MYL12B Antibody

What is MYL12B and what cellular functions does it regulate?

MYL12B (myosin light chain 12B, regulatory) is a regulatory subunit of myosin that plays a crucial role in both smooth muscle and nonmuscle cell contractile activity. It functions primarily through phosphorylation mechanisms that regulate myosin II biophysical properties . MYL12B is involved in several key cellular processes:

• Regulation of cell contraction via phosphorylation

• Actin polymerization in vascular smooth muscle

• Cytoskeletal organization and stress fiber formation

• Cell locomotion and migration

• Cytokinesis and receptor capping

MYL12B is located in multiple cellular compartments, including the cytoskeleton (specifically in myosin II complexes and stress fibers), cytosol, and can be found in extracellular exosomes. It is also present in specialized cellular regions such as the apical part of cells, brush border, cell cortex, and Z discs .

What are the standard applications for MYL12B antibodies in laboratory research?

MYL12B antibodies have been validated for multiple research applications according to manufacturer specifications and published literature:

For optimal results in each application, researchers should titrate the antibody concentration in their specific testing system, as results can be sample-dependent .

How should MYL12B antibodies be stored and handled to maintain reactivity?

Proper storage and handling of MYL12B antibodies are critical for maintaining their reactivity and specificity:

• Store at -20°C (most common recommendation across manufacturers)

• Antibodies are typically stable for 12 months after shipment when properly stored

• Aliquoting is often unnecessary for -20°C storage, though some smaller size presentations (e.g., 20μL) may contain BSA (0.1%)

• Avoid repeated freeze/thaw cycles to maintain antibody integrity

• Most formulations contain PBS with sodium azide (typically 0.02%) and glycerol (40-50%) for stability

When receiving shipped antibodies, it's recommended to store them immediately at the recommended temperature upon receipt .

What are the key considerations when selecting between polyclonal and monoclonal MYL12B antibodies?

The choice between polyclonal and monoclonal MYL12B antibodies depends on specific experimental requirements:

Polyclonal MYL12B Antibodies:

• Recognize multiple epitopes on the MYL12B protein

• Often provide stronger signal due to binding of multiple antibodies per target molecule

• Useful for detection of denatured proteins in applications like Western blot

• Examples include Proteintech 10324-1-AP (rabbit) and Abcam ab197929 (rabbit)

Monoclonal MYL12B Antibodies:

• Recognize a single epitope with high specificity

• Provide consistent results between experiments with less batch-to-batch variation

• May have higher specificity for distinguishing between closely related proteins (e.g., MYL12A vs MYL12B)

• Examples include Santa Cruz sc-130331 (mouse IgG2b, clone 29) and Abcam ab137063 (rabbit, clone EPR9331)

For experiments requiring differentiation between MYL12A and MYL12B, researchers should select antibodies specifically validated for this purpose. Some antibodies, like Santa Cruz sc-376606 (clone A-10), are designed to detect both MYL12A and MYL12B proteins .

How can researchers effectively validate MYL12B antibody specificity for their experimental system?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For MYL12B antibodies, consider these validation approaches:

• Western blot with positive and negative controls:

  • Use tissues known to express MYL12B (e.g., mouse skeletal muscle, heart tissue, and rat skeletal muscle)

  • Compare band sizes to the expected molecular weight (18-20 kDa for MYL12B)

  • Consider using CRISPR/Cas9 knockout samples as negative controls (available from vendors)

• Cross-reactivity testing:

  • Test antibody against samples from multiple species if working with non-human models

  • Most MYL12B antibodies have validated reactivity with human, mouse, and rat samples

• Peptide competition assay:

  • Pre-incubate antibody with purified MYL12B protein to block specific binding

  • Compare results with and without peptide competition

• Immunoprecipitation followed by mass spectrometry:

  • For definitive validation, perform IP with the antibody and identify pulled-down proteins by MS

  • Verify that MYL12B is among the identified proteins

• Orthogonal method verification:

  • Compare results with alternative detection methods (e.g., RNA expression, tagged protein expression)

What are the recommended protocols for detecting phosphorylated MYL12B versus total MYL12B?

Detecting phosphorylated versus total MYL12B requires specific methodological considerations:

For Total MYL12B Detection:

• Standard western blotting protocols with general MYL12B antibodies (e.g., Proteintech 10324-1-AP, Abcam ab197929)

• Typical dilutions range from 1:500-1:3000 for WB applications

• Observed molecular weight is usually 18-20 kDa

For Phosphorylated MYL12B Detection:

• Use phospho-specific antibodies such as anti-Mylc2b (phospho S18) antibody (Abcam ab63479)

• Anti-phospho-myosin light chain 2 antibody from Cell Signaling Technology (#3671) has been validated for this purpose

• When detecting phosphorylated MYL12B in tissue/plasma samples:

  • Include phosphatase inhibitors in lysis buffers

  • Use Phos-tag™ SDS-PAGE for enhanced separation of phosphorylated forms

  • Consider using lambda phosphatase treatment as a negative control

Research has shown that phosphorylated MYL12B, rather than total MYL12B, may serve as a more effective biomarker for certain conditions such as sepsis-associated kidney injury (SAKI) . Two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry has been used to successfully identify and distinguish phosphorylated forms of MYL12B .

How does MYL12B phosphorylation impact cellular processes in different tissue contexts?

MYL12B phosphorylation has tissue-specific effects that regulate distinct cellular functions:

In Smooth Muscle and Non-muscle Cells:

• Phosphorylation at Thr18/Ser19 enhances Actin-activated myosin ATPase activity

• This modification is essential for muscle contraction and cellular movements

• Phosphorylation triggers actin polymerization in vascular smooth muscle

• Contributes to regulation of cell shape and cytoskeletal reorganization

In Auditory Development:

• MYL12 phosphorylation by smooth muscle myosin light chain kinase (smMLCK) contributes to apical constriction-like cellular shape change of hair cells

• This process appears to be related to the development of auditory sensory epithelium

In Inflammatory Conditions:

• Phosphorylated MYL12B has been identified as a potential plasma biomarker for sepsis-associated kidney injury (SAKI)

• 2D-DIGE analysis showed that while total MYL12B levels remained unchanged between SAKI and control kidney tissues, phosphorylated MYL12B was significantly increased

Research has identified two groups of residues on MYL12B that can be phosphorylated by distinct kinases, each having contrasting effects on myosin II biophysical properties . This dual regulation mechanism allows for fine-tuned control of cell contractility in different physiological contexts.

What is the role of MYL12B in inflammatory diseases and immune cell recruitment?

Recent research has revealed important functions of MYL12B in inflammatory processes:

As CD69 Ligand in Inflammatory Diseases:

• MYL12A, MYL12B, and MYL9 have been identified as novel ligands for CD69

• This interaction plays a crucial role in recruiting activated CD69+ T cells to inflamed tissues

• Blocking CD69-MYL9/12 interaction reduces allergic airway inflammation in mouse asthma models

• In airway inflammation, MYL9/12 proteins are predominantly found on the vascular surface

In Inflammatory Bowel Disease (IBD):

• Myl9/12 are involved in the pathogenesis of IBD and may represent a new therapeutic target

• Studies have shown that CD69-expressing cells are preferentially located near Myl9/12-positive vessels in inflamed tissues

• Inhibiting the CD69-MYL9/12 interaction decreases leukocyte infiltration and improves airway inflammation

Experimental Methods to Study MYL12B in Inflammation:

• Immunohistochemistry using confocal microscopy with specific antibody combinations:

  • Anti-MYL9/12 (F-6) antibody combined with markers for CD69 and vascular structures (e.g., von Willebrand factor)

  • For human samples: von Willebrand factor (Abcam), anti-Myl9/12 (F-6), and AlexaFluor 647-labeled human CD69 (FN50, BD)

• Quantification methods include counting CD69-expressing cells near Myl9/12-positive or -negative vessels and normalizing to total DAPI-positive cells

What are the technical challenges in distinguishing between MYL12A and MYL12B in experimental systems?

Distinguishing between MYL12A and MYL12B presents significant technical challenges due to their high sequence homology and similar properties:

Key Challenges:

• Protein Similarity: MYL12A and MYL12B have highly similar structures and molecular weights (both approximately 20 kDa)

• Antibody Cross-Reactivity: Many commercially available antibodies recognize both MYL12A and MYL12B (e.g., Santa Cruz sc-376606, sc-28329, sc-48414)

• Functional Redundancy: The proteins share many biological functions, complicating functional validation approaches

• Similar Phosphorylation Sites: Both contain regulatory phosphorylation sites that affect their activities in comparable ways

Recommended Approaches for Differentiation:

• Gene-Specific Targeting:

  • Use CRISPR/Cas9 knockout or RNA interference specifically targeting either MYL12A or MYL12B

  • MYL12B-specific CRISPR/Cas9 plasmids are commercially available (sc-418405)

• Transcript-Level Discrimination:

  • RT-qPCR with gene-specific primers (e.g., Bio-Rad's Unique Assay IDs: dRnoCPE5148326 for MYL12A and dRnoCPE5151868 for MYL12B)

  • RNA-seq analysis to specifically quantify each transcript

• Selective Antibodies:

  • Some antibodies are designed to specifically recognize MYL12B, such as Santa Cruz sc-130331 (clone 29)

  • Validation through western blotting in systems with known differential expression

• Recombinant Expression:

  • Express tagged versions of each protein (e.g., His-tagged MYL12B) for use as positive controls or to develop more specific antibodies

For experimental approaches requiring absolute specificity, researchers should consider using genetic approaches (CRISPR/Cas9 or siRNA) to manipulate expression of individual genes rather than relying solely on antibody-based discrimination.

How can researchers effectively use MYL12B antibodies in multiplex immunofluorescence applications?

Multiplex immunofluorescence with MYL12B antibodies requires careful planning:

Protocol Optimization:

• Antibody Panel Selection:

  • When studying inflammation: combine anti-MYL9/12 (F-6) with anti-CD69 and anti-von Willebrand factor antibodies

  • For cytoskeletal studies: pair MYL12B antibodies with actin markers and other myosin components

  • Consider antibody species compatibility to avoid cross-reactivity

• Antigen Retrieval Optimization:

  • For IHC applications with MYL12B antibodies, use TE buffer pH 9.0 for antigen retrieval

  • Alternative: citrate buffer pH 6.0 can also be effective

• Validated Fluorescent Secondary Antibodies:

  • AlexaFluor 488-labeled anti-rabbit antibodies

  • AlexaFluor 647-labeled anti-goat antibodies

  • AlexaFluor 546 anti-sheep IgG antibodies

• Counterstaining:

  • DAPI for nuclear visualization

  • CellMask Deep Red for cytoplasm staining

Data Analysis Approaches:

• Use ImageJ software for quantitative analysis of multiplex images

• For co-localization studies, calculate Pearson's correlation coefficient between MYL12B and other markers

• For tissue infiltration studies, count the number of positive cells within defined areas (e.g., 200 μm square fields)

What are common problems encountered when using MYL12B antibodies and how can they be resolved?

Researchers may encounter several challenges when working with MYL12B antibodies:

Problem: Weak or No Signal in Western Blot
Potential Solutions:

• Optimize antibody dilution (try 1:500-1:2000 range)

• Verify sample preparation (use tissues with known high expression: skeletal muscle, heart tissue)

• Increase protein loading (start with 40 μg of total protein)

• Extend exposure time (up to 20 seconds has been effective)

• Ensure proper transfer of proteins in the 18-20 kDa range to membrane

Problem: High Background in Immunohistochemistry
Potential Solutions:

• Use more dilute antibody concentrations (1:200-1:500)

• Extend blocking step duration

• Use more stringent washing conditions

• Test alternative antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

Problem: Cross-Reactivity with MYL12A or MYL9
Potential Solutions:

• Select MYL12B-specific antibodies when available (e.g., sc-130331)

• Include appropriate controls (knockout/knockdown samples)

• Validate results with orthogonal methods (e.g., RT-qPCR)

Problem: Inconsistent Results Between Experiments
Potential Solutions:

• Standardize sample preparation protocols

• Use the same lot of antibody when possible

• Include positive controls in each experiment

• Consider using recombinant MYL12B protein as a standard

How can researchers accurately determine phosphorylation status of MYL12B in complex biological samples?

Accurately determining MYL12B phosphorylation status requires specialized techniques:

1. Phospho-Specific Antibody Approaches:

• Use antibodies specifically targeting phosphorylated residues:

  • Anti-Mylc2b (phospho S18) antibody (ab63479)

  • Anti-phospho-myosin light chain 2 (Cell Signaling #3671)

• Always include appropriate controls:

  • Positive control: sample treated with phosphatase inhibitors

  • Negative control: sample treated with lambda phosphatase

2. Advanced Electrophoresis Techniques:

• Two-dimensional differential gel electrophoresis (2D-DIGE) can separate phosphorylated from non-phosphorylated forms

• Phos-tag™ SDS-PAGE enhances mobility shifts of phosphorylated proteins

• Use CyDye labeling for quantitative comparison:

  • Cy3 and Cy5 for experimental samples

  • Cy2 for internal standards

3. Mass Spectrometry-Based Approaches:

• Enrich for phosphopeptides using:

  • Titanium dioxide (TiO2) chromatography

  • Immobilized metal affinity chromatography (IMAC)

• Analyze using MALDI-TOF/TOF MS to identify phosphorylation sites

4. Kinase Activity Assays:

• In vitro kinase assays using purified MYL12B (e.g., nHis-tagged MYL12B)

• Monitor phosphorylation by relevant kinases (MLCK, ROCK)

• Detect phosphorylation by western blot or mass spectrometry

Research has demonstrated that phosphorylated MYL12B levels may change independently of total MYL12B levels in certain pathological conditions, highlighting the importance of specifically monitoring phosphorylation status .

What are the best practices for quantifying MYL12B expression levels across different experimental models?

Accurate quantification of MYL12B expression requires systematic approaches:

For Protein-Level Quantification:

• Western Blot Quantification:

  • Always include loading controls (β-actin, GAPDH)

  • Use standard curves with recombinant MYL12B protein

  • Analyze band intensity with appropriate software (ImageJ, Image Studio Lite)

  • Report results as relative expression normalized to controls

• ELISA-Based Quantification:

  • Commercial MYL12B proteins show activity in functional ELISA

  • Example: Human MYL12A at 2μg/mL binding to Anti-MYL9 recombinant antibody (EC50: 5.325-6.456 ng/mL)

• Mass Spectrometry-Based Proteomics:

  • Use stable isotope labeling (SILAC, TMT) for accurate quantification

  • Include internal standards for absolute quantification

  • Account for post-translational modifications in analysis

For Transcript-Level Quantification:

• RT-qPCR Analysis:

  • Use validated primer sets for MYL12B

  • Normalize to stable reference genes (e.g., Tbp)

  • Report results using the 2^-ΔΔCt method or standard curves

• Digital PCR:

  • Provides absolute quantification without standard curves

  • Less susceptible to inhibitors than traditional qPCR

Best Practices Across Experimental Models:

• Tissue Samples:

  • Process samples consistently (flash freezing, consistent lysis protocols)

  • Account for tissue heterogeneity

  • Verify with immunohistochemistry for spatial distribution

• Cell Lines:

  • Standardize cell culture conditions and passage numbers

  • Account for confluence effects on cytoskeletal proteins

  • Use early passage cells when possible

• Animal Models:

  • Match for age, sex, and genetic background

  • Control for circadian variations

  • Consider tissue-specific expression patterns

• Statistical Analysis:

  • Use appropriate statistical tests for the experimental design

  • Report both biological and technical replicates

  • Consider power analysis to determine sample size

How might MYL12B be utilized as a biomarker or therapeutic target in inflammatory and autoimmune diseases?

Based on current research, MYL12B shows promise as both a biomarker and therapeutic target:

As a Biomarker:

• Phosphorylated MYL12B has been identified as a potential plasma biomarker for sepsis-associated kidney injury (SAKI)

• Increased MYL9/12 expression has been observed in inflammatory lesions of nasal polyps in patients with eosinophilic chronic rhinosinusitis

• The CD69-MYL9/12 interaction could serve as a biomarker for inflammatory cell infiltration into tissues

As a Therapeutic Target:

• Blocking CD69-MYL9/12 interaction reduces allergic airway inflammation in mouse asthma models

• Inhibiting this interaction decreases leukocyte infiltration and improves inflammation outcomes

• MYL9/12 has been identified as a potential therapeutic target for patients suffering from inflammatory bowel disease (IBD)

Future Research Directions:

• Development of Specific Inhibitors:

  • Design small molecules or peptides that specifically disrupt CD69-MYL9/12 interactions

  • Target MYL12B phosphorylation pathways (MLCK, ROCK) in a tissue-specific manner

• Validation in Additional Disease Models:

  • Expand studies to other inflammatory conditions (rheumatoid arthritis, multiple sclerosis)

  • Investigate the role of MYL12B in cancer progression and metastasis

  • Explore connections to myocardial injury and cardiac disorders

• Advanced Diagnostic Applications:

  • Develop assays to detect phosphorylated MYL12B in biofluids as diagnostic tools

  • Create multiplex panels combining MYL12B with other inflammatory markers

  • Investigate the utility of MYL12B as a prognostic indicator for treatment response

The growing understanding of MYL12B's role in regulating inflammation through CD69 interactions provides a strong foundation for these future applications. Continued research into tissue-specific functions and regulatory mechanisms will be essential for translating these findings into clinical applications.

What emerging technologies might enhance our understanding of MYL12B function in cellular mechanics and tissue homeostasis?

Several cutting-edge technologies show promise for advancing MYL12B research:

1. Advanced Imaging Technologies:

• Super-resolution microscopy (STORM, PALM, SIM) to visualize MYL12B dynamics at nanoscale resolution

• Lattice light-sheet microscopy for long-term, low-phototoxicity imaging of MYL12B in living cells

• Förster resonance energy transfer (FRET) sensors to monitor MYL12B phosphorylation and protein interactions in real-time

• Correlative light and electron microscopy (CLEM) to connect MYL12B localization with ultrastructural features

2. Genetic Engineering Approaches:

• CRISPR/Cas9 gene editing for generating tissue-specific knockout models of MYL12B

• CRISPR activation/interference (CRISPRa/CRISPRi) systems for temporal control of MYL12B expression

• Knock-in of fluorescent tags at endogenous loci to track native MYL12B without overexpression artifacts

3. Single-Cell Analysis Methods:

• Single-cell RNA sequencing to identify cell populations with differential MYL12B expression

• Single-cell proteomics to measure MYL12B levels and phosphorylation states in heterogeneous samples

• Spatial transcriptomics to map MYL12B expression patterns within tissue contexts

4. Biophysical Techniques:

• Atomic force microscopy (AFM) to measure changes in cellular mechanics associated with MYL12B activity

• Traction force microscopy to quantify how MYL12B phosphorylation affects cellular force generation

• Optical tweezers to measure molecular forces in myosin-actin interactions regulated by MYL12B

5. Structural Biology Approaches:

• Cryo-electron microscopy of myosin complexes containing MYL12B

• Hydrogen/deuterium exchange mass spectrometry to analyze conformational changes upon MYL12B phosphorylation

• X-ray crystallography of MYL12B in complex with binding partners like CD69

These technologies, particularly when used in combination, will provide unprecedented insights into the spatial and temporal dynamics of MYL12B function in complex biological systems, potentially revealing new therapeutic targets and diagnostic applications.