MYO3B Antibody, FITC conjugated

What is MYO3B Antibody, FITC conjugated and what are its primary research applications?

MYO3B Antibody, FITC conjugated is a fluorescently-labeled polyclonal antibody specifically targeting the human Myosin-IIIb protein (EC 2.7.11.1). The antibody is typically raised in rabbits using recombinant Human Myosin-IIIb protein fragments (commonly the 1110-1276AA region) as the immunogen . This reagent has been validated for applications such as ELISA, with reactivity specifically against human samples .

The primary research applications include:

• Fluorescent detection of MYO3B protein in immunofluorescence studies examining actin-based cellular structures

• Investigation of signal transduction pathways involving MYO3B

• Study of actin protrusion formation and elongation mechanisms, particularly in cell types exhibiting specialized actin-rich structures

• Examination of interactions between MYO3B and other cytoskeletal proteins like Espin that cooperatively regulate actin dynamics

The FITC conjugation enables direct visualization without requiring secondary antibodies, simplifying experimental workflows in fluorescence microscopy. The fluorescein component is typically excited by the 488 nm laser line, with emission collected at approximately 530 nm, making it compatible with standard fluorescence imaging systems .

How should MYO3B Antibody, FITC conjugated be stored to maintain optimal activity?

Proper storage of MYO3B Antibody, FITC conjugated is critical for maintaining its immunoreactivity and fluorescent properties. Based on manufacturer recommendations and general antibody handling principles, the following storage guidelines should be implemented:

• Temperature conditions: Store at -20°C or -80°C for long-term preservation . The choice between these temperatures may depend on anticipated frequency of use and manufacturer specifications.

• Avoid repeated freeze-thaw cycles: Each freeze-thaw cycle can cause protein denaturation and fluorophore degradation, reducing both antibody functionality and fluorescence intensity .

• Buffer composition considerations: The antibody is typically supplied in a stabilizing buffer containing 50% Glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as a preservative . This formulation helps maintain antibody stability during freeze-thaw transitions.

• Aliquoting strategy: Upon receipt, divide the antibody into small single-use aliquots before freezing to minimize freeze-thaw damage. The volume of each aliquot should be determined based on typical experimental needs.

• Light protection: FITC is susceptible to photobleaching, so storing the antibody in amber or opaque tubes and minimizing exposure to light during handling will help preserve fluorescent signal intensity .

• Working solution handling: When preparing diluted working solutions, maintain cold conditions (4°C) and use within 24 hours for optimal performance.

Following these guidelines will help ensure consistent antibody performance across experiments and maximize the usable lifetime of this research reagent.

What experimental controls should be implemented when using MYO3B Antibody, FITC conjugated?

When designing experiments using MYO3B Antibody, FITC conjugated, implementing appropriate controls is essential for data validation and interpretation. The following control strategies should be considered:

• Specificity controls:

  • Negative control tissues/cells known not to express MYO3B

  • Isotype control: FITC-conjugated non-specific IgG from the same host species (rabbit) at matching concentration

  • Peptide competition assay: Pre-incubation of the antibody with excess immunizing peptide (Human Myosin-IIIb protein, 1110-1276AA region) to confirm binding specificity

• Technical controls:

  • Unstained samples to assess autofluorescence levels

  • Secondary antibody-only controls (for protocols incorporating additional detection steps)

  • Fixed but otherwise untreated samples to establish background fluorescence baseline

• Expression validation controls:

  • Parallel detection with an alternative MYO3B antibody recognizing a different epitope

  • Correlation with mRNA expression using techniques like fluorescent in situ hybridization (FISH) to confirm expression patterns

  • Western blot analysis to confirm antibody specificity and target protein molecular weight

• Fluorescence controls:

  • Fluorescence minus one (FMO) controls for multicolor flow cytometry applications

  • Photobleaching controls to account for signal decay during imaging

• Biological function controls:

  • Comparison with MYO3A expression patterns in the same samples, as these proteins have distinct but related functions

  • Co-localization with known interaction partners like Espin to confirm biologically relevant detection

What is the biological function of MYO3B protein in cellular processes?

MYO3B (Myosin-IIIb) is a motor protein with significant roles in cytoskeletal organization and cellular morphogenesis. Understanding its biological functions provides important context for antibody-based investigations:

• Actin protrusion regulation: MYO3B plays a critical role in the formation and elongation of actin-based cellular protrusions, including stereocilia and other stable actin-rich structures . These structures are essential for specialized cellular functions such as sensory perception in hair cells.

• Molecular transport: MYO3B functions as a transporter protein, carrying cargo molecules along actin filaments from the base to the tips of cellular protrusions . This transport activity contributes to the proper localization of proteins required for the maintenance of actin-based structures.

• Partnership with actin-bundling proteins: MYO3B works cooperatively with actin-bundling proteins, particularly Espin, to regulate the morphogenesis of apical extensions in specialized cells . This interaction is crucial for establishing and maintaining the proper ultrastructure of these cellular projections.

• Signal transduction: MYO3B is involved in signal transduction pathways , potentially linking mechanical or chemical signals to cytoskeletal responses and cellular morphology adaptations.

• Functional distinction from MYO3A: Unlike MYO3A, MYO3B lacks an extended tail domain with additional actin-binding motifs . Consequently, MYO3B exhibits approximately half the motor activity and reduced ATPase activity compared to MYO3A . This functional difference results in distinct contributions to actin protrusion dynamics.

Understanding these biological roles provides essential context for designing experiments and interpreting results when using MYO3B antibodies for research purposes. The specific localization and function of MYO3B make it an important target for studies of cellular architecture and specialized cellular structures.

How does FITC conjugation affect antibody performance in research applications?

FITC (fluorescein isothiocyanate) conjugation introduces specific considerations that can impact antibody performance in research applications. Understanding these effects is crucial for experimental design and data interpretation:

• Conjugation chemistry: FITC molecules covalently attach to primary amines (lysine residues) on the antibody structure . Typically, between 3-6 FITC molecules are conjugated to each antibody molecule for optimal performance .

• Effects on antibody binding:

  • Excessive conjugation (>6 FITC molecules per antibody) can sterically hinder antigen binding sites

  • Under-conjugation results in insufficient fluorescence signal for detection

  • Conjugation may alter the antibody's isoelectric point, potentially affecting binding kinetics and non-specific interactions

• Fluorescence properties:

  • FITC exhibits maximum excitation at 488 nm and emission at approximately 530 nm

  • The fluorophore is susceptible to photobleaching during extended imaging sessions

  • FITC fluorescence is pH-sensitive, with optimal signal at slightly alkaline pH (7.5-8.5)

• Technical considerations:

  • Signal-to-noise ratio may be lower than with indirect detection methods (primary + secondary antibody)

  • Autofluorescence from biological samples often overlaps with FITC emission spectrum

  • Higher antibody concentrations may be needed compared to unconjugated primary antibodies

• Experimental advantages:

  • Eliminates potential cross-reactivity issues from secondary antibodies

  • Enables direct single-step staining protocols

  • Facilitates multicolor imaging when combined with antibodies carrying spectrally distinct fluorophores

When working with MYO3B Antibody, FITC conjugated, researchers should consider these factors and potentially adjust protocols to accommodate the specific characteristics of this conjugate. For example, using anti-fade mounting media, optimizing antibody concentration through titration experiments, and implementing appropriate controls to account for background fluorescence will help maximize experimental success.

How can I optimize immunofluorescence protocols using MYO3B Antibody, FITC conjugated for studying actin-based cellular protrusions?

Optimizing immunofluorescence protocols for studying actin-based protrusions with MYO3B Antibody, FITC conjugated requires careful consideration of several technical factors:

• Sample preparation optimization:

  • Fixation method: For actin-rich structures, 4% paraformaldehyde (PFA) preservation generally maintains structural integrity better than methanol fixation

  • Permeabilization: Use 0.1% Triton X-100 for balanced membrane permeabilization while preserving delicate actin structures

  • Antigen retrieval: Test whether gentle antigen retrieval (citrate buffer, pH 6.0) improves MYO3B detection without disrupting actin architecture

• Co-visualization strategies:

  • Phalloidin counterstaining: Use far-red fluorescent phalloidin (not green) to avoid spectral overlap with FITC when visualizing the actin cytoskeleton

  • Co-staining with Espin antibodies (red or far-red fluorophores) to examine MYO3B-Espin interactions at actin protrusion tips

  • Nuclear counterstaining with DAPI provides orientation reference without interfering with FITC emission spectrum

• Protocol refinements:

  • Antibody titration: Perform serial dilutions (1:50 to 1:500) to determine optimal signal-to-noise ratio

  • Extended incubation: Consider overnight incubation at 4°C to improve antibody penetration into dense actin structures

  • Blocking optimization: Test 5% BSA vs. 10% normal serum from unrelated species to minimize background

• Imaging considerations:

  • Z-stack acquisition: Capture the full three-dimensional structure of actin protrusions

  • Deconvolution: Apply computational deconvolution to improve resolution of fine actin-based structures

  • Confocal microscopy: Utilize optical sectioning to eliminate out-of-focus fluorescence from dense samples

• Specialized approaches for difficult samples:

  • Pre-extraction protocol: Brief treatment with 0.5% Triton X-100 before fixation to remove soluble cytoplasmic proteins and enhance visualization of cytoskeletal-associated MYO3B

  • Sample orientation: For polarized cells with apical extensions, careful sectioning and orientation facilitates visualization of apical junctional complexes where MYO3B concentrates

By systematically optimizing these parameters, researchers can achieve high-quality immunofluorescence visualization of MYO3B in the context of actin-based cellular protrusions, enabling detailed analysis of its distribution and co-localization with functional partners.

What are the key considerations when comparing MYO3A and MYO3B functions using specific antibodies?

When designing experiments to compare MYO3A and MYO3B functions using specific antibodies, researchers should address several important considerations to ensure valid and informative results:

• Structural and functional distinctions:

  • MYO3A contains an extended tail domain with an additional actin-binding motif that is absent in MYO3B

  • MYO3A exhibits approximately 2-fold faster motor activity with enhanced ATPase activity and higher actin affinity compared to MYO3B

  • These differences contribute to distinct roles in actin protrusion formation and elongation

• Antibody selection strategy:

  • Choose antibodies targeting non-homologous regions to ensure isoform specificity

  • Validate cross-reactivity profiles against recombinant proteins of both isoforms

  • Consider using antibodies targeting the extended tail region unique to MYO3A for definitive discrimination

• Experimental design approaches:

  • Comparative localization: Use differently labeled antibodies (e.g., FITC-MYO3B and TRITC-MYO3A) in co-localization studies to map differential distribution

  • Functional perturbation: Compare effects of dominant-negative constructs (e.g., Myo3b-DN) on cellular protrusion morphology

  • Cargo transport analysis: Investigate differential transport of binding partners like ESPN1 and ESPNL by each myosin isoform

• Data interpretation framework:

  • Quantitative morphometric analysis of actin protrusion length, area, and density in cells expressing either MYO3A or MYO3B

  • Assessment of protrusion dynamics and stability over time, as MYO3A more effectively stabilizes and extends actin protrusions

  • Statistical comparison of protein localization patterns along the length of actin protrusions

• Control implementations:

  • Genetic knockout or knockdown controls to verify antibody specificity

  • Rescue experiments reintroducing wild-type or mutant forms to confirm functional relationships

  • Chimeric constructs (e.g., MYO3A tail fused to MYO3B motor) to dissect domain-specific contributions to function

This systematic approach allows researchers to clearly delineate the distinct roles of MYO3A and MYO3B in actin dynamics and cellular morphogenesis, while avoiding potential pitfalls in antibody-based discrimination between these related but functionally distinct proteins.

How can MYO3B Antibody, FITC conjugated be used in quantitative fluorescence studies of actin dynamics?

Implementing MYO3B Antibody, FITC conjugated in quantitative fluorescence studies requires rigorous methodology to generate reliable measurements of actin dynamics. The following approaches can enhance quantitative accuracy:

• Fluorescence calibration strategies:

  • Establish quantitative reference standards using calibrated fluorescent beads

  • Implement internal intensity controls in each experiment to normalize between samples

  • Account for FITC photobleaching rates by measuring fluorescence decay curves under standardized imaging conditions

• Quantitative imaging protocols:

  • Maintain consistent exposure settings, gain, and offset across all comparative samples

  • Acquire images below pixel saturation to ensure linear signal response

  • Implement flat-field correction to account for illumination non-uniformity

  • Standardize z-stack acquisition parameters when analyzing three-dimensional structures

• Colocalization analysis approaches:

  • Calculate Pearson's correlation coefficient between MYO3B-FITC and actin markers

  • Perform Manders' overlap coefficient analysis for measuring MYO3B association with actin structures

  • Apply intensity correlation analysis to assess spatial relationships between MYO3B and its binding partners like Espin

• Dynamic measurement techniques:

  • Time-lapse imaging to track MYO3B-positive actin protrusion formation and elongation rates

  • Fluorescence recovery after photobleaching (FRAP) to measure MYO3B mobility within actin structures

  • Single-particle tracking of MYO3B-enriched regions to analyze movement along actin filaments

• Quantitative phenotype assessment:

  • Measure apical extension size in cells with normal versus disrupted MYO3B function

  • Quantify the area covered by MYO3B-positive actin protrusions using standardized thresholding

  • Compare protrusion density, length, and lifetime between experimental conditions

• Data analysis considerations:

  • Apply appropriate background subtraction methods consistently across samples

  • Implement batch processing pipelines to ensure identical analysis parameters

  • Use intensity ratio measurements rather than absolute intensity values when possible

  • Verify normal distribution of data and apply appropriate statistical tests

By implementing these methodological approaches, researchers can transform qualitative observations into quantitative measurements, enabling robust statistical analysis of MYO3B's role in actin dynamics and cellular morphogenesis.

What techniques can be used to verify interactions between MYO3B and its binding partners in actin dynamics?

Verifying the interactions between MYO3B and its binding partners requires a multi-technique approach that addresses both physical associations and functional relationships. The following methodologies provide complementary evidence for these interactions:

• Immunoprecipitation-based approaches:

  • Co-immunoprecipitation (Co-IP) using MYO3B Antibody to pull down protein complexes, followed by immunoblotting for binding partners like Espin

  • Reciprocal Co-IP with Espin antibodies to confirm the interaction from both perspectives

  • Proximity-dependent biotinylation (BioID) using MYO3B-BirA* fusion proteins to identify proximal proteins in living cells

• Microscopy-based interaction analyses:

  • Dual-color immunofluorescence using MYO3B Antibody, FITC conjugated and red/far-red labeled antibodies against binding partners

  • Super-resolution microscopy (STORM, PALM) to resolve nanoscale co-localization beyond the diffraction limit

  • Förster Resonance Energy Transfer (FRET) between appropriately labeled MYO3B and binding partners to detect direct molecular proximity (<10 nm)

• Functional interaction assays:

  • Dominant-negative approaches using Myo3b-DN constructs to disrupt endogenous MYO3B function and observe effects on binding partner localization

  • Rescue experiments in MYO3B-depleted cells to determine essential domains for binding partner interactions

  • Live-cell imaging of fluorescently tagged MYO3B and binding partners to track co-transport along actin filaments

• Biochemical characterization:

  • In vitro binding assays using purified recombinant proteins to establish direct interactions

  • Actin co-sedimentation assays to measure MYO3B-dependent recruitment of binding partners to actin filaments

  • Analytical ultracentrifugation to characterize the stoichiometry and affinity of protein complexes

• Genetic manipulation approaches:

  • CRISPR/Cas9-mediated genome editing to generate MYO3B mutants lacking specific interaction domains

  • Examine localization of binding partners like Espin in cells expressing Myo3b-DN, which can reduce Espin apical staining

  • Compare binding partner distribution in wild-type versus espin-/- backgrounds to assess interdependence

By integrating evidence from multiple techniques, researchers can build a comprehensive understanding of how MYO3B interacts with binding partners to regulate actin dynamics in cellular protrusions.

What are the technical challenges in using MYO3B Antibody, FITC conjugated in tissues with high autofluorescence?

Working with MYO3B Antibody, FITC conjugated in tissues with high autofluorescence presents substantial technical challenges that require specialized approaches to obtain reliable results:

• Sources of interfering autofluorescence:

  • Lipofuscin in aged or fixed tissues emits broadly in the same spectral range as FITC

  • Elastin and collagen contribute significant green autofluorescence in connective tissues

  • Fixative-induced fluorescence, particularly with aldehyde-based fixatives like formaldehyde

  • NADH and flavin coenzymes in metabolically active tissues

• Pre-treatment strategies:

  • Sudan Black B treatment (0.1-0.3% in 70% ethanol) to quench lipofuscin autofluorescence

  • Sodium borohydride treatment (0.1-1% in PBS) to reduce aldehyde-induced fluorescence

  • Photobleaching of samples with extended exposure to excitation light before antibody application

  • Copper sulfate treatment (1-10mM CuSO4 in 50mM ammonium acetate) to reduce autofluorescence

• Alternative detection approaches:

  • Consider antibody conjugation to fluorophores with longer emission wavelengths (e.g., Cy3, Cy5) where autofluorescence is typically lower

  • Implement tyramide signal amplification to enhance specific signal relative to background

  • Explore enzyme-based detection methods (e.g., HRP-DAB) as alternatives to fluorescence for highly autofluorescent tissues

• Imaging strategies to improve signal discrimination:

  • Spectral unmixing to computationally separate FITC signal from autofluorescence based on spectral signatures

  • Time-gated detection exploiting the longer fluorescence lifetime of FITC compared to autofluorescence

  • Narrow bandpass filters to selectively capture FITC emission while excluding broader autofluorescence spectra

• Analytical approaches:

  • Implement autofluorescence subtraction using matched unstained control sections

  • Employ ratio imaging between FITC channel and autofluorescence channel

  • Use machine learning algorithms trained to distinguish specific antibody labeling from autofluorescence patterns

• Tissue-specific considerations:

  • For neuronal tissues, consider specialized fixation protocols that preserve MYO3B epitopes while minimizing fixative-induced fluorescence

  • In epithelial tissues where MYO3B is often studied , preserve apical structures while implementing autofluorescence reduction techniques

By systematically addressing these challenges, researchers can overcome autofluorescence limitations and obtain reliable data regarding MYO3B distribution and function even in challenging tissue types.