MYL7 Antibody

What is MYL7 and why is it significant as a research target?

MYL7, also known as myosin regulatory light chain 7 or myosin light chain 2a (MLC-2a), is a calcium-binding protein essential for heart development. It functions as part of a hexameric complex composed of two heavy chains and four light chains and is predominantly expressed in adult atrial muscle . Its significance stems from:

• Role as a crucial regulator of muscle contraction and relaxation through calcium binding

• Function as an important molecular marker for cardiac chamber specification

• Predominant expression in atrial cardiomyocytes, making it useful for distinguishing atrial from ventricular cells

• High conservation across species (95% amino acid homology between human and mouse)

MYL7's expression pattern in the heart is primarily restricted to the atrium, making it an excellent marker for atrial cardiomyocytes in developmental and disease studies .

Selection between monoclonal and polyclonal MYL7 antibodies should be based on your specific experimental requirements:

Monoclonal antibodies (e.g., sc-365255):

• Offer high specificity to a single epitope of MYL7

• Provide consistent results across different lots

• Ideal for applications requiring high reproducibility such as quantitative Western blots

• Better for detecting specific conformational states of MYL7

Polyclonal antibodies (e.g., 17283-1-AP, ab127001):

• Recognize multiple epitopes on MYL7

• Generally provide stronger signals by binding multiple sites per target molecule

• Better for applications like immunoprecipitation and detecting denatured proteins

• More effective for detecting MYL7 in fixed tissues for IHC

For critical experiments, validation with both antibody types may provide complementary data. Recent recombinant antibody options (e.g., 81570-1-RR) combine advantages of both types with improved consistency .

What are the recommended fixation and antigen retrieval methods for MYL7 immunohistochemistry?

Successful MYL7 immunohistochemistry requires proper fixation and antigen retrieval:

Fixation:

• 4% paraformaldehyde (PFA) for 10 minutes at 37°C has been validated for cell cultures and tissue sections

• For whole-mount preparations (e.g., zebrafish embryos), longer fixation with paraformaldehyde may be necessary

Antigen Retrieval:

• TE buffer pH 9.0 is strongly recommended as the primary method

• Alternative: citrate buffer pH 6.0 if TE buffer does not yield optimal results

• Permeabilization with 0.1% Triton X-100 for 10 minutes followed by blocking with DPBS/0.1% Triton X/1% BSA

Following antigen retrieval, overnight incubation with primary antibody at 4°C has shown optimal results for detection of MYL7 in cardiac tissues .

How can I validate the specificity of an MYL7 antibody?

Validating MYL7 antibody specificity requires a multi-faceted approach:

• Positive control tissues: Use heart tissue (specifically atrial tissue) where MYL7 is abundantly expressed

• Molecular weight confirmation: Verify band detection at the expected molecular weight:

  • Native MYL7: 19 kDa (observed)

  • Full protein has 175 amino acids

• Negative controls:

  • Use tissues known to lack MYL7 expression

  • Include isotype control antibodies in parallel experiments

  • Apply secondary antibody alone to assess background

• Knockdown validation: If available, use MYL7 knockdown models (siRNA, CRISPR) to confirm signal reduction

• Cross-reactivity assessment: Test against related myosin light chains (e.g., MLC2v) to ensure specificity

Advanced validation has been performed for some commercial antibodies using orthogonal RNAseq approaches that correlate protein detection with RNA expression patterns .

How can MYL7 antibodies be used for purification of atrial cardiomyocytes?

Purification of atrial cardiomyocytes using MYL7 antibodies involves:

• Direct FACS approach:

  • Dissociate cardiac tissue into single-cell suspension using enzymatic digestion

  • Perform fixation and permeabilization for intracellular MYL7 staining

  • Use fluorophore-conjugated MYL7 antibodies (e.g., PE, FITC, or Alexa Fluor conjugates)

  • Gate for MYL7-positive cells using flow cytometry

• Reporter system approach:

  • Generate MYL7-GFP reporter cell lines as demonstrated in research using iPSCs

  • Sort MYL7-GFP positive cells using FACS without antibody staining

  • This approach allows for isolation of viable cells for downstream functional assays

• Magnetic separation method:

  • Use MYL7 antibodies conjugated to magnetic beads

  • Perform negative selection to remove non-atrial cell populations first

  • Follow with positive selection using anti-MYL7 antibodies

Research has shown successful isolation of MYL7-positive cardiomyocytes with >90% purity when combining reporter systems with flow cytometry sorting, enabling downstream applications including transcriptomic and epigenomic analyses .

What strategies exist for co-localization studies using MYL7 antibodies with other cardiac markers?

Effective co-localization studies using MYL7 antibodies require careful planning:

• Antibody compatibility considerations:

  • Choose primary antibodies raised in different host species (e.g., rabbit anti-MYL7 with mouse anti-cardiac troponin)

  • For antibodies from the same species, use directly conjugated primary antibodies or sequential staining protocols

• Multi-color immunofluorescence optimization:

  • Use different fluorophores with minimal spectral overlap

  • Consider brightness hierarchy (assign brightest fluorophores to least abundant targets)

  • Validated combinations include:

    • MYL7 (green channel) with MLC2v (red channel) for atrial vs. ventricular differentiation

    • MYL7 with transcription factors (e.g., HAND2) for developmental studies

• Advanced imaging approaches:

  • Super-resolution microscopy for detailed sarcomere structure analysis

  • Live-cell imaging using MYL7-GFP reporter systems

  • 3D reconstruction of confocal z-stacks for tissue architecture analysis

Researchers have successfully used MYL7 co-localization with MLC2v to precisely delineate atrial and ventricular boundaries during cardiac development and in disease models .

How can MYL7 antibodies be integrated into genomic and epigenomic studies?

Integration of MYL7 antibodies into genomic and epigenomic studies enables targeted analysis of cardiac cell populations:

• Cell sorting for downstream analysis:

  • Use MYL7 antibodies or MYL7-reporter systems to purify atrial cardiomyocytes

  • Perform bulk RNA-seq, ATAC-seq, or ChIP-seq on isolated populations

  • Example: Researchers successfully performed ATAC-seq on FACS-purified MYL7-GFP+ cells to identify regulatory elements

• ChIP applications:

  • Use MYL7 antibodies to identify protein-DNA interactions

  • Combine with next-generation sequencing to map genome-wide binding sites

  • Look for transcription factors that regulate MYL7 expression

• Single-cell multi-omics:

  • Integrate antibody-based sorting with single-cell technologies

  • Compare chromatin accessibility patterns between MYL7+ and MYL7- populations

Researchers have utilized MYL7-reporter systems to identify enhancer elements controlling cardiac-specific gene expression, including identifying 22 enriched open chromatin regions upstream of the hand2 transcription start site in myl7 reporter-expressing cells .

How should I troubleshoot cross-reactivity issues between MYL7 and related myosin light chains?

Cross-reactivity troubleshooting for MYL7 antibodies requires systematic analysis:

• Potential cross-reactivity sources:

  • MYL7 shares sequence homology with other myosin light chains, particularly MLC2v (ventricular isoform)

  • Antibodies generated against full-length MYL7 may recognize conserved domains

• Methodological solutions:

  • Western blot analysis using recombinant MYL7 and related proteins to assess specificity

  • Peptide competition assays to confirm epitope specificity

  • Use antibodies targeting unique regions (e.g., N-terminal unique sequences)

  • Consider antibodies validated by orthogonal methods (e.g., RNAseq correlation)

• Validation in knockout/knockdown models:

  • Test antibody in MYL7 knockout or knockdown models

  • Validated CRISPRi systems targeting MYL7 can serve as excellent controls

  • Compare staining patterns with mRNA expression data (in situ hybridization)

When working with antibodies showing potential cross-reactivity, include appropriate controls and consider using multiple antibodies targeting different epitopes to confirm findings.

What are the considerations for using MYL7 antibodies in studying cardiac differentiation from stem cells?

Using MYL7 antibodies in stem cell differentiation studies requires consideration of temporal and developmental factors:

• Temporal expression patterns:

  • MYL7 expression begins early in cardiac differentiation

  • Use time-course immunostaining to track differentiation progression

  • Combine with other stage-specific markers (e.g., GATA4, NKX2.5, cardiac troponins)

• Optimization for iPSC-derived cardiomyocytes:

  • For iPSC-derived cardiomyocytes, perform staining from day 9-15 post-differentiation

  • Consider reporter systems (MYL7-GFP) for live monitoring of differentiation

  • Use FACS with MYL7 antibodies to quantify differentiation efficiency

• Assessment of subtype specification:

  • MYL7 as marker for atrial-like cardiomyocytes

  • Co-staining with MLC2v to distinguish atrial vs. ventricular differentiation

  • Quantitative immunofluorescence to assess relative expression levels

• Functional correlation:

  • Correlate MYL7 expression patterns with electrophysiological properties

  • Calcium imaging in conjunction with MYL7 immunostaining

Researchers have successfully used dual-edited MYL7-GFP/dCas9 iPSC lines to enable genetic screens in differentiated cardiomyocytes, providing a platform for investigating factors controlling cardiac subtype specification .

How can I design experiments to investigate MYL7 phosphorylation states using specific antibodies?

Investigating MYL7 phosphorylation requires specialized experimental approaches:

For detecting dynamics of phosphorylation, time-course experiments following stimulation with agents affecting cardiac contractility can provide insights into regulatory mechanisms.

What approaches are effective when MYL7 antibody results contradict transcriptomic data?

When facing contradictions between MYL7 protein detection and RNA expression:

• Systematic validation:

  • Confirm antibody specificity using multiple approaches (different antibodies, different applications)

  • Verify RNA data using alternative methods (qPCR, in situ hybridization)

  • Consider potential post-transcriptional regulation (miRNAs, RNA binding proteins)

• Technical considerations:

  • Evaluate protein extraction efficiency (membrane-associated proteins can be challenging)

  • Assess protein stability and half-life (some proteins persist after mRNA degradation)

  • Check for post-translational modifications affecting epitope recognition

• Biological explanations:

  • Temporal discrepancies between mRNA and protein expression

  • Cell-type specific translation efficiency

  • Protein localization issues (nuclear vs. cytoplasmic)

• Integrated approaches:

  • Combine immunofluorescence with RNA fluorescence in situ hybridization (RNA-FISH)

  • Perform targeted proteomics with RNA-seq on the same samples

  • Consider single-cell multi-omics to resolve heterogeneity issues

Enhanced validation technologies like orthogonal RNAseq have been developed specifically to address these contradictions, correlating protein detection with RNA expression patterns at single-cell resolution .