MYH14 Antibody

What is MYH14 and why is it significant in cellular research?

MYH14, also known as KIAA2034 and NMHC II-C, belongs to the class II nonmuscle myosins family. It plays crucial roles in various cellular processes including cytokinesis, cell shape maintenance, organelle translocation, ion-channel gating, and cytoskeleton reorganization . MYH14 encodes one of the heavy chains of class II nonmuscle myosins with a calculated molecular weight of approximately 228 kDa, though some antibodies detect it at around 245 kDa .

Despite being relatively low in abundance in certain cell types, MYH14 has profound impacts on cell architecture, behavior, and mechanics. It has been identified as a mechanoresponsive protein that accumulates in response to mechanical stress, making it particularly relevant in cancer research and mechanobiology studies . Additionally, MYH14 has been implicated in hearing function, with mutations in the gene linked to autosomal dominant hearing impairment (DFNA4) .

What are the recommended applications and dilutions for MYH14 antibodies?

MYH14 antibodies have been validated for multiple experimental applications with specific recommended dilution ranges:

For optimal results, it is strongly recommended to titrate the antibody concentration in each specific testing system as sensitivity can vary depending on sample type, detection method, and experimental conditions .

How should MYH14 antibodies be stored and handled to maintain efficacy?

Proper storage and handling of MYH14 antibodies is critical for experimental reproducibility:

Most commercial MYH14 antibodies are supplied in liquid form with specific buffer compositions, typically PBS with 0.02% sodium azide and 50% glycerol at pH 7.3-7.4 . These antibodies should be stored at -20°C where they remain stable for approximately one year after shipment.

For optimal preservation:

• Aliquot antibodies upon receipt to minimize freeze-thaw cycles, which can degrade antibody quality

• Some formulations (20μl sizes) contain 0.1% BSA as a stabilizer

• Aliquoting is generally unnecessary for -20°C storage according to manufacturer guidelines

• Thaw aliquots completely before use and mix gently to ensure homogeneity

• Avoid prolonged exposure to room temperature or light

For working dilutions, prepare fresh solutions on the day of use whenever possible, as diluted antibodies may lose activity over time, especially at higher temperatures.

What are the critical steps for successful Western blot detection of MYH14?

Detecting MYH14 by Western blot requires attention to several critical factors:

Sample Preparation and Protein Extraction:

• MYH14 has been successfully detected in various tissues including human skeletal muscle, mouse colon tissue, and kidney tissues from both mouse and rat

• Cell lines including COLO 320, HEK-293, and HeLa have shown positive detection

• Standard lysis buffers are typically sufficient, though gentle lysis methods help preserve cytoskeletal structure

Gel Electrophoresis Considerations:

• Due to MYH14's high molecular weight (228 kDa calculated, observed at similar size), use low percentage gels (6-8%) or gradient gels

• For distinguishing between myosin isoforms, specialized polyacrylamide gels with high glycerol concentration have been successfully employed

Antibody Incubation Parameters:

• Primary antibody dilutions typically range from 1:1000-1:5000 for standard Western blot

• Some antibodies may require higher concentrations (1:500-1:2000) depending on the specific clone

• Overnight incubation at 4°C often yields better results than shorter incubations

Detection Optimization:

• Use appropriate molecular weight markers that extend to high molecular weight range

• Extended transfer times may be necessary for efficient transfer of this large protein

• Enhanced chemiluminescence (ECL) detection systems provide suitable sensitivity

How can researchers optimize immunohistochemistry protocols for MYH14 detection?

For successful immunohistochemical detection of MYH14:

Antigen Retrieval:

• Heat-induced epitope retrieval with TE buffer at pH 9.0 is strongly recommended

• Alternative retrieval may be performed with citrate buffer at pH 6.0 if necessary

• Optimization of retrieval time and temperature is essential for balancing antigen exposure and tissue preservation

Antibody Dilution and Incubation:

• Recommended dilutions range from 1:400 to 1:1600

• Longer incubation times (overnight at 4°C) often improve specific staining while reducing background

• Use of amplification systems may enhance detection in tissues with lower expression

Controls and Validation:

• Human stomach cancer tissue has been validated for positive MYH14 detection

• Include both positive and negative controls in each experimental run

• Consider dual staining with other cytoskeletal markers to provide context

Signal Development and Visualization:

• Both chromogenic and fluorescence-based detection systems have been successfully employed

• When using DAB as chromogen, carefully optimize development time to avoid over-staining

• Counterstaining protocols should be optimized to provide context without obscuring specific signal

What methodological approaches are available for studying MYH14 in hearing impairment models?

Research into MYH14's role in hearing impairment has employed several specialized approaches:

Gene Expression Analysis:

• RT-PCR has confirmed MYH14 expression in mouse cochlea, with a specific 768 bp fragment amplified from cochlear cDNA

• Comparison of expression levels between wild-type and disease models provides insight into pathological mechanisms

Genetic Mutation Screening:

• DHPLC (Denaturing High-Performance Liquid Chromatography) has been used for initial screening of MYH14 mutations

• Direct sequencing of PCR products showing abnormal chromatographic profiles can identify specific mutations

• Known pathogenic mutations include a nonsense mutation (S7X) and missense mutations affecting evolutionarily conserved residues

CRISPR/Cas9 Knockout Models:

• Successful generation of MYH14 knockout mice using CRISPR/Cas9 technology targeting exon 9 of the Myh14 gene

• Target sequence 5′-CCTGAAGAAAGAGCGCAATA-3′ has been validated for effective knockout

• Verification of knockout through Western blot analysis of cerebellum tissue and immunocytochemistry on cochlea whole mounts

Functional Hearing Assessments:

• Noise exposure experiments have revealed that Myh14−/− mice show increased vulnerability to high-intensity noise compared to control mice

• This model supports the hypothesis that MYH14 plays a protective role in noise-induced hearing loss

How can MYH14 antibodies be used to investigate mechanotransduction in cancer progression?

MYH14 has emerged as an important factor in cancer mechanobiology, particularly in pancreatic cancer:

Expression Analysis in Cancer Tissues:

• MYH14 and other mechanoresponsive proteins (MYH9, α-actinin 4, filamin B) are highly expressed in pancreatic cancer compared to healthy ductal epithelia

• Interestingly, less responsive sister paralogs like myosin IIB (MYH10) show lower expression differential or disappear with cancer progression

Functional Role Assessment:

• Despite its low abundance, MYH14 promotes metastatic behavior in cancer cells

• MYH14 contributes to altered cellular contractility and deformability, providing cancer cells with mechanical flexibility to disseminate through different microenvironments

Pharmacological Manipulation:

• The compound 4-hydroxyacetophenone (4-HAP) increases MYH14 assembly, causing cell stiffening

• This intervention decreases cell dissemination, induces cortical actin belts in spheroids, and slows retrograde actin flow

• In vivo studies show 4-HAP reduces liver metastases in human pancreatic cancer-bearing nude mice

Experimental Design Considerations:

• Immunofluorescence staining can reveal changes in MYH14 localization during cancer progression

• Co-staining with other cytoskeletal components provides context for understanding mechanical changes

• Mechanical testing of cells following manipulation of MYH14 levels or activity offers insight into functional consequences

These findings suggest that targeting mechanoresponsive proteins like MYH14 may represent a novel strategy for inhibiting metastasis in pancreatic cancer.

What approaches can be used to examine MYH14 interactions with other cytoskeletal components?

Investigating MYH14's interactions within the cytoskeletal network requires specialized techniques:

Co-immunoprecipitation Studies:

• MYH14 antibodies have been validated for immunoprecipitation, particularly from mouse kidney tissue

• This approach can identify direct binding partners of MYH14

• Typical protocols use 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

Proximity Labeling:

• BioID or APEX2-based proximity labeling can identify proteins in close spatial relationship with MYH14

• These approaches are particularly valuable for identifying transient or context-dependent interactions

Confocal Microscopy and Co-localization:

• Dual immunofluorescence staining can reveal spatial relationships between MYH14 and other cytoskeletal proteins

• Super-resolution microscopy techniques (STORM, PALM, SIM) provide enhanced resolution of co-localization

• Live-cell imaging with fluorescently tagged proteins can capture dynamic interactions

Functional Interference Studies:

• Comparing phenotypes between MYH14 knockout and knockdowns of interacting proteins

• Using specific inhibitors or activators of potential partner proteins

• Competition experiments with peptides derived from interaction domains

Biomechanical Measurements:

• Atomic force microscopy to measure changes in cell stiffness when MYH14 and potential partners are manipulated

• Traction force microscopy to quantify alterations in force generation

• Microfluidic deformation assays to assess changes in whole-cell mechanics

How can researchers use CRISPR/Cas9 technology to develop effective MYH14 knockout models?

The development of MYH14 knockout models using CRISPR/Cas9 requires careful planning and validation:

Guide RNA Design and Validation:

• Target selection in exon 9 of the Myh14 gene has been validated

• The specific guide sequence 5′-CCTGAAGAAAGAGCGCAATA-3′ has demonstrated efficacy

• sgRNA can be produced by in vitro transcription using T7 promoter and the MEGAshortscript kit

Component Preparation:

• Cas9 mRNA synthesis from pST1374-N-NLS-flag-linker-cas9 using mMESSAGE mMACHINE T7 kit

• Purification of both sgRNA and Cas9 mRNA with MEGAclear kit

• Elution in RNase-free water for optimal quality

Delivery Method:

• Microinjection into pronuclear stage mouse embryos has shown success

• The CBA/CaJ strain background has been validated for MYH14 knockout generation

Mutation Verification Strategy:

• Genotyping using sequence analysis of PCR products

• TA cloning of PCR products for detailed characterization of mutations

• Off-target analysis to confirm specificity of CRISPR editing

Protein-Level Validation:

• Western blot analysis of tissues with high MYH14 expression (e.g., cerebellum)

• Immunocytochemistry on relevant tissues (e.g., cochlea whole mounts)

• Functional assays to confirm phenotypic consequences of knockout

Successfully generated Myh14−/− mouse models have been instrumental in understanding MYH14's role in hearing function, particularly its protective effect against noise-induced hearing loss.

How can researchers validate the specificity of MYH14 antibodies?

Thorough validation is essential for ensuring the reliability of MYH14 antibody experiments:

Genetic Models for Validation:

• Use of MYH14 knockout tissues/cells as negative controls provides the gold standard for specificity testing

• The CRISPR/Cas9-generated Myh14−/− mouse model offers an ideal negative control system

• Western blot analysis of cerebellum tissue from Myh14−/− mice has confirmed complete absence of MYH14 signal, validating antibody specificity

Peptide Competition Assays:

• Preincubation of antibody with excess immunogen peptide should abolish specific signal

• This approach can be applied across applications (WB, IHC, IF)

• Multiple MYH14 antibodies are raised against specific peptides, facilitating this validation approach

Cross-Reactivity Assessment:

• Compare staining patterns across species (human, mouse, rat) where sequence homology is known

• Test against tissues with known differential expression of MYH14 versus other myosin isoforms

• Evaluate recognition patterns of closely related isoforms using recombinant proteins

Multi-antibody Validation:

• Use multiple antibodies targeting different epitopes of MYH14

• Consistent results across different antibodies increase confidence in specificity

• Comparing polyclonal and monoclonal antibodies can provide complementary information

Orthogonal Techniques:

• Correlate protein detection with mRNA expression (RT-PCR, RNA-seq)

• Mass spectrometry confirmation of immunoprecipitated proteins

• Correlation with fluorescently tagged MYH14 in transfection studies

What are common technical challenges when detecting MYH14 in different sample types?

Researchers face several technical challenges when working with MYH14:

High Molecular Weight Detection Issues:

• The large size of MYH14 (228 kDa) requires optimization of transfer conditions in Western blotting

• Incomplete transfer can result in weak or absent signals

• Solutions include extended transfer times, lower percentage gels, and specialized transfer buffers

Variable Expression Levels:

• MYH14 is expressed at lower levels than other myosin isoforms in many tissues

• This necessitates optimization of detection sensitivity

• Loading higher protein amounts or using signal amplification systems may be required

Complex Tissue Architecture:

• In tissues like cochlea, the specialized localization of MYH14 (e.g., in apical junctional complexes) requires careful sectioning and imaging

• Whole mount preparations have proven successful for cochlear immunostaining

• Confocal microscopy may be necessary to resolve specific subcellular localization

Isoform Cross-Reactivity:

• Distinguishing MYH14 from other myosin heavy chains can be challenging

• Specialized electrophoresis conditions with high glycerol concentration help separate myosin isoforms

• Careful antibody selection targeting unique epitopes minimizes cross-reactivity

Fixation Sensitivity:

• Cytoskeletal proteins can be sensitive to fixation conditions

• For immunohistochemistry, antigen retrieval with TE buffer at pH 9.0 is recommended for MYH14

• Alternative retrieval with citrate buffer at pH 6.0 may be necessary for some tissue types

How should researchers interpret discrepancies in MYH14 detection between different experimental methods?

When faced with conflicting results across different detection methods:

Molecular Weight Variations:

• The calculated molecular weight of MYH14 is 228 kDa, but observed weights may vary between 228-245 kDa depending on the antibody and detection system

• Post-translational modifications can alter migration patterns

• Alternative splicing produces multiple isoforms of MYH14 with different sizes (167 kDa/202 kDa/227 kDa/228 kDa/232 kDa)

Discrepancies Between Protein and mRNA Levels:

• Post-transcriptional regulation can lead to differences between mRNA and protein abundance

• MYH14 protein levels may not directly correlate with transcript levels due to differences in translation efficiency or protein stability

• Integrating both protein (antibody-based) and transcript (PCR-based) data provides more comprehensive understanding

Different Antibody Epitopes:

• Antibodies targeting different regions of MYH14 may give discordant results

• Conformation-dependent epitopes may be differentially accessible in various applications

• Comparing results from antibodies recognizing different epitopes can provide complementary information

Application-Specific Factors:

• Denaturation in Western blotting versus native conformation in immunostaining

• Fixation and permeabilization effects on epitope accessibility

• Context-dependent protein interactions masking epitopes

When encountering discrepancies, systematic validation using multiple techniques, antibodies, and controls is essential for accurate interpretation.

How are MYH14 antibodies being used to understand noise-induced hearing loss?

MYH14 antibodies have been instrumental in characterizing the role of this protein in auditory function:

Expression Pattern Analysis:

• Immunohistochemistry using MYH14 antibodies has revealed specific localization in the cochlea, particularly in or near the reticular lamina

• MYH14 immunoreactivity is concentrated in apical junctional complexes (AJCs) of cochlear cells

Knockout Model Validation:

• MYH14 antibodies have confirmed complete absence of protein in Myh14−/− mouse models

• Western blot analysis of cerebellum (where MYH14 expression is high) and immunocytochemistry on cochlea whole mounts have validated knockout efficiency

Structural Integrity Assessment:

• Immunofluorescence studies of cochlear structures before and after noise exposure

• Evaluation of morphological changes in Myh14−/− mice compared to controls

• Analysis of MYH14 redistribution in response to acoustic trauma

Functional Correlations:

• Combining antibody-based protein detection with functional hearing measurements

• Correlating MYH14 expression patterns with susceptibility to noise-induced hearing loss

• Examining potential compensatory mechanisms in MYH14-deficient tissues

These studies have revealed that Myh14−/− mice are more vulnerable to high-intensity noise compared to control mice, suggesting MYH14 plays a protective role in noise-induced hearing loss .

What roles does MYH14 play in cancer progression and how can antibodies help characterize these functions?

MYH14 has emerged as a significant factor in cancer mechanobiology:

Differential Expression Analysis:

• Immunohistochemistry with MYH14 antibodies shows increased expression in pancreatic cancer compared to healthy ductal epithelia

• Unlike some paralogs that disappear with cancer progression, MYH14 expression is maintained or increased

Mechanical Properties Assessment:

• MYH14 contributes to altered cellular contractility and deformability in cancer cells

• These mechanical changes facilitate dissemination through diverse microenvironments

• Immunofluorescence allows visualization of MYH14 distribution changes during metastatic progression

Therapeutic Target Validation:

• The compound 4-hydroxyacetophenone (4-HAP) targets MYH14 assembly

• MYH14 antibodies help visualize changes in protein organization following treatment

• These studies revealed that 4-HAP increases MYH14 assembly, stiffening cells and decreasing their metastatic potential

Structural Reorganization Studies:

• Immunofluorescence analysis of MYH14 in spheroid models shows formation of cortical actin belts following treatment

• This reorganization correlates with reduced invasive capacity

• Co-staining with other cytoskeletal components provides context for understanding mechanical changes

These findings suggest that targeting mechanoresponsive proteins like MYH14 represents a promising strategy for inhibiting metastasis in pancreatic cancer.

How can affinity improvements be made to existing MYH14 antibodies for enhanced detection sensitivity?

Recent advances in antibody engineering provide avenues for improving MYH14 detection:

Epitope Selection Optimization:

• Advanced computational prediction methods can identify optimal epitopes for antibody generation

• Machine learning approaches like MutAb can predict the effect of residue mutations on antibody affinity without requiring antigen-antibody complex structures

• These tools outperform traditional methods when applied to antibodies against novel targets

Affinity Maturation Strategies:

• Directed evolution approaches through display technologies (phage, yeast, or mammalian display)

• Site-directed mutagenesis targeting complementarity-determining regions (CDRs)

• Computational design of affinity-enhancing mutations

Format Modifications:

• Development of recombinant antibody fragments (Fab, scFv) with improved tissue penetration

• Creation of bispecific formats targeting MYH14 and complementary markers

• Incorporation of detection tags for simplified visualization or purification

Signal Amplification Approaches:

• Polymer-conjugated secondary detection systems

• Tyramide signal amplification for immunohistochemistry

• Proximity ligation assays for detecting MYH14 interactions with enhanced sensitivity

These approaches can overcome current challenges in detecting low-abundance MYH14 in certain tissues or experimental conditions, enabling more sensitive and specific analysis of this important cytoskeletal protein.