Investigating MYO18A's association with the Golgi apparatus and role in membrane trafficking requires specialized methodological approaches that leverage FITC-conjugated antibodies in combination with advanced imaging and biochemical techniques:

High-resolution microscopy approaches:

• Super-resolution microscopy (STED, STORM) to visualize Golgi-associated MYO18A at sub-diffraction resolution

• Correlative light-electron microscopy (CLEM) to precisely localize MYO18A relative to Golgi ultrastructure

• Live-cell confocal imaging with FITC-MYO18A antibody fragments to track dynamics

• Ratiometric imaging of MYO18A:Golgi marker distribution during vesicle budding events

Perturbation experimental designs:

• Brefeldin A treatment to disrupt Golgi and monitor MYO18A redistribution

• Nocodazole/cytochalasin treatments to assess cytoskeletal requirements for MYO18A-Golgi association

• Temperature blocks (20°C, 15°C) to arrest trafficking at different stages

• siRNA-mediated knockdown of trafficking regulators to identify cooperative interactions

Biochemical fractionation strategies:

• Density gradient fractionation to isolate Golgi-enriched membranes and quantify associated MYO18A

• Protease protection assays to determine MYO18A topology relative to membranes

• IP-MS from isolated Golgi fractions to identify MYO18A interactors

• In vitro reconstitution of MYO18A-dependent vesicle budding

Cargo trafficking assays:

• Quantitative measurement of VSV-G or other cargo protein transport rates with/without MYO18A perturbation

• RUSH system (Retention Using Selective Hooks) to synchronize and visualize cargo release

• Dual-color live imaging of MYO18A and cargo proteins

• Correlating MYO18A localization with sites of vesicle fission

These methodological approaches provide complementary insights into MYO18A's role in providing tensile force for vesicle budding from the Golgi and its contribution to Golgi membrane trafficking and architecture .

What are the emerging techniques and future directions for studying MYO18A's interactions with the actin cytoskeleton in cell migration and cancer metastasis?

Emerging techniques are revolutionizing the study of MYO18A's interactions with the actin cytoskeleton in cell migration and potential roles in cancer metastasis. These cutting-edge approaches extend beyond traditional antibody applications:

Advanced live-cell imaging techniques:

• Lattice light-sheet microscopy for 3D visualization of MYO18A-actin dynamics with reduced phototoxicity

• FRET/FLIM imaging to measure direct MYO18A-actin interactions in living cells

• Expansion microscopy combined with FITC-MYO18A antibody staining for super-resolution insights

• Optogenetic control of MYO18A activity to precisely manipulate function during migration

Tension sensing approaches:

• Force-sensitive FRET biosensors to measure MYO18A-generated forces

• Molecular tension microscopy to visualize force transmission at cell protrusions

• Traction force microscopy correlated with MYO18A localization

• Atomic force microscopy to measure mechanical properties of MYO18A-rich regions

Single-molecule techniques:

• Single-molecule tracking of MYO18A to measure diffusion coefficients and binding kinetics

• In vitro reconstitution of MYO18A-actin networks with purified components

• Optical tweezers to measure MYO18A-generated forces on individual actin filaments

• DNA-PAINT super-resolution to precisely map MYO18A distribution relative to actin structures

Multi-omics integration:

• Spatial transcriptomics combined with MYO18A protein localization

• Phosphoproteomics to identify regulatory modifications during migration

• Proximity labeling (BioID, APEX) to map the MYO18A interactome

• CRISPR-Cas9 screening for synthetic interactions with MYO18A in migration

Translational research directions:

• Correlation of MYO18A expression with migration potential in patient-derived cancer models

• Development of inhibitors targeting MYO18A's roles in metastasis

• Investigation of MYO18A as a biomarker for invasive cancer phenotypes

• Study of MYO18A in mechanosensing and response to extracellular matrix rigidity

These emerging approaches are enabling researchers to dissect MYO18A's precise functions in modulating lamellar actomyosin retrograde flow and its contributions to cell protrusion and migration, with potential implications for understanding cancer metastasis mechanisms .

How should researchers select the most appropriate techniques for their specific MYO18A investigations based on research questions and available resources?

Selecting appropriate techniques for MYO18A research requires systematic evaluation of research questions, available resources, and technical considerations. This decision framework guides researchers toward optimal methodological choices:

Decision matrix for technique selection:

• Define primary research objective:

  • Protein localization → Immunofluorescence with FITC-conjugated antibodies

  • Protein quantification → Western blot, ELISA, flow cytometry

  • Protein interactions → IP, proximity labeling, FRET

  • Dynamic behavior → Live-cell imaging, FRAP

  • Functional analysis → siRNA/CRISPR with rescue experiments

• Consider sample type constraints:

  • Fixed tissues → IHC with antigen retrieval (TE buffer pH 9.0)

  • Live cells → Flow cytometry, live-cell compatible antibody fragments

  • Cell lysates → Western blot, IP with optimized high MW protocols

  • Subcellular fractions → Western blot, mass spectrometry

• Evaluate available resources:

  • Core facility access → Super-resolution, mass spectrometry

  • Budget constraints → Prioritize validated antibodies over newer techniques

  • Time limitations → Balance technique complexity with timeline requirements

  • Technical expertise → Consider collaboration for specialized techniques

• Assess technical requirements:

  • Sensitivity needs → Signal amplification strategies for low-abundance targets

  • Resolution requirements → Match imaging technique to biological question

  • Quantitative vs. qualitative data → Select appropriate analytical approaches

  • Throughput requirements → Consider automated image acquisition/analysis

This structured approach ensures researchers select the most appropriate techniques for their specific MYO18A investigations, balancing scientific rigor with practical considerations to maximize research impact and resource efficiency .

What are the critical quality control considerations for publications involving MYO18A antibodies?

Essential antibody validation documentation:

• Complete antibody information (catalog number, clone, lot, RRID)

• Evidence of antibody specificity (knockdown/knockout controls, peptide competition)

• Demonstration of expected molecular weight (230 kDa/190 kDa for MYO18A)

• Cross-reactivity testing for multi-species applications

Experimental protocol transparency:

• Detailed fixation/permeabilization methods (critical for epitope preservation)

• Complete antibody dilution and incubation conditions

• Comprehensive imaging parameters (exposure, gain, offset settings)

• Processing and analysis algorithms with parameter settings

Controls and reproducibility:

• Technical replicates across multiple experiments

• Biological replicates using different cell sources/tissue samples

• Positive and negative controls clearly presented in figures

• Independent verification using alternative detection methods

Quantification and statistical approach:

• Objective quantification methods for fluorescence intensity

• Appropriate statistical tests for sample size

• Blinded analysis procedures when applicable

• Raw data availability in public repositories

Limitations acknowledgment:

• Discussion of potential antibody limitations

• Consideration of alternative interpretations

• Transparent reporting of inconsistent or conflicting results

• Clear delineation between observation and interpretation