MYL9 Recombinant Monoclonal Antibody

What is MYL9 and what are its primary biological functions?

MYL9 (Myosin Regulatory Light Chain 9) functions as a versatile regulatory subunit of myosin II in both muscle and non-muscle cells. Its primary biological roles include regulating muscle contraction, controlling cell motility, and participating in various cellular processes associated with changes in cell shape and movement. The phosphorylation of MYL9 and its interactions with other proteins are central to its functions in these processes . MYL9 is also implicated in cytokinesis, receptor capping, and cell locomotion, as documented in multiple studies (PubMed:11942626, PubMed:2526655). Additionally, in myoblasts, it may regulate PIEZO1-dependent cortical actomyosin assembly involved in myotube formation .

How are MYL9 recombinant monoclonal antibodies produced?

The production of MYL9 recombinant monoclonal antibodies involves a multi-step process:

• Integration of MYL9 antibody genes into plasmid vectors

• Introduction of these engineered vectors into suitable host cells using exogenous protein expression techniques

• Production of antibodies by the host cells

• Purification via affinity chromatography

This process ensures the generation of highly specific antibodies with consistent performance across batches . The recombinant production method offers advantages over traditional hybridoma techniques, including improved reproducibility and reduced batch-to-batch variation.

What applications are MYL9 recombinant monoclonal antibodies validated for?

MYL9 recombinant monoclonal antibodies have been validated for multiple research applications:

These applications have been confirmed through rigorous testing, with specific detection limits and binding characteristics documented .

What is the optimal protocol for Western blot analysis using MYL9 antibodies?

For optimal Western blot analysis with MYL9 antibodies, the following protocol is recommended based on validated experimental data:

• Prepare protein lysates (40 μg of protein per lane is typically sufficient)

• Separate proteins using 4-20% SDS-PAGE gel

• Transfer proteins to a PVDF membrane

• Block with 5% non-fat dry milk (NFDM) in TBST for 1 hour at room temperature

• Incubate with primary MYL9 antibody at 1:1,000 dilution in 5% NFDM/TBST for 2 hours at room temperature

• Wash with TBST

• Incubate with an appropriate HRP-conjugated secondary antibody (e.g., Goat Anti-Rabbit IgG-HRP) at 1:100,000 dilution for 1 hour at room temperature

• Visualize using a suitable detection method

This protocol has been demonstrated to produce clear bands at approximately 19 kDa in various tissue lysates, including HeLa cells, mouse colon, rat colon, and rat uterus tissues .

How should immunohistochemistry be performed with MYL9 antibodies?

For effective immunohistochemistry using MYL9 antibodies, follow this validated protocol:

• Prepare paraffin-embedded tissue sections

• Perform heat-mediated antigen retrieval using Tris-EDTA buffer (pH 9.0) for 20 minutes

• Block tissues in 1% BSA for 20 minutes at room temperature

• Wash with ddH₂O and PBS

• Probe with primary MYL9 antibody at 1:1,000 dilution for 1 hour at room temperature

• Detect using an HRP-conjugated compact polymer system

• Develop with DAB as the chromogen

• Counterstain tissues with hematoxylin

• Mount with DPX

This protocol has been successfully used for detecting MYL9 in human colon tissue, with specific cellular localization patterns observed .

What are the common issues encountered when using MYL9 antibodies and how can they be resolved?

Several technical challenges may arise when working with MYL9 antibodies:

• Non-specific binding or high background:

  • Solution: Optimize blocking conditions (try different blocking agents like 3-5% BSA or 5% normal serum)

  • Increase washing steps and duration

  • Titrate antibody concentration to determine optimal dilution

• Weak or no signal in Western blot:

  • Solution: Ensure sufficient protein loading (40 μg/lane recommended)

  • Verify transfer efficiency

  • Consider longer primary antibody incubation (overnight at 4°C)

  • Check for protein degradation in your samples

• Inconsistent IHC staining:

  • Solution: Optimize antigen retrieval conditions (Tris-EDTA buffer at pH 9.0 has been validated)

  • Control fixation time of tissues

  • Ensure consistent section thickness

These troubleshooting approaches are based on experimental evidence from successful MYL9 antibody applications .

How can researchers validate the specificity of MYL9 antibodies?

Validating antibody specificity is crucial for reliable research results. For MYL9 antibodies, consider these validation approaches:

• Knockout validation: Use wild-type and MYL9 knockout samples in parallel. The antibody ab191393 has been validated using human MYL9 knockout HeLa cell lines, showing clear loss of signal in knockout samples .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples. Specific binding should be blocked by the peptide.

• Cross-reactivity testing: Test the antibody against multiple species samples if performing cross-species studies. The homology between human, mouse, and rat MYL9 is high, but validation is still recommended .

• Multiple antibody approach: Confirm results using antibodies targeting different epitopes of MYL9. Several clones are available, including JE34-63 (targeting aa 51-100), EPR13012(2), and SR1294 (targeting a synthetic peptide derived from human MYL9) .

What are the appropriate storage conditions for MYL9 antibodies to maintain activity?

To maintain optimal antibody activity and prevent degradation, proper storage is essential:

• Store MYL9 antibodies at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles, which can lead to decreased activity

• Some preparations are provided in PBS, pH 7.4, with 150mM NaCl and 50% glycerol to help maintain stability during freeze-thaw cycles

• For short-term use (1-2 weeks), antibodies can be stored at 4°C

• Always centrifuge briefly before opening antibody vials to collect liquid at the bottom

Following these storage recommendations will help maintain antibody activity and extend the usable life of your reagents .

How do phosphorylation states of MYL9 affect antibody recognition?

• Phosphorylation-state specific antibodies may be necessary for studying active vs. inactive MYL9

• MYL9 can be phosphorylated at multiple sites, including Ser19 and Thr18, with different functional outcomes

• When analyzing phosphorylation-dependent functions, phosphatase inhibitors should be included in lysis buffers

• For functional studies, correlation between phosphorylation status and observed cellular phenotypes should be established

Understanding these considerations is essential for advanced research into MYL9's role in mechanotransduction and cellular contractility .

What experimental designs are recommended for studying MYL9 interactions with other proteins?

For investigating MYL9 protein interactions, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): MYL9 antibodies have been validated for immunoprecipitation at a 1:50 dilution, making them suitable for Co-IP studies to identify binding partners .

• Proximity Ligation Assay (PLA): For detecting in situ protein interactions within cells or tissues with spatial resolution.

• FRET/BRET analysis: For studying dynamic interactions in living cells.

• Pull-down assays: Using recombinant MYL9 as bait to identify novel interacting partners.

• Cross-linking studies: To capture transient or weak interactions.

When designing these experiments, consider the cellular localization of MYL9 (cytoplasm, cytoskeleton, cell cortex) and the potential impact of experimental conditions on protein interactions .

How can researchers effectively use MYL9 antibodies in multiplex immunofluorescence studies?

For multiplex immunofluorescence studies involving MYL9:

• Selection of compatible antibodies: Choose MYL9 antibodies raised in different host species from your other target antibodies to avoid cross-reactivity.

• Antibody panel design:

  • Consider the subcellular localization of MYL9 (cytoplasm, cytoskeleton) when selecting other markers

  • Include appropriate controls for each antibody in the panel

  • Validate each antibody individually before multiplexing

• Signal amplification strategies: For low-abundance targets, consider using signal amplification systems compatible with multiplexing.

• Spectral unmixing: For complex panels, use imaging systems with spectral unmixing capabilities to resolve overlapping fluorophore emissions.

The rabbit monoclonal antibodies (clones JE34-63, EPR13012(2), and SR1294) are particularly suitable for such applications due to their high specificity and sensitivity .

What considerations should be made when using MYL9 antibodies for cross-species studies?

When planning cross-species studies using MYL9 antibodies:

• Sequence homology analysis: The interspecies sequence homology shows high conservation between human, mouse (62% homology), and rat (61% homology) MYL9 .

• Epitope-specific validation: Antibodies targeting the region aa 51-100 of human MYL9 (like clone JE34-63) have been validated for cross-reactivity with mouse and rat samples .

• Application-specific testing: An antibody that works for Western blot in one species may not work for IHC in another. Validate each application separately.

• Positive controls: Include positive control samples from each species in your experiments.

• Dilution optimization: Optimal working dilutions may vary between species; titration experiments are recommended.

Western blot analysis has confirmed cross-reactivity of certain MYL9 antibodies with mouse colon, rat colon, and rat uterus tissue lysates, demonstrating their utility in comparative studies .

How are MYL9 antibodies being used in mechanobiology research?

MYL9's role in cell contractility and mechanotransduction makes its antibodies valuable tools in mechanobiology research:

• Cellular response to substrate stiffness: MYL9 phosphorylation status can be monitored using specific antibodies to assess how cells respond to different mechanical environments.

• Force-induced cytoskeletal reorganization: Immunofluorescence with MYL9 antibodies can reveal changes in actomyosin distribution under mechanical stress.

• Mechanosensitive signaling pathways: In myoblasts, MYL9 may regulate PIEZO1-dependent cortical actomyosin assembly involved in myotube formation, suggesting roles in mechanosensitive development processes .

• 3D culture systems: MYL9 antibodies can be used to compare cytoskeletal organization between 2D and 3D culture environments.

Researchers should select antibodies validated for their specific application (WB, IHC, IF) when designing mechanobiology experiments .

What methodological considerations are important when using MYL9 antibodies in live-cell imaging?

While most applications of MYL9 antibodies focus on fixed cells or tissues, adaptations for live-cell imaging require special considerations:

• Antibody fragment preparation: Consider using Fab fragments derived from MYL9 antibodies to improve cell penetration and reduce interference with protein function.

• Cell delivery methods: Microinjection, cell-penetrating peptides, or temporary permeabilization may be necessary for antibody delivery.

• Functional impact assessment: Ensure that antibody binding does not interfere with MYL9 function, particularly phosphorylation events.

• Alternative approaches: Consider genetic approaches using fluorescently-tagged MYL9 constructs as alternatives to antibody-based detection in live cells.

• Fixation comparison studies: Always validate live-cell observations with parallel fixed-cell experiments using validated antibody protocols.

These methodological considerations will help researchers obtain reliable data while minimizing artifacts in dynamic cellular processes involving MYL9 .