MYO3B Antibody, Biotin conjugated

What is MYO3B and what cellular functions does it perform?

MYO3B (Myosin-IIIb) is a 151.8 kDa protein belonging to the STE Ser/Thr protein kinase family. The canonical human MYO3B comprises 1341 amino acid residues with up to seven different isoforms reported. It functions as an actin-based motor with protein kinase activity and exhibits subcellular localization in the cytoplasm. MYO3B is predominantly expressed in retina, kidney, and testis tissues. This protein serves as a marker for identifying Midbrain-Derived Inhibitory Neurons and plays crucial roles in signal transduction pathways . Recent research has also implicated MYO3B in cancer progression, particularly in endometrial cancer, through modulation of calcium homeostasis and RhoA/ROCK1 signaling pathways .

What are the optimal storage conditions for MYO3B antibodies?

The MYO3B antibody, especially in its biotin-conjugated form, requires specific storage conditions to maintain functionality. Upon receipt, store the antibody at -20°C or -80°C to preserve activity. Avoid repeated freeze-thaw cycles as these can degrade antibody performance. The antibody is typically supplied in a liquid form with a buffer composition consisting of 0.03% Proclin 300 preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 . This formulation helps maintain structural integrity and binding capacity during storage. For working solutions, refrigeration at 4°C for up to two weeks is generally acceptable, though longer-term storage should revert to freezing temperatures.

What applications are most appropriate for biotin-conjugated MYO3B antibodies?

The biotin-conjugated MYO3B antibody is particularly well-suited for ELISA applications . The biotin conjugation allows for amplified signal detection through streptavidin-based detection systems. While the product information specifically mentions ELISA, similar biotin-conjugated antibodies can also be effectively employed in immunohistochemistry, flow cytometry, and immunofluorescence microscopy with appropriate protocol modifications. The antibody's polyclonal nature (raised in rabbits against recombinant human Myosin-IIIb protein spanning amino acids 1110-1276) provides recognition of multiple epitopes, enhancing sensitivity in detection applications . For researchers requiring exceptionally high specificity, consideration of the antibody's cross-reactivity profile is essential when designing experiments.

How is MYO3B expression quantified in tissue samples?

Quantification of MYO3B expression in tissue samples typically employs a semi-quantitative scoring system based on immunohistochemical staining. This approach classifies staining intensity as none (0 points), low (1 point), medium (2 points), or high (3 points). The proportion of positive tissue is additionally scored as 0-25% (1 point), 26-50% (2 points), 51-75% (3 points), and 76-100% (4 points). The final expression score is calculated by multiplying these two metrics, resulting in grades 0 (0-3 points), 1 (4-6 points), 2 (6-9 points), and 3 (9-12 points). For analytical purposes, grades 0-1 are typically classified as low expression, while grades 2-3 represent high expression . This standardized approach enables consistent comparison of MYO3B levels across different tissue samples and experimental conditions.

What controls should be included when using MYO3B antibodies in research?

When designing experiments with MYO3B antibodies, several controls are essential to ensure result validity. For functional studies involving gene knockdown or overexpression, appropriate control groups must include: (1) untreated controls, (2) negative control vectors (sh-NC for knockdown studies; pcDNA-NC for overexpression studies), and (3) experimental intervention groups (sh-MYO3B for knockdown; pcDNA-MYO3B for overexpression) . For immunodetection assays, positive controls should include tissues known to express MYO3B (retina, kidney, or testis) while negative controls might include tissues with minimal expression or samples processed without primary antibody. When validating antibody specificity, blocking peptide controls or samples from MYO3B knockout models provide definitive validation. For quantitative PCR studies measuring MYO3B transcript levels, housekeeping genes such as GAPDH serve as essential internal controls for normalization .

How should MYO3B antibodies be validated for specificity?

Thorough validation of MYO3B antibodies requires a multi-step approach. First, antibodies should undergo Western blot validation against the recombinant antigen fragment and known MYO3B-expressing cell lines, with attention to band size (expected 151.8 kDa for canonical isoform). Second, knockdown validation through siRNA or shRNA targeting MYO3B should demonstrate reduced signal intensity proportional to transcript reduction measured by qPCR. For example, strategies using multiple shRNA constructs (sh-MYO3B-1 through sh-MYO3B-6) allow selection of optimal knockdown efficiency . Third, immunohistochemical distribution patterns should align with known tissue expression profiles, especially in retina, kidney, and testis . Fourth, immunoprecipitation followed by mass spectrometry can confirm antibody specificity by identifying peptides matching MYO3B sequence. Finally, cross-reactivity against closely related proteins (particularly MYO3A) should be assessed to ensure discrimination between these paralogs .

What is the optimal protocol for MYO3B immunohistochemistry in tissue sections?

For optimal MYO3B immunohistochemical detection in formalin-fixed, paraffin-embedded tissues, follow this validated protocol: Begin with 5 μm tissue sections that undergo deparaffinization through standard xylene and ethanol series. Perform antigen retrieval using citrate buffer (pH 6.0) under pressure or heat-induced conditions. Block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes at room temperature. Apply protein blocking with normal serum (typically 5-10% in PBS) for 30-60 minutes. Incubate sections with primary MYO3B antibody (recommended dilution 1:600 based on published protocols) overnight at 4°C . The next day, apply appropriate HRP-labeled secondary antibody (e.g., goat anti-rabbit at 1:100 dilution) for 30 minutes at 37°C. Develop signal using DAB substrate and counterstain with hematoxylin. This protocol has been successfully employed for detecting MYO3B in endometrial cancer tissues and provides clear visualization of MYO3B expression patterns with minimal background staining .

How can MYO3B function be assessed in cellular models?

Functional assessment of MYO3B in cellular models employs several complementary approaches targeting either gain or loss of function. For knockdown studies, shRNA constructs targeting MYO3B (sh-MYO3B) can be transfected into cells using lipid-based transfection reagents such as Lipofectamine 2000 . Conversely, overexpression studies utilize pcDNA-MYO3B constructs. Following transfection, MYO3B function can be evaluated through multiple assays: (1) Cell proliferation using CCK8 assays, (2) Apoptosis assessment via Annexin V-APC/PI flow cytometry, (3) Intracellular calcium measurement using Fluo-4 AM fluorescent probe and flow cytometry, (4) Cell migration via scratch assays, and (5) Invasion potential through Transwell assays . For mechanistic studies, pharmacological modulators of downstream pathways can be incorporated, such as calmodulin agonists (CALP-2, 57.9 μM) or antagonists (W-7, 28 μM), as well as RhoA pathway modulators (U-46619 agonist at 0.1 μM or Y27632 inhibitor at 10 μM) .

How does MYO3B contribute to cancer progression mechanisms?

Recent research has illuminated MYO3B's role in promoting cancer progression, particularly in endometrial cancer. MYO3B exerts its oncogenic effects through multiple interrelated mechanisms. First, MYO3B modulates intracellular calcium homeostasis, which subsequently activates the RhoA/ROCK1 signaling pathway . This activation leads to reorganization of the actin cytoskeleton, enhancing cancer cell migration and invasion capabilities. Through careful experimental design utilizing both knockdown and overexpression approaches, researchers have demonstrated that MYO3B suppression inhibits cell proliferation, migration, and invasion, while increasing apoptosis rates. Conversely, MYO3B overexpression produces opposite effects . The molecular mechanisms involve altered expression of downstream effectors including RhoA, RhoB, RhoC, ROCK1, LIMK, phosphorylated LIMK, cofilin, and phosphorylated cofilin - all critical regulators of cytoskeletal dynamics and cellular motility . These findings suggest MYO3B may represent a potential therapeutic target in endometrial cancer treatment strategies.

What methods detect interactions between MYO3B and calcium signaling pathways?

Investigating MYO3B's interactions with calcium signaling pathways requires specialized methodological approaches. Flow cytometry using the Fluo-4 AM fluorescent calcium probe provides quantitative assessment of intracellular calcium levels in response to MYO3B modulation . This approach allows for single-cell resolution of calcium dynamics. Complementary to this, pharmacological manipulation using calmodulin agonists (CALP-2 at 57.9 μM) or antagonists (W-7 at 28 μM) helps establish causal relationships between MYO3B, calcium signaling, and downstream cellular effects . For protein-level interactions, co-immunoprecipitation of MYO3B with calcium-regulated proteins such as calmodulin can demonstrate direct physical interactions. Western blot analysis quantifying the expression levels of calcium-related signaling proteins before and after MYO3B knockdown or overexpression further elucidates the molecular mechanisms. For dynamic visualization, confocal microscopy with dual labeling of MYO3B and calcium-binding proteins allows spatial correlation of these factors within cellular compartments.

How can researchers distinguish between different MYO3B isoforms?

Distinguishing between the seven reported MYO3B isoforms requires carefully designed experimental approaches. At the RNA level, isoform-specific RT-PCR utilizing primers that span unique exon-exon junctions can identify specific splice variants. The canonical MYO3B variant and variant 2, which differs in the tail domain, can be distinguished using primers targeting these regions . For protein-level discrimination, Western blot analysis using antibodies targeting different domains can help identify specific isoforms. For instance, the 4J10 mMyo3B antibody, raised against the tail domain (amino acids 1104-1132), can detect multiple isoforms because its immunoreactivity does not require the N-terminal half of the tail domain or the region deleted in variant 2 . For highest resolution discrimination, mass spectrometry analysis of immunoprecipitated MYO3B can identify peptide fragments unique to specific isoforms. Functional studies comparing the activities of different isoforms require cloning of each specific variant into expression vectors, followed by transfection and phenotypic analysis.

What approaches examine MYO3B's role in RhoA/ROCK1 signaling pathways?

Investigating MYO3B's influence on RhoA/ROCK1 signaling requires multiple complementary techniques. Western blot analysis represents the foundation of this research, quantifying expression levels of pathway components including RhoA, RhoB, RhoC, ROCK1, LIMK, p-LIMK, cofilin, and p-cofilin in response to MYO3B modulation . Antibody dilutions range from 1:1000 to 1:5000 depending on the specific target . Immunofluorescence staining provides spatial information on these signaling components within cellular compartments. Functional assessment incorporates pharmacological modulators, with the RhoA agonist U-46619 (0.1 μM) and the RhoA inhibitor Y27632 (10 μM) allowing researchers to determine whether RhoA activation rescues the effects of MYO3B knockdown or whether RhoA inhibition blocks the effects of MYO3B overexpression . Pull-down assays specific for active (GTP-bound) RhoA can measure direct activation status. For in vivo validation, xenograft mouse models using cells with modified MYO3B expression (e.g., stable sh-MYO3B transfected Ishikawa cells) demonstrate the physiological relevance of these pathways in tumor formation and progression .

What factors influence detection sensitivity when using biotin-conjugated MYO3B antibodies?

Multiple factors impact detection sensitivity with biotin-conjugated MYO3B antibodies. First, endogenous biotin in tissues can cause high background signal, necessitating blocking steps with avidin/biotin blocking kits before antibody application. Second, antibody concentration significantly affects signal-to-noise ratio; for ELISA applications, optimization through titration is essential . Third, detection system selection is critical - streptavidin-HRP typically offers superior sensitivity compared to avidin-based systems due to lower non-specific binding. Fourth, incubation conditions including temperature, time, and buffer composition require optimization; overnight incubation at 4°C generally provides improved sensitivity compared to shorter room-temperature incubations. Fifth, sample preparation quality directly impacts results; proper fixation (for tissues) or lysis conditions (for cells) preserves antigen integrity. Sixth, the detection substrate selection (luminescent vs. colorimetric) dramatically affects lower detection limits. Finally, signal amplification strategies, such as tyramide signal amplification, can enhance detection of low-abundance MYO3B expression by orders of magnitude.

How can researchers minimize background in immunohistochemistry with MYO3B antibodies?

Background reduction in MYO3B immunohistochemistry requires systematic optimization. First, thorough blocking with appropriate sera (matching the host species of the secondary antibody) at 5-10% concentration effectively reduces non-specific binding. For biotin-conjugated antibodies, additional blocking with avidin/biotin blocking kits is essential to neutralize endogenous biotin. Second, antibody dilution optimization is critical; although published protocols suggest 1:600 for MYO3B antibodies in IHC , each laboratory should establish optimal working concentrations. Third, wash steps should be thorough (three 5-minute washes between each step) using PBS with 0.05-0.1% Tween-20 to remove unbound antibody. Fourth, endogenous peroxidase blocking with 3% hydrogen peroxide must be complete to prevent non-specific signal development. Fifth, antigen retrieval methods should be optimized; citrate buffer (pH 6.0) under pressure generally provides good results with MYO3B. Sixth, secondary antibody specificity is crucial; using highly cross-adsorbed secondary antibodies reduces cross-reactivity. Finally, counterstain intensity should be adjusted to provide contrast without obscuring specific MYO3B staining.

How should researchers approach cross-reactivity with MYO3A in experimental systems?

Addressing potential cross-reactivity between MYO3B and its paralog MYO3A requires careful experimental design. First, antibody selection is critical; antibodies raised against the tail domain (amino acids 1104-1132 of mMyo3B) provide specificity due to sequence divergence between MYO3A and MYO3B in this region . Second, validation through parallel detection using isoform-specific antibodies helps distinguish true signal from cross-reactivity. For example, anti-mMyo3A antibodies raised against exon 30 (amino acids 1140-1424) specifically target MYO3A epitopes . Third, peptide competition assays using soluble MYO3A or MYO3B peptides can demonstrate antibody specificity by selectively blocking binding. Fourth, genetic approaches using siRNA or shRNA specific to either MYO3A or MYO3B can confirm signal source through selective knockdown. Fifth, recombinant protein expression systems expressing either MYO3A or MYO3B serve as definitive positive controls. Sixth, immunoprecipitation followed by mass spectrometry can unambiguously identify the captured protein. Finally, expression pattern analysis in tissues with differential expression of these paralogs (e.g., specific retinal layers) provides physiological evidence for antibody specificity .

What emerging roles for MYO3B have been identified in neuronal systems?

Recent research has revealed MYO3B's significant functions in neuronal systems. MYO3B serves as a reliable marker for Midbrain-Derived Inhibitory Neurons, providing researchers with a tool for identifying these specialized neuronal populations . Its expression in retinal tissues suggests important roles in visual processing and photoreceptor function . The protein's function as an actin-based motor with protein kinase activity indicates involvement in neuronal cytoskeletal organization, potentially influencing neurite outgrowth, growth cone navigation, and synaptic plasticity. MYO3B's seven isoforms may serve distinct neuronal functions, with differential expression patterns across developmental stages or neuronal subtypes . Understanding these roles has important implications for neurological disorders where cytoskeletal dynamics or protein kinase activity is dysregulated. Current research is investigating whether MYO3B functions similarly to its paralog MYO3A in photoreceptor cells, potentially compensating for MYO3A defects in certain contexts and explaining why Myo3A knockout mice lack severe phenotypes observed in human MYO3A mutations .

How do in vivo models advance understanding of MYO3B function in disease progression?

In vivo models have significantly advanced our understanding of MYO3B's role in disease, particularly cancer progression. Xenograft mouse models using Ishikawa (IK) cells with stable MYO3B knockdown (sh-MYO3B) demonstrate reduced tumor growth compared to negative controls (sh-NC) . The experimental design involves injecting cell suspensions (1 × 10^7 cells/mouse) into the right axilla of mice and monitoring tumor development over 28 days. These models reveal that MYO3B suppression inhibits tumor growth in vivo, validating findings from in vitro studies . Immunohistochemical analysis of tumor tissues shows altered expression of proliferation markers (Ki-67) and pathway components (ROCK1, RhoA, F-actin) in response to MYO3B modulation . These models enable assessment of MYO3B's effects in the complex microenvironment of whole organisms, accounting for factors like vascularization, immune interactions, and tissue architecture that cannot be replicated in cell cultures. Proper ethical oversight, as illustrated by compliance with institutional animal control committees (e.g., approval code KYLL-2024-118), ensures responsible research while advancing understanding of MYO3B's pathophysiological roles .

What technical advances have improved detection of low-abundance MYO3B in research samples?

Technical innovations have significantly enhanced detection of low-abundance MYO3B in research samples. Biotin conjugation represents one such advance, allowing signal amplification through streptavidin-based detection systems, providing greater sensitivity than direct detection methods . Purification techniques have also improved; contemporary MYO3B antibodies undergo rigorous purification (>95% purity through Protein G methods), reducing non-specific interactions . Developments in immunohistochemical scoring systems provide standardized quantification approaches, with clear gradations from none (0 points) to high (3 points) staining intensity, enabling detection of subtle expression differences . Signal amplification technologies, particularly tyramide signal amplification, can enhance detection sensitivity by orders of magnitude through enzymatic deposition of fluorescent or chromogenic substrates. Advances in digital imaging and analysis software enable quantification of weak signals through computational enhancement and background reduction algorithms. Combined methodological approaches—integrating protein detection (Western blot, immunohistochemistry) with transcript analysis (RT-PCR)—provide complementary data streams that collectively confirm low-level expression patterns .

What potential therapeutic applications target MYO3B signaling pathways?

Emerging research suggests several promising therapeutic applications targeting MYO3B signaling pathways. In endometrial cancer, where MYO3B promotes proliferation and invasion, direct inhibition of MYO3B expression through RNA interference approaches (shRNA) demonstrates anti-tumor effects both in vitro and in vivo . MYO3B's role in calcium signaling suggests calcium modulators may offer therapeutic potential; the calmodulin antagonist W-7 (28 μM) counteracts the pro-tumor effects of MYO3B overexpression . Targeting downstream effectors represents another promising strategy; the RhoA inhibitor Y27632 (10 μM) blocks the oncogenic effects of MYO3B overexpression by inhibiting the RhoA/ROCK1 pathway . The protein's kinase domain presents opportunities for small-molecule inhibitor development, potentially allowing selective targeting of MYO3B's catalytic activity. For neurological applications, modulating MYO3B may offer potential in conditions where neuronal cytoskeletal dynamics are dysregulated. As with many emerging therapeutic targets, combinatorial approaches may yield synergistic benefits; for example, simultaneously targeting MYO3B expression and downstream pathways could provide enhanced efficacy in cancer treatment strategies.