MYH6 Antibody, HRP conjugated

What is MYH6 and why is it significant in cardiac research?

MYH6 encodes the alpha chain of cardiac myosin, a muscle motor protein with critical actin-binding regions (aa 657-679 and 759-773). This gene plays essential roles in embryonic and adult heart development and muscle contraction. Defects in MYH6 are associated with atrial septal defect 3, hypertrophic cardiac myopathy, and sick sinus syndrome . In adult cardiac tissue, the beta chain isoform (MYH7) predominates in ventricular tissue, while atrial tissue contains both isoforms in variable proportions. Force development is significantly faster in atrial tissue, where MYH6 is more prevalent, with correspondingly higher ATP consumption .

What are the molecular and structural characteristics of MYH6 protein?

MYH6 protein (also called Myosin-6) has a molecular weight of approximately 230 kDa . It functions as part of a hexameric complex consisting of two heavy chain subunits, two light chain subunits, and two regulatory subunits . The heavy chain contains distinct functional domains including motor domains with ATPase activity and actin-binding regions. The protein belongs to the TRAFAC class myosin-kinesin ATPase superfamily . Western blot analysis typically reveals MYH6 as a band at approximately 230 kDa under reducing conditions .

How do monoclonal and polyclonal MYH6 antibodies differ in research applications?

Monoclonal MYH6 antibodies:

• Provide consistent lot-to-lot reproducibility with recognition of a single epitope

• Examples include clone 940344 (MAB8979) and clone 3-48 (NB300-284)

• Offer high specificity, particularly important for distinguishing between MYH6 and MYH7

• Typically produce cleaner background in immunohistochemistry applications

Polyclonal MYH6 antibodies:

• Recognize multiple epitopes, potentially increasing detection sensitivity

• Available in various host species including rabbit (such as Abbexa's polyclonal antibody)

• May provide stronger signals in applications like Western blot

• Show greater tolerance to minor protein denaturation or modifications

The selection should be based on the specific experimental requirements, with monoclonals preferred for precise localization studies and polyclonals often advantageous for detection of denatured proteins .

What are the validated applications for HRP-conjugated MYH6 antibodies?

HRP-conjugated MYH6 antibodies have been primarily validated for ELISA applications , but depending on the specific product, may also be suitable for:

The direct HRP conjugation streamlines workflows by eliminating the need for secondary antibody incubation steps while potentially reducing non-specific binding .

What tissue and sample types have been validated with MYH6 antibodies?

MYH6 antibodies have been successfully validated in multiple sample types:

• Human tissues:

  • Heart (both atrium and ventricle) showing specific localization to cardiomyocyte cytoplasm

  • Prostate tissue (in cancer research investigations)

• Cell models:

  • BG01V human embryonic stem cells differentiated into cardiomyocytes

  • Human iPSC-derived cardiomyocytes

• Animal tissues:

  • Mouse heart tissue (immunohistochemistry)

  • Rat heart (Western blot analysis of atrium and ventricle)

  • Canine cardiac tissues

The antibodies show cytoplasmic staining patterns consistent with the structural role of MYH6 in cardiac muscle tissue .

What are the optimal storage and handling conditions for HRP-conjugated MYH6 antibodies?

For maximum stability and performance of HRP-conjugated MYH6 antibodies:

• Storage temperature:

  • Long-term: -20°C to -70°C (typically in 50% glycerol)

  • Short-term (1 month): 2-8°C under sterile conditions

• Stability considerations:

  • Avoid repeated freeze-thaw cycles (aliquot upon first thaw)

  • Typical shelf-life: 12 months from date of receipt at recommended storage temperature

  • Buffer composition often includes 0.01M PBS, pH 7.4, with 50% glycerol as cryoprotectant

• Handling precautions:

  • Briefly centrifuge before opening the vial to collect contents

  • Protect from prolonged exposure to light to preserve HRP activity

  • Use manual defrost freezers rather than auto-defrost models

How should researchers optimize immunohistochemistry protocols for MYH6 detection?

Optimizing MYH6 immunohistochemistry protocols requires attention to several key factors:

• Antigen retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • For paraffin-embedded sections, complete antigen unmasking is critical due to the large size of the protein

• Antibody dilution:

  • Starting dilution ranges of 1:50-1:200 for IHC-P applications

  • Perform titration experiments to determine optimal concentration for your specific tissue

• Special considerations:

  • When using mouse-derived antibodies (like clone 3-48) on mouse tissues, employ Mouse-on-Mouse blocking reagents to reduce background

  • For HRP-conjugated antibodies, include hydrogen peroxide treatment (0.3% H₂O₂ in methanol for 10-30 minutes) to quench endogenous peroxidase activity

  • Counterstaining with hematoxylin provides good nuclear contrast against the cytoplasmic MYH6 staining

• Controls:

  • Positive control: Human heart atrium tissue shows robust MYH6 expression

  • Negative control: Omitting primary antibody or using non-cardiac tissues like liver

What are the common challenges in Western blotting for MYH6 and how can they be addressed?

Western blotting for MYH6 presents several technical challenges due to its high molecular weight (~230 kDa):

For MYH6 Western blotting, reducing conditions are typically recommended, and human heart atrium lysate serves as an excellent positive control .

How can researchers distinguish between MYH6 and MYH7 in cardiac tissue samples?

Distinguishing between these highly similar cardiac myosin isoforms requires careful experimental design:

• Antibody selection:

  • Choose antibodies validated for isoform specificity

  • Some antibodies (like clone 3-48) recognize both alpha and beta heavy chains

  • For exclusive MYH6 detection, select antibodies raised against unique epitopes not present in MYH7

• Tissue selection strategy:

  • Atrial tissue contains higher proportions of MYH6

  • Ventricular tissue expresses predominantly MYH7

  • Compare expression patterns between these tissues as internal controls

• Experimental approaches:

  • Dual immunofluorescence labeling with isoform-specific antibodies

  • Correlation with mRNA expression (RT-PCR or in situ hybridization)

  • Sequential immunoprecipitation to deplete cross-reactive components

  • For Western blot, run positive controls for both isoforms to confirm band separation

• Distinguishing features:

  • Force development is much faster in atrial tissue (MYH6-rich)

  • ATP consumption is higher in MYH6-dominant tissues

How are MYH6 antibodies used to monitor cardiomyocyte differentiation from stem cells?

MYH6 antibodies serve as important tools for tracking cardiomyocyte differentiation:

• Developmental monitoring:

  • MYH6 expression indicates cardiac differentiation in human embryonic stem cells

  • Allows assessment of differentiation efficiency in iPSC cardiomyocyte protocols

  • Enables temporal tracking of sarcomere formation and maturation

• Validated methods:

  • Immunofluorescence: MYH6 was detected in BG01V human embryonic stem cells differentiated into cardiomyocytes using NorthernLights™ 557-conjugated secondary antibodies

  • Flow cytometry: Quantification of MYH6-positive population percentage during differentiation

  • Western blot: Assessment of expression levels relative to other cardiac markers

• Experimental design considerations:

  • Co-staining with other cardiac markers (troponin T, α-actinin) improves characterization

  • MYH6 antibodies enable identification of early versus mature cardiomyocytes

  • Counterstaining with DAPI facilitates assessment of differentiation efficiency

What is the emerging role of MYH6 in cancer research and how can antibodies contribute?

Recent studies have revealed an unexpected role for MYH6 in cancer biology:

• Tumor suppressor function:

  • MYH6 shows lower expression in prostate cancer tissues compared to normal controls

  • Lower MYH6 gene expression correlates with worse clinical outcomes in prostate cancer patients

  • In vitro and in vivo studies demonstrate that overexpressed MYH6 suppresses proliferation and migration of prostate cancer cells

• Mechanistic insights:

  • MYH6 appears to function by downregulating KIT Proto-Oncogene

  • This relationship was established through RNA-seq analysis and confirmed with rescue assays

• Research applications of MYH6 antibodies:

  • Immunohistochemical assessment of MYH6 expression in tumor versus normal tissues

  • Western blot analysis to confirm overexpression or knockdown in experimental models

  • Immunoprecipitation studies to investigate protein-protein interactions with oncogenic pathways

  • Potential development of prognostic markers based on MYH6 expression patterns

What are the current limitations of MYH6 antibodies and emerging strategies to address them?

Despite their utility, current MYH6 antibodies present several research challenges:

• Cross-reactivity issues:

  • Many antibodies cross-react with MYH7 due to sequence homology

  • Some also detect beta heavy chain of slow human skeletal muscle

  • Solution: Development of antibodies targeting unique sequence regions; complementary use of mRNA-based detection methods

• Specificity validation:

  • Limited knockout/knockdown validation data for many commercial antibodies

  • Recommendation: Perform comprehensive validation using multiple detection methods and appropriate controls

• Application limitations:

  • Many HRP-conjugated MYH6 antibodies are only validated for ELISA

  • Solution: Researcher-conducted validation for additional applications with proper controls

• Emerging approaches:

  • Development of proximity ligation assays for improved specificity

  • CRISPR/Cas9-based gene tagging provides alternative to antibody-based detection

  • Mass spectrometry-based approaches for absolute quantification

  • Single-cell technologies for correlating protein expression with functional parameters

What controls should be included when working with HRP-conjugated MYH6 antibodies?

For rigorous experimental design with HRP-conjugated MYH6 antibodies:

• Positive controls:

  • Human heart atrium tissue shows robust MYH6 expression

  • BG01V human embryonic stem cells differentiated into cardiomyocytes

  • Human heart atrium lysate for Western blot applications

• Negative controls:

  • Omission of primary antibody while maintaining all other steps

  • Non-cardiac tissues such as liver or lung samples

  • Blocking peptide competition assays to confirm specificity

  • Isotype control antibodies (same host species and isotype)

• Procedural controls for HRP-conjugated antibodies:

  • Endogenous peroxidase quenching verification

  • Substrate-only controls to assess endogenous enzymatic activity

  • Dilution series to determine optimal signal-to-noise ratio

• Validation approaches:

  • Comparison with unconjugated primary + HRP-secondary antibody approach

  • Correlation with gene expression data (RT-PCR, RNA-seq)

  • Parallel testing with alternative antibody clones targeting different epitopes

How can researchers determine the optimal dilution for MYH6 antibodies in different applications?

Systematic titration experiments are essential for optimizing MYH6 antibody performance:

• Western blot optimization:

  • Initial dilution range: 1:500-1:2000

  • Load consistent amounts of positive control (human heart lysate)

  • Evaluate signal-to-noise ratio at each dilution

  • Consider exposure time variations to account for detection sensitivity

• Immunohistochemistry dilution strategy:

  • Start with manufacturer's recommended range (typically 1:50-1:300)

  • Prepare serial dilutions (e.g., 1:50, 1:100, 1:200, 1:400)

  • Evaluate staining intensity, background, and specificity

  • For HRP-conjugated antibodies, adjust substrate development time at each dilution

• ELISA optimization:

  • Prepare broader dilution range (1:500-1:10,000)

  • Include standard curves at each antibody dilution

  • Calculate signal-to-noise ratio and determine lower limit of detection

  • Consider coating concentration optimization in parallel

• Documentation and standardization:

  • Record detailed experimental conditions with each optimization

  • Document lot numbers for reproducibility in future experiments

  • Consider secondary antibody titration in parallel (for non-conjugated formats)

  • Evaluate performance across different sample types when applicable

What are the best practices for sample preparation to ensure optimal MYH6 detection?

Sample preparation significantly impacts MYH6 detection quality:

• Tissue fixation and processing:

  • For FFPE sections: 10% neutral buffered formalin fixation for 24-48 hours

  • Optimal section thickness: 4-6 μm for IHC applications

  • Complete deparaffinization and rehydration before staining

  • For frozen sections: flash-freezing in OCT compound after minimal fixation

• Cell preparation for immunocytochemistry:

  • Fixation with 4% paraformaldehyde (10-20 minutes at room temperature)

  • Gentle permeabilization (0.1-0.3% Triton X-100 for 10 minutes)

  • For stem cell-derived cardiomyocytes, fixation timing affects sarcomere preservation

• Protein extraction for Western blot:

  • For heart tissue: Use specialized extraction buffers containing high salt (0.5M NaCl)

  • Include protease inhibitor cocktail to prevent degradation

  • Sample homogenization on ice to preserve protein integrity

  • Western Blot Buffer Group 1 has been validated for MYH6 detection

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Pressure cooker or microwave-based retrieval methods are both effective

  • Optimization of retrieval time (10-30 minutes) based on tissue type and fixation duration

Implementing these methodological approaches will significantly improve the quality and reliability of research using MYH6 antibodies, particularly HRP-conjugated variants.